Studies in Natural Products Chemistry Volume 32 Bioactive Natural Products (Part L)
Studies in Natural Products Chemistry edited by Atta-ur-Rahman
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Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidation (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure and Chemistry (Part B) Stereoselective Synthesis (Part F) Stereoselective Synthesis (Part G) Stereoselective Synthesis (Part H) Bioactive Natural Products (Part A) Stereoselective Synthesis (Part I) Structure and Chemistry (Part C) Stereoselective Synthesis (Part J) Structure and Chemistry (Part D) Stereoselective Synthesis (Part K) Structure and Chemistry (Part E) Structure and Chemistry (Part F) Bioactive Natural Products (Part B) Bioactive Natural Products (Part C) Bioactive Natural Products (Part D) Bioactive Natural Products (Part E) Bioactive Natural Products (Part F) Bioactive Natural Products (Part G) Bioactive Natural Products (Part H) Bioactive Natural Products (Part I) Bioactive Natural Products (Part J) Bioactive Natural Products (Part K) Studies in Natural Products Chemistry: Cumulative Indices Vol. 1-30 Bioactive Natural Products (Part L)
Studies in
Natural Products Chemistry Volume 32 Bioactive Natural Products (Part L)
Edited by
Atta-ur-Rahman H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
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FOREWORD Natural product chemistry continues to expand to exciting new frontiers of great importance in medicine. Advances in spectroscopic techniques such as NMR and mass spectroscopy coupled with new developments in high throughput screening techniques have opened up new horizons for discovery of bioactive substances. The pharmaceutical industry has been using these developments to a great extent which is reflected from the growing number of patents based on pharmacophores derived from natural sources. Natural product chemistry offers some significant advantages in comparison to combinatorial synthetic methods now being increasingly used for preparing bioactive compounds of different structural types. The wide range of structures found in terrestrial and marine organisms offer exciting opportunities for the discovery of new pharmacophores which can reveal novel mechanisms to tackle diseases. Volume 32 of "Studies in Natural Products Chemistry" contains 23 comprehensive articles written by international authorities in various fields of natural product chemistry ranging from immunosuppressant and antimalarial compounds to bioactive substances useful in cancer and neural diseases. It is hoped that the present volume, which is the 32nd of the Series, which I initiated in 1988 will again be of great interest to research scientists and scholars working in the exciting field of new drug discovery.
I would like to express my thanks to Mr. Liaquat Raza and Ms. Qurat-ul-Ain Fatima for their assistance in the preparation of the index. I am also grateful to Mr. Wasim Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.
Atta-ur-Rahman Ph.D. (Cantab.), Sc.D. (Cantab.) Federal Minister/ Chairman Higher Education Commission Government of Pakistan
July 2005
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Vll
PREFACE The study of natural products, or "Nature's Combinatorial Library" has had a long history as a source of drugs. In the anticancer area, for example, vinblastine and vincristine, etoposide, paclitaxel (Taxol™), docetaxel, topotecan, irinotecan, the anthracyclines, the bleomycins and the mitomycins are all clinically used natural products or natural product derivatives. In addition to these compounds other natural products or natural product analogs are in advanced clinical trials as anticancer agents, including several epothilones and the halichondrin analog E7389. A review of all new small chemical entities introduced as drugs between 1981 and 2002 showed that 33% of these were natural products or derivatives of natural products, and the figure rises to 49% if synthetic compounds based on natural product models are included (Newman et al., J. Nat. Prod. 2003, 66, 1022). In spite of this impressive record of success, the study of natural products as potential Pharmaceuticals or agrochemicals has lost some favor, particularly within the pharmaceutical industry, as resources were diverted to the newer technique of combinatorial chemistry and other new areas of research. Fortunately, the study of bioactive natural products continues to flourish in universities, research institutes, and selected pharmaceutical companies around the world, and this latest volume in the wellestablished series "Studies in Natural Products Chemistry" bears eloquent testimony to the continued vitality of natural products research. The opening chapter sets the stage with a review of the synthesis of the immunosuppressant FR901483. One of the potential problems with natural products as Pharmaceuticals is that of drug supply, but this chapter demonstrates that moderately complex compounds can be synthesized efficiently. An even more dramatic example of the use of synthesis is that of the halichondrin analog E7389 referred to above, and drug supply is thus less of an issue than it was in the past. The marine environment has become an important source of new structures and new activities, and this is reflected in the next three chapters, which review bioactive natural products from South African marine invertebrates, bioactive marine sesterterpenoids, and antimalarial leads from marine organisms. The ready accessibility of plants ensures that these sources of bioactivity will continue to be thoroughly investigated, and there are chapters covering saponins, iridoids, sesquiterpenoids, cucurbitacins, phthalides, polyisoprenylated benzophenones, and simple benzophenones. Several chapters review the constituents of specific plant genera or families; these include Trypterygium wilfordii, Erythrina, Aristolochia, and the Solanaceae family. It is interesting to note that even well-known compounds and compound classes can provide novel bioactivities. Thus the iridoid geniposide can inhibit angiogenesis, certain cucurbitacins have anti-inflammatory properties, and both garcinol and some withanolides have cancer chemopreventive activity. It is also encouraging to note that a derivative of the well-known compound triptolide is in Phase I clinical trials as an anticancer agent. Of course, not all bioactivities are beneficial; many Illicium sesquiterpenoids are neurotoxic, and aristolochic acid from Aristolochia sp. is responsible for the symptoms of Chinese herb neuropathy recognized in 1992. Microbial sources are making an increasingly important contribution to bioactive natural products, and these sources are represented by chapters on griseofulvin and other
vi halogenated compounds and on bioactive alkaloids from fungi, and by a chapter on metabolites from extremophiles collected in the Berkeley acid mine waste pit. The use of extremophile organisms opens up exciting possibilities for new structures and new activities; in this case novel polyketide-terpenoid metabolites were isolated from a Penicillium sp. with selective cytotoxicity in the National Cancer Institute's 60-cell line screen. The remaining three chapters cover an assortment of topics, from studies of plants used in Bantu medical and magic practices to metabolites from oomycete phytopathogens to isoflavones as functional food components. The variety of the compounds and activities covered in this volume is renewed evidence of the structural and pharmacological diversity of natural products and of the strength of natural products research. This work thus celebrates the truth, to paraphrase Mark Twain, that "reports of the death of natural products research have been greatly exaggerated". The reader is invited to join in the celebration.
David G. I. Kingston Department of Chemistry Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061, USA
IX
CONTENTS Foreword
v
Preface
vii
Contributors
xi
Synthesis of immunosuppressant FR901483 and biogenetically related TAN1251 alkaloids JOSEP BONJOCH AND FAIZA DIABA
3
Bioactive natural products from southern african marine invertebrates MICHAEL T. DAVIES-COLEMAN
61
Bioactive marine sesterterpenoids SALVATORE DE ROSA AND MAYA MITOVA
109
Antimalarial lead compounds from marine organisms ERNESTO FATTORUSSO AND ORAZIO TAGLIALATELA-SCAFATI
169
Bioactive saponins with cancer related and immunomodulatory activity: Recent developments MARIE-ALETH LACAILLE-DUBOIS
209
Chemical and biological aspects of iridoid bearing plants of temperate region NEERAJ KUMAR, BIKRAM SINGH, V.K. KAUL AND P.S. AHUJA
247
Iridoids and secoiridoids from Oleaceae JOSE A. PEREZ, JOSE M. HERNANDEZ, JUAN M. TRUJILLO AND HERMELO L6PEZ
303
Pharmacological activities of iridoids biosynthesized by route II MARINA GALVEZ, CARMEN MARTIN-CORDERO AND MARIA JESUS AYUSO
365
Chemistry and neurotrophic activity of .seco-prezizaane- and anislactone-type sesquiterpenes from Illicium species YOSHIYASU FUKUYAMA AND JIAN-MEI HUANG
395
New insights into the bioactivity of cucurbitacins JOSE LUIS RIOS, JOSE M. ESCANDELL AND M. CARMEN RECIO
429
Griseofulvin and other biologically active halogen containing compounds from fungi T. REZANKA AND J. SPIZEK
471
Bioactive alkaloids of fungal origin HIDEO HAYASHI
549
Chemistry and biological activities of naturally occurring phthalides GE LIN, SUNNY SUN-KIN CHAN, HOI-SING CHUNG AND SONG-LIN LI
611
Chemistry and biological activity of polyisoprenylated benzophenone derivatives OSMANY CUESTA-RUBIO, ANNA LISA PICCINELLI AND LUCA RASTRELLI
671
The benzophenones: Isolation, structural elucidation and biological activities SCOTT BAGGETT, EUGENE P. MAZZOLA AND EDWARD J. KENNELLY
721
Bioactive compounds from Tripterygium wilfordii RENSHENG XU, JOHN M. FIDLER AND JOHN M. MUSSER
773
Bioactive natural compounds from medico-magic plants of bantu area BLANDINE AKENDENGUE, GUY JOSEPH LEMAMY, HENRI BOUROBOU BOUROBOU AND ALAIN LAURENS
803
Bioactive non-alkaloidal constituents from the genus Erythrina RUNNER R.T. MAJINDA, CORNELIUS C.W. WANJALA AND BENARDF. JUMA
821
Chemical constituents and pharmacology of Aristolochia species TIAN-SHUNG WU, AMOORU G. DAMU, CHUNG-REN SU AND PING-CHUNG KUO
855
Chemistry and bioactivity of withanolides from South American Solanaceae ADRIANA S. VELEIRO, JUAN C. OBERTI AND GERARDO BURTON
1019
Bioactive secondary metabolites related to life-cycle development of oomycete phytopathogens MD. TOFAZZAL ISLAM AND SATOSHI TAHARA
1053
Bioprospecting in the Berkeley PIT: Bioactive metabolites from acid mine waste extremophiles ANDREA A. STIERLE AND DONALD B. STIERLE
1123
Isoflavones as functional food components F.R. MARIN, J.A. PEREZ-ALVAREZ AND C. SOLER-RIVAS
1177
Subject Index
1209
XI
CONTRIBUTORS P.S. Ahuja
Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India
Blandine Akendengue
Departement de Pharmacologie, Faculte de Medecine, Universite des Sciences de la Sante, B.P. 7464 Libreville, Gabon
Maria Jesus Ayuso
Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain
Scott Baggett
Department of Biological Sciences, Lehman College and The Graduate Center, The City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA
Josep Bonjoch
Laboratori de Quimica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028Barcelona, Spain
Henri Bourobou Bourobou
Centre National de Recherche Scientifique et Technologique Herbier National du Gabon, B.P. 13354, Libreville, Gabon, France
Gerardo Burton
Departamento de Quimica Organica and UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2 Ciudad, Universitaria C1428EGA Buenos Aires, Argentina
Sunny Sun-Kin Chan
Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P.R. China
Hoi-Sing Chung
Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P.R. China
Osmany Cuesta-Rubio
Instituto de Farmacia y Alimentos (IFAL), Universidad de La Habana, Ave. 23, No. 21425, CP 13600 La Lisa, Ciudad de La Habana, Cuba
Amooru G. Damu
Department of Chemistry, University, Tainan, Taiwan
Michael T. DaviesColeman
Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa
National
Cheng
Kung
Xll
Salvatore De Rosa
Istituto di Chimica Biomolecolare del C.N.R. Via Campi Flegrei, 34, 80078 Pozzuoli (NA), Italy
FaTza Diaba
Laboratori de Quimica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028Barcelona, Spain
Jose E. Escandell
Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Vicent Andres Estelles s/n. 46100 Burjassot, Valencia, Spain
Ernesto Fattorusso
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli "Federico II", Via D. Montesano, 49, 1-80131, Naples, Italy
John M. Fidler
Pharmagenesis Inc., Palo Alto, CA 94304, USA
Yoshiyasu Fukuyama
Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 7708514, Japan
Marina Galvez
Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain
Hideo Hayashi
Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
Jose M. Hernandez
Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain
Jian-Mei Huang
Beijing University of Chinese Medicine, Beijing 100029, China
MD. Tofazzal Islam
Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 0608589, Japan
Benard F. Juma
Department of Chemistry, University of Botswana, P/Nbag UB 00704, Gaborone, Botswana
V.K. Kaul
Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India
xi
Edward J. Kennelly
Department of Biological Sciences, Lehman College and The Graduate Center, The City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA
Neeraj Kumar
Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India
Ping-Chung Kuo
Department of Chemistry, University, Tainan, Taiwan
Marie-Aleth LacailleDubois
Laboratoire de Pharmacognosie, Unite de Molecules d'Interet Biologique, UMIB EA 3660, Faculte de Pharmacie, Universite de Bourgogne, BP 87900, 21079 Dijon Cedex, France
Alain Laurens
Laboratoire de Pharmacognosie, UMR 8076 CNRS, Faculte de Pharmacie, Universite Paris XI, rue JeanBaptiste Clement, 92296 Chatenay-Malary, France
Guy Joseph Lemamy
Departement de Chimie-Biochimie, Faculte de Medecine, Universite des Sciences de la Sante, B.P. 7464 Libreville, Gabon, France
Song-Lin Li
Institute of Nanjing Military Command for Drug Control, No. 293, Zhongshan Eastern Road, Nanjing 210002, P.R. China
GeLin
Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P.R. China
Hermelo Lopez
Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain
Runner R.T. Majinda
Department of Chemistry, University of Botswana, P/Nbag UB 00704, Gaborone, Botswana
F.R. Marin
Departamento de Quimica-Fisica Aplicada (Area de Tecnologia de Alimentos), Facultad de Ciencias, Universidad Autonoma de Madrid, 2804, Madrid, Spain
Carmen Martin-Cordero
Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain
National
Cheng
Kung
XIV
Eugene P. Mazzola
University of Maryland-FDA Joint Institute for Food Safety & Applied Nutrition, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
Maya Mitova
Institute of Organic Chemistry with Centre Phytochemistry, B.A.N., 1113 Sofia, Bulgaria
John H. Musser
Pharmagenesis Inc., Palo Alto, CA 94304, USA
Juan C. Oberti
Departamento de Quimica Organica and IMBIV, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, 5000 Cordoba, Argentina
Jose A. Perez
Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain
J.A. Perez-Alvarez
Departamento de Technologia Agroalimentaria (Division de Tecnologia de Alimentos), Escuela Politecnica Superior de Orihuela, Universidad Miguel Hernandez de Elche, 03312, Orihuela (Alicante), Spain
Anna Lisa Piccinelli
Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084, Fisciano, Salerno, Italy
Luca Rastrelli
Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084, Fisciano, Salerno, Italy
M. Carmen Recio
Departament de Farmacologie, Facultat de Farmacia, Universitat de Valencia, Vicent Andres Estelles s/n. 46100 Burjassot, Valencia, Spain
T. Rezanka
Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Prague 4, Czech Republic
Jose Luis Rios
Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Vicent Andres Estelles s/n. 46100 Burjassot, Valencia, Spain
Bikram Singh
Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India
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C. Soler-Rivas
Departamento de Quimica-Fisica Aplicada (Area de Tecnologia de Alimentos), Facultad de Ciencias, Universidad Autonoma de Madrid, 2804, Madrid, Spain
J. Spizek
Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Prague 4, Czech Republic
Andrea A. Stierle
Department of Chemistry and Geochemistry, Montana Tech of the University of Montana, Butte, Montana
Donald B.Stierle
Department of Chemistry and Geochemistry, Montana Tech of the University of Montana, Butte, Montana
Chung-Ren Su
Department of Chemistry, University, Tainan, Taiwan
Orazio TaglialatelaScafati
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli "Federico II", Via D. Montesano, 49, 1-80131, Naples, Italy
Satoshi Tahara
Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 0608589, Japan
Juan M. Trujillo
Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain
Adriana S. Veleiro
Departamento de Quimica Organica and UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2 Ciudad, Universitaria C1428EGA Buenos Aires, Argentina
Cornelius C.W. Wanjala
Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya
Tian-Shune Wu
Department of Chemistry, University, Tainan, Taiwan
Rensheng Xu
Pharmagenesis Inc., Palo Alto, CA 94304, USA
National
National
Cheng
Cheng
Kung
Kung
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Bioactive Natural Products
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
SYNTHESIS OF IMMUNOSUPPRESSANT FR901483 AND BIOGENETICALLY RELATED TAN1251 ALKALOIDS JOSEP BONJOCH and FAIZA DIABA Laboratori de Quimica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028-Barcelona ABSTRACT: The review covers the synthetic studies of FR901483 and the biogenetically related TAN 1251 alkaloids.
1. INTRODUCTION The immunosuppressant FR901483 (1, Figure 1) was isolated from the fermentation broth of Cladobotrym sp. No. 11231 by a Fujisawa group in 1996 [1]. The structure was determined X-ray crystallographically, and the absolute configuration was not assigned until Snider achieved the enantiocontrolled total synthesis in 1999 [2]. From a structural point of view, the most conspicuous feature of 1 is an azatricyclic ring system consisting of the combination of the morphan and indolizine nuclei sharing the piperidine ring, namely, 5-azatricyclo[6.3.1.01>5]dodecane. Furthermore, there is a phosphate ester residue, which is essential for the activity of FR901483. OMe
HO. 8
i-J
2J^/3 '1
..
'NHMe
OMe
2
. 10 1
MeHN
(HO) 2 P-O
OMe
OPO(OH)2
FR901483
Figure 1. On the left, FR901483 structure with the replacement nomenclature numbering used in this review. On the right, FR901483 structure showing the heterocyclic system numbering (octahydro-l//-7,10a-methanopyrrolo[l,2-a]azocine) used by several authors.
FR901483 exerts potent immunosuppressive activity in vitro and significantly prolongs graft survival time in the rat skin allograft model, apparently by inhibition of purine nucleotide biosynthesis. This compound is likely to function by a different mechanism from that of cyclosporin A or tacrolimus (FK506), an important feature given the drug-associated side effects of both drugs. It is thought that the role of
CO2H
OH OH
Adenylsuccinate synthetase
inosine monophosphate
H
0H
OH Adenylsuccinate lyase
adenylsuccinate
OH OH adenosine monophosphate
Scheme 1. Adenosine biosynthesis
FR901483 in suppressing the immune system results from an antimetabolite activity whereby adenylosuccinate synthetase and/or adenylosuccinate lyase are inhibited. These enzymes function as key catalysts in the de novo purine nucleotide biosynthetic pathway. Addition of adenosine or deoxyadenosine (but not deoxyguanosine, deoxycytidine, uridine or thymidine) results in elimination of the immunosuppressive activity of FR901483. Thus, FR901483 may inhibit one of the key steps for adenosine biosynthesis (Scheme 1). The TAN1251 series of compounds have a novel tricyclic skeleton containing a l,4-diazabicyclo[3.2.1]octane and a spiro-fused cyclohexanone. It can be believed that they are biosynthetically related to the FR901483, which is probably biosynthesized from modified tyrosine dimer I by oxidative coupling to close the pyrrolidine ring and further elaboration to provide keto aldehyde II (Scheme 2). An intramolecular aldol reaction of the keto aldehyde will lead to the tricyclic skeleton of FR901483, while dienamine formation from the secondary amine and aldehyde will provide TAN1251C (4), which can be isomerized to TAN1251A (2) or reduced to TAN1251D (5).
RO
OMe
'NHMe
P.
FR901483
Scheme 2. Possible Biosynthesis of TAN1251 Compounds and FR901483
TAN1251A-D (2-5, Figure 2) are a series of alkaloids isolated in 1991 from a Penicillium thomii RA-89 fermentation broth at Takeda Industries [3]. TAN1251A (2) and TAN1251B (3) are muscarinic antagonists of value as mydriatic or antispasmodic/antiulcer agents that inhibit the acetylcholine-induced contraction of Guinea pig ileum with ED50 values of 8.0 and 10.0 nM, respectively. The relative stereochemistry of TAN1251B (3) was determined by X-ray crystallographic analysis, and the absolute configuration was established by analysis of the CD of the dibenzoate of the diol obtained by reduction of the a-hydroxy ketone moiety [4]. Since TAN1251A (2) is converted to 3 by Penicillium thomii RA-89, it has the same absolute stereochemistry. The absolute configuration of TAN1251C (4) and the absolute and relative configuration of TAN1251D (5) were assigned on the basis of their synthesis by Snider (see section 5.2).
TAN1251A(2) X = H TAN1251B(3) X = OH Figure 2. TAN1251 alkaloids
TAN1251C (4)
TAN1251D(5)
2. AN OVERVIEW OF THE FR901483 SYNTHESES Four total syntheses of FR901483 have been reported so far: the enantiocontrolled approaches of Snider [2], Sorensen [5], and Ciufolini [6], and Funk's synthesis in the racemic series [7]. Furthermore, Brummond has communicated a partial total synthesis [8] and Wardrop reported a formal synthesis of the desmethylamino analog, which had been described by Snider [9,10]. Additionally, Kibayashi and Bonjoch and co-workers have reported other synthetic entries to the azatricyclic core of FR901483, which has also been reported by Brummond in her initial studies [11-14]. An overview of FR901483 synthetic studies is outlined in Table 1. The synthetic strategies developed to reach FR901483 deserve a brief general comment. The major stumbling blocks in the synthesis of the target alkaloid are the generation of the spirocenter at C(l) and the assembling of the bridged framework of the alkaloid. The synthetic strategies adopted for the construction of its skeleton are outlined in Scheme 3, in which for the sake of clarity the substituents in the tricyclic framework have been omitted. All the total syntheses use an aldol process from a functionalized l-azaspiro[4.5]decanone to construct
the azatricyclic core of this target. In Brummond's synthesis the ring closure is the result of a Mannich reaction, which is the last step in a domino process that starts from a cyclohexanone derivative. In addition to the synthetic approaches described in the total syntheses of FR901483, four other methodologies to achieve its azatricyclic Table 1. Main Features of Syntheses in the FR901483 Field Main Author (Year)
Ref
Precursor of Tricyclic Framework
Bond Formed
Process-type
Total Syntheses Snider (1999) Sorensen (2000) Ciufolini(2001) Funk (2001) Brummond (2003)
azaspirodecanone azaspirodecanone azaspirodecanone cyclohexanone cyclohexanone
C(7)-C(8) C(7)-C(8) C(7)-C(8) C(7)-C(8) C(3)-C(4)
aldol aldol aldol aldol Mannich
Desmethylamino Derivative Snider (1998) 9 Wardrop(2001) 10
azaspirodecanone azaspirodecanone
C(7)-C(8) C(7)-C(8)
aldol radical
Azatricyclic Core Kibayashi(1997) Brummond (2001) Kibayashi(2001) Bonjoch (2003)
2-azabicyclononane cyclohexanone 2-azabicyclononane azaspirodecanone
C(4)-N(5) C(3)-C(4) C(2)-C(3) C(7)-C(8)
reductive amination Mannich metathesis Pd coupling
11 12 13 14
R = Boc; X = O R = Boc; X = H, H R = Ts; X = O
Snider (1999) Sorensen (2000) Ciufolini (2001)
Funk (2001)
Brummond (2003)
CH(OMe)2 OH
OTIPS
Skeleton syntheses Bonjoch (2003)
Wardrop (2001) OSiMe, CHO H
Kibayashi (1997) (2001)
Scheme 3. Construction of the Framework of FR901483
Form
(±) (±) (±) (±)
skeleton have been reported. In two of them, the bridge framework is assembled by ring closure of an azaspiro[4.5]decanone either by radical cyclization (Wardrop) or palladium-promoted coupling of a vinyl halide and ketone enolate (Bonjoch). Meanwhile, in Kibayashi's two approaches the pyrrolidine ring is the last to be constructed from 1-substituted 2azabicyclo[3.3.1]nonanes either by an intramolecular iV-alkylation or a ring-closing metathesis reaction. Intermolecular processes intramolecular CH2=CHCO2Et O'
"t>
inter- or intramolecular
Snider (1999)
intermolecular
Intramolecular processes CH2=CHCH2MgBr Bonjoch (2003)
Sorensen (2000) Ciufotini (2001) Wardrop (2001)
y CO2Me
+
Phl(OCOR)2
R'-Met
Kibayashi (1997,2001)
CH(OMe)2
+ OTIPS
Funk (2001)
Brummond (2003)
Scheme 4. Generation of C(l) quaternary center of FR901483
The other key step in the synthetic approaches to FR901483 is the elaboration of the quaternary spirocenter at C(l) (Scheme 4). Sorensen, Ciufolini, and Wardrop all use an oxidative spirocyclization of nitrogen tethered phenol derivatives, employing hypervalent iodine reagents, to generate the spirocenter at C(l). In contrast, in the other approaches the formation of the quaternary stereocenter does not coincide with the
closure of the azaspiranic ring, which is performed at a more advanced stage of the synthesis. Snider and our team worked with nitrones and imines derived from 1,4-cyclohexanediones to generate the quaternary stereocenter through a dipolar 1,3-cycloaddition with ethyl acrylate and a nucleophilic addition with allylmagnesium bromide, respectively. Starting from acyclic precursors, Funk also prepared 1-alkyl-laminocyclohexane derivatives by means of an intermolecular DielsAlder reaction. On the other hand, both Kibayashi and Brummond generated l-alkyl-2-azabicyclo[3.3.1]nonane derivatives using an intermolecular nucleophilic attack or an intramolecular Aza-Cope rearrangement starting from bridged azabicyclic iminium salts. 3. TOTAL SYNTHESES OF FR901483 3.1 Snider's approach through 1,3-dipolar cycloaddition and intramolecular aldol reactions Snider published the synthesis of desmethylamino FR901483 in 1998 [9] and one year later, the first total synthesis of natural (-)-FR901483 [2]. The disconnective analysis is outlined in Scheme 5. The key step in the retrosynthetic analysis is the disassembly of the azatricyelic structure of 1 by a retro-aldol process, which leads to aldehyde 6. In the forward sense, while this approach should provide efficient access to the ring system, OMe Aldol cyclization NHMe 10
(HO) 2 P-O FR901483(1)
f
1,3-dipolar cycloaddition
J CO2Me
(I
o 7
CO2Et
8
Ar = p-OMeC 6 H 4
Scheme 5. Snider's retrosynthetic analysis of FR901483
9
the process can be stereochemically troublesome since the aldol reaction of 6 could result in eight products. Enolization of the ketone can occur to either carbon C(8) or C(10), addition to the aldehyde can occur to give either the equatorial or axial alcohol and enolization of the aldehyde could convert 6 to a diastereomer that could result in four additional aldol products. Keto aldehyde 6 should be easily formed from lactam acetal ester 7, which should be readily available from isoxazolidine 8. The latter can be constructed by a convergent 1,3-dipolar cycloaddition of nitrone 9 with ethyl aery late. This strategy to spirocyclic lactams is precedented with nitrones derived from TV-benzylhydroxylamine [15]. Concerned about the possible stereochemical complexity of the aldol reaction of 6, Snider decided to first investigate this step in the racemic series using a model lacking the protected iV-methylamino substituent. 3.1.1 Synthesis of(±)-desmethylamino FR901483 The starting material for the synthesis was the hydroxylamine rac-10 whose synthesis from p-methoxybenzaldehyde and hydantoin in five steps is outlined in Scheme 6.
LNaOH 2.HCI OMe 1.NH2OH.HCI 2.TsOH, EtOH
MeCT^
O
A
/\^CO2Et
^J
BH3.NMe3 HCI, EtOH
OH
I
^
II
NH0H rao10
Scheme 6. Synthesis of racemic hydroxylamine rac-10
Condensation of hydroxylamine rac-10 with 1,4-cyclohexanedione monoethylene acetal provided nitrone rac-9, which was treated with ethyl acrylate to give 74% of a 8-9:1 mixture of isoxazolidine rac-S and its diastereomer. Reduction of this mixture with hydrogen afforded 8-9:1 of a mixture of lactam rac-1 and its diastereomer (Scheme 7). After crystallization, X-ray analysis of the major product confirmed that its stereochemistry is the one required for carrying out the synthesis of FR901483. The excellent stereoselectivity observed in the 1,3-dipolar cycloaddition of nitrone rac-9 was attributed to the substitution pattern in the stereogenic center adjacent to the nitrogen atom. These substituents stabilize one chair conformer relative to the other and thus allow a good
10
stereocontrol in the process involving an equatorial attack by and endo ester group on the nitrone moiety. At this stage, the hydroxyl group of rac-1 was removed to give the lactam 11 by converting it to a tosylate, followed by formation of the corresponding iodide, and subsequent reduction with Bu3SnH. Then, reduction of 11 with LiBH4 and further acid hydrolysis provided the corresponding keto alcohol, which was converted to ketoaldehyde 12 with Dess Martin reagent. It is worth noting that an aldol reaction in 12 can only give four aldol products, rather than the eight possible from 6, since 12 is a racemic compound (throughout this review the prefix rac- is only used if the compound also appears in its enantiopure form).
EtOH NHOH rac-10
rac-9
Ar = p-OMeC6H4
Ar,
ArN 45 psi H 2 , Pd/C AcOH
EtO
EtO2
CO2Et
(74%)
(91%) rac-7
rao8
I.TsCI, DMAP, Et3N 2. Nal, acetone
1.LiBH4, THF 2. HCI, AcOH, H2O 3. Dess-Martin
3. Bu3SnH, AIBN (65%) (95%)
Scheme 7. Synthesis of ketoaldehyde 12
When the aldol reaction was carried out with NaOMe in MeOH, a protic solvent, the desired compound 13 with the right stereochemistry was obtained in 51% yield together with epi-13 (equatorial OH) and 14, the latter resulting from the regioenolate at C(10) of the starting ketone 12. However, when the reaction was achieved with K0M3u in toluene, an aprotic solvent, only epi-13 (79%) and epi-14 (22%) were isolated, both with an equatorial hydroxyl group (Scheme 8). From these results it is plausible to believe that compounds resulting from enolization at C(8) are favored because the /?-methoxybenzyl group in the resulting 13 and epi-13 is equatorially located, whereas in compounds originating from
11
the enolate at C(10), i.e. 14 and epi-14, this substituent is in an axial disposition. Moreover, the solvent in which the reaction was carried out was a decisive factor in the configuration of the hydroxyl group. In MeOH, axial sodium alkoxide was stabilized by chelation with the solvent and therefore the axial derivative was the main product, while in toluene equatorial potassium alkoxide can only be stabilized by chelation with the ketone and hence only epi-13 and epi-14 with an equatorial OH were isolated.
12 NaOMe, MeOH 25 °C, 15min KOf-Bu, toluene 25 °C, 25 min
13
ep/-13
-
70%
14
"
epi-14
22%
Scheme 8. Aldol reaction on 12
Both 13 and ephl3 can be elaborated to desmethylamino FR901483. Reaction of 13 with LiAlH4 reduced the amide and the ketone from the less hindered exo face to stereoselectively give alcohol 15 with the wrong endo configuration. On the other hand, inversion of the configuration of the hydroxyl group at C(7) inepi-13 was accomplished by reaction with />-nitrobenzenesulfonylchloride, followed by treatment of the resulting nosylate with CsOAc [16] to yield an acetate, which after reduction with LiAlH4 gave the same alcohol 15 (Scheme 9). Inversion of the configuration at C(9) was accomplished by an initial reaction of alcohol 15 with/?-nitrobenzenesulfonylchloride to selectively form the nosylate of the less hindered equatorial alcohol. The axial alcohol at C(7) was protected as the TBDMS ether and the resulting compound was treated with CsOAc to afford the desired acetate 16 (69%) together with the readily separable elimination product 17. Elaboration of 16 to desmethylamino FR901483 was accomplished by hydrolysis of the acetate to give the alcohol, which was converted to the corresponding dibenzyl phosphite ester [17], which in turn, was oxidized to the phosphate ester 18. Hydrolysis of the TBDMS group and debenzylation of the diphosphate ester afforded the FR901483 analog 19, which was converted to the dipotassium salt to avoid confusion in NMR analysis due to totally or partially protonated samples in the tertiary amine.
12
1./>NO2PhSO2CI 2. CsOAc, 18-crown-6
LiAIH 4 ,THF -78 °C to 65 °C
ep/-13
13
3. LiAIH4
15 ,Ar 1./>NO2PhSO2CI 2. TBDMSOTf 15
TBDMSO,.
3. CsOAc, 18-crown-6 OAc 17
16 1. K2CO3, MeOH 2. Tetrazole, (BnO)2PN(/-Pr)2
,Ar
™
TBDMSQ,
3. m-CPBA OPO(OBn)2
18 Scheme 9. Completion of the synthesis of 19
3.1.2 Synthesis of(-)-FR901483 When Snider started the synthesis of enantiopure FR901483, its absolute configuration was unknown, but he estimated that it might originate from L- rather than D-tyrosine and hence started the enantiocontrolled synthesis of FR901483 using (S)-hydroxylamine 10. The latter was prepared from iV-Boc-L-tyrosine through methylation, cleavage of the Boc group, and conversion of the resulting methyl (5)-O-methyltyrosine to hydroxylamine 10 by Grundke's procedure [18] in 73% yield (Scheme 10). The synthesis was pursued using the methodology developed in the desmethyamino series. The stereoselectivity (6:1) in the formation of isoxazolidine 8 was slightly lower than in the racemic series, probably due to the change of the ethyl ester of rac-10 to the less demanding methyl ester in 10. Hydrogenolysis of the 6:1 mixture of isoxazolidine 8 and the diastereomer gave the lactam 7 and its epimer. The two diastereoisomers were separated after conversion of the corresponding tosylates to the azide 23 and its epimer. Next, 23 was converted to iV-methylcarbamate 24 by hydrogenolysis in the presence of Boc2O [19] and then methylation [20]. Reduction of the ester in 24 with
13 O2Me
EtO2C,,
T9
O,Me
NH 2
NHOH
Ss/Nk^--Ar / \ T | ] CO2Me
1. anisaldehyde
OMe
2. m-CPBA 3.NH2OH.HCI
, (86%)
2. (77%)
(73%)
Ar
45psi H2, Pd/C, AcOH
1-TsCI ^
CO2Me
. NaN3 (78%)
\ < j S " Af f ]I I CO2Me O
X
n 23
Ar = p-OMeC6H4
1. KOf-Bu,f-BuOH 25 °C, 30 min
1.LiBH 4 , THF 2. HCI, AcOH, H2O (97%) N(Boc)Me
3. Boc2O, Et3N (95%) 4. Dess Martin
2. TFA, CH 2 CI 2
6 (PG = Boc) OMe
OMe
NHMe
25 (36%)
OMe
NHMe
26 (16%)
27 (5%)
Scheme 10. Preparation of ketoaldehyde 6 and its aldol reaction
UBH4 followed by an acid hydrolysis gave a deprotected keto alcohol, in which the secondary amine was again protected with BOC2O. Finally, the aldehyde intermediate 6 was obtained using the Dess Martin reagent. The aldol reaction of 6 proceeded analogously to that of the model ketoaldehyde 12, although in lower yield. Treatment of crude 6 with KtBuO in ?-BuOH followed by a cleavage-step of the Boc group provided 41% of a unseparated mixture of the desired aldol adduct 25 and the bad regioisomer 27, compound 26 being isolated in 16% yield. The use of NaOMe in MeOH in this process did not improve the yield of the desired compound 25.
14
Reduction of the 7:1 mixture of 25 and 27 with LiAlH4 afforded the corresponding diol mixture, which after benzyloxycarbonylation and purification give carbamate 28 (52% from 25). For the inversion of the equatorial alcohol at C(9), 28 was chemoselectively converted to its nosylate and the axial alcohol at C(7) protected as the triethylsilyl ether to give 29. The use of TBDMS protecting group, used in the synthesis of 19, was discarded due to its reluctance to undergo cleavage for steric reasons. Compound 29 was treated with CsOAc and 18-crown-6 to displace the nosylate to give acetate 30, obtained together with the undesired elimination compound 31. The remaining steps to reach FR901483 were carried out in virtually the same conditions that were developed in the synthesis of 19. Nevertheless, some processes had to be modified to deal with the problems caused by the presence of the methylamino group at C(3). The acetate 29 was hydrolyzed with K2CO3 in MeOH and then treated with OMe
OMe 1.LiAIH4,THF -78°C->65°C
HQ,
1.p-NO2PhSO2CI
•NHMe 2. Et3N, CbzCI
2. TESOTf
(52%) 28
25
.OMe
,Cbz
CsOAc, 18-crown-6
OMe
TESQ,
OAc
29
30 (70%)
31 (20%)
R = SO 2 PhNO 2 OMe 1. K2CO3, MeOH 1.TBAF 2. HCI
2. (BnO)2PN(/Pr)2, tetrazole; then f-BuOOH
Cbz OPO(OBn)2
32
Scheme 11. Snider's synthesis of (-)-FR901483
3. H2, Pd
FR901483.HCI
15
(BnO)2PN(/-Pr)2 and tetrazole to yield the corresponding phosphite ester. This was converted to the phosphate 32 with MBuOOH [21] instead of mCPB A since the latter reagent easily causes the oxidation of the tertiary amine to the corresponding amine oxide. Finally, hydrolysis of the TES group with TBAF, protonation of the tertiary amine with excess of HC1 and a hydrogenolysis process that allows debenzylation of the phosphate ester as well as deprotection of the secondary amine provided the monohydrochloride salt of 1. In summary, Snider developed the first synthesis of (-)-FR901483 (1) in 2% overall yield from O-methyltyrosine methyl ester in 22 steps establishing the absolute configuration of the natural product. The strategy relies on a 1,3-dipolar cycloaddition from a nitrone, followed by transformation of the resulting bicyclic isoxazolidine into the azaspiranic ring and then by an intramolecular aldol reaction to give the tricyclic ring of the target. The synthesis is completed with adjustments of the oxidation level and configuration as well as the formation of the phosphate unit. 3.2 The approaches of Sorensen and Ciufolini. Synthesis of l-azaspiro[4.5]decan-8-ones from two tyrosine units The other two enantioselective syntheses of FR901483 reported by Sorensen and Ciufolini (sections 3.2.2 and 3.2.3) as well as an approach to the demethylamino derivative by Wardrop (section 4.1) have in common the elaboration of the l-azaspiro[4.5]decan-8-one intermediates by means of an oxidative azacyclization from a nitrogen-tethered phenol derivatives. In the following section an overview of this process is given. 3.2.1 The hypervalent iodine oxidation of nitrogen-tethered phenol derivatives, a straightforward entry to l-azaspiro[4.5]decanediones Phenolic oxidation with organohypervalent iodine reagents [22] constitutes a powerful tool for the synthesis of functionalized 1azaspirodecanes, a process which formally involves generation of an aryloxenium ion and intramolecular capture of this intermediate by Nnucleophiles. An alternative way to achieve the same type of azabicyclic compounds is a dearomatizing spirocyclization triggered by an electrophilic center pendant to the aromatic ring, such as an Nacylnitrenium ion. A particularly direct synthetic entry to subtarget IV would materialize if a phenolic amide or amine such as Ilia or Hlb could be induced to undergo oxidative cyclization to spirodienone IV, which could then be reduced to valuable synthetic intermediates. (Scheme 12).
16
Hr
III
%
Phl(OAc)2 [DIB]
path a; X = O path b; X = H, H
IV
Scheme 12. The spirocyclization of nitrogen-tethered phenols
The desirability of the transformation of Scheme 12 was recognized as early as 1987, when Kita published a pioneering study of the oxidation of phenolic amides with iodobenzene bis(trifluoroacetate) (PIFA) [23]. However, this intramolecular spirocyclization fails due to the propensity of the nucleophilic oxygen atom of the amide to intercept the electrophilic intermediate arising through activation of the phenol (Scheme 13). The preferential formation of spirolactones is probably due to an electronic effect. In most cases non-nucleophilic solvents are required to prevent solvent participations [24]. Bn
Phl(OCOCF3)2 [PIFA]
-i
H,0
Scheme 13. Oxidative Ospirocyclization of phenolic amides
The transformation depicted in Scheme 12 using synthetic equivalents of phenol-tethered amides (path "a") was first carried out successfully by Ciufolini [25,26], who later applied this methodology to his synthesis of FR901483 (3.2.3). Variations of this process starting from JVmethoxyamides were reported by Kikugawa [27-30] and Wardrop (4.1 and 5.3). These transformations involve formation of jV-acylnitrenium ions rather than aryl oxidation and subsequent trapping. On the other hand, a more straightforward route (path "b") using amines was reported for the first time by Sorensen [5] in his FR901483 synthesis (3.2.2). Ciufolini went on to establish an efficient oxidative spirocyclization of phenolic sulfonamides [31]. Ciufolini was able to harness the effects responsible for the reactivity of the oxygen atom of an amide in the oxidative cyclizations by engaging an oxazoline, in which the nitrogen atom acts as the nucleophile [25].
17
Thus, oxidation of oxazoline derivatives of phenolic compounds 33 with IBD in trifluoroethanol leads to spirocyclic amides 34 [26]. The low yield of compound 34b is attributable to the carbamate carbonyl of 33b competing effectively with the oxazoline nitrogen to capture the electrophilic intermediate obtained by activation of the phenol (Scheme 14).
Phl(OAc)2 [DIB] OAc TFE
a b c
H NHBoc NHTs
R, H Bn Bn
Yield 42% 22% 41%
Scheme 14. Oxidative spirocyclization of phenolic oxazolines
On the other hand, the azaspirocyclization of N-acyl-Nalkoxynitrenium ions, which can be generated under mild conditions by the treatment of iV-methoxyamides with iodine (III) reagents, constitutes a very efficient synthetic entry to l-azaspiro[4.5]decanediones.
OMe Phl(CF3CO2)2 [PIFA]
1. f-BuOCI 2. Ag2CO3/TFA
a. inCH 2 CI 2 (72%) ref 10 b. in TFEA (80%) ref 28
(83%) ref27
OMe
Phl(TsO)(OH) [HTIB] (82%) ref 29
OMe
PIFA (77%) ref 30, isolated the OMe A/-Phth derivative
NPhth
38
Scheme 15. Synthesis of azaspirodecanediones from ,/V-methoxyphenylamides
In 1989 Kikugawa reported the intramolecular ipso attack of a nitrenium ion generated from the JV-chloro-./V-methoxyamide of anisole 35, to give the l-methoxy-l-azaspiro[4.5]decadienone 36 in 83% yield [27]. The same author went on to improve this process, which had already been done by Wardrop [10]. Both described the cyclization of the JV-methoxyamide 35 using PIFA in trifluoroethanol (TFEA) [28] and
18
CH2CI2 [10] in good yields (Scheme 15). Kikugawa has also reported the cyclization of the 7V-methoxy-(4-halophenyl)amides 3 7 with [hydroxy(tosyloxy)iodo]benzene (HTIB), the fluoro derivative resulting in the best yield of azaspirodecadienone 36 (82%) [29]. Moreover, Kikugawa has described the cyclization of both 4-methoxy and 4-fluoro derivatives of the corresponding jV-phtalimide analogs (i.e. 38) using the hypervalent iodine reagents PIFA and HTIB, respectively, the azaspirodienone being isolated in 77% and 79% yield [30]. It is worth noting that Wardrop has also successfully explored the Nacylnitrenium route applied to a- and ^-substituted 3-(methoxyphenyl)N-methoxypropionamides (e.g.39) [32]. Using PIFA as the oxidant agent, a 71-face selective azaspirocyclization was found, compound 40 being isolated with an excellent diastereoselectivity (Scheme 16).
PIFA
OMe
MO
^
0 M e
». -78 °C (75%, dr >96%)
Scheme 16. yV-acylnitrenium ion-promoted diastereoselective spirocyclization
Last but not least, the oxidative cyclization of phenolic secondary amines (cf. Illb -> IVb, X = iV-alkyl) is possible, but it remains problematic. This challenging transformation may rightfully be termed the "Sorensen reaction" as it was introduced in the synthesis of FR901483 by this author (see 3.2.2). Yields are often unsatisfactory, and the spirocyclic targets IVb are accompanied by a host of byproducts, although Honda [33] has successfully applied this transformation in his synthesis of TAN1251A (5.5). Such shortcomings are magnified when the amino group in the substrate is primary (Illb, X = NH), rendering the transformation entirely unsuitable for multistep synthetic operations. Ciufolini [31] reported that sulfonamide substrates (Illb, X = NSO2R) can be used to efficiently carry out these hitherto problematic oxidative transformations. Treatment of homotyramine sulfonamides with iodobenzene diacetate (DIB) in hexafluoroisopropanol induces oxidative spirocyclization in high yield, although if a protected amino substituent is included, the process proceeds less efficiently (Scheme 17).
19
Oxidative azaspiroannulation IVb
TS
>r H Phl(OAc)2 (CF3)2CHOH (60%)
Scheme 17. Oxidative spirocyclization of phenolic sulfonamides
3.2.2 Sorensen's synthesis via oxidative cyclization of an amino-tethered phenol Sorensen's synthetic analysis of FR901483 is shown in Scheme 18. The cornerstone of his strategy is the idea that exposure of aminophenol 41 to an appropriate oxidant, such as iodobenzene diacetate, could give rise to 42. This appealing ring-closure would create the azaspiro[4.5]decane substructure of target 1 and would not be complicated by the formation of diastereoisomers. His goal was to effect intramolecular C-N bond N-, 3
per1 Oxidative azaspiroannulation
HQ,,
Aldol to
cyclization
Scheme 18. Sorensen's retrosynthetic analysis of FR901483
FR901483(1)
20
formations with substrates possessing the natural oxidation state at C(3). To construct the remaining ring and C(7) stereocenter of 1, Sorensen used an intramolecular aldol reaction that had been precedented in the syntheses of 2-azabicyclo[3.3.1]nonanes from amide derivatives [2,34], but not from (3-aminoaldehydes. Finally, from 44 Sorensen explored an uncommon strategy for introducing the conspicuous phosphate ester of 1. The starting materials for the synthesis were the known methyl (S)-Omethyltyrosine and the protected aminoaldehyde 45, which was prepared following the multi-step sequence depicted in Scheme 19 (69% yield from the known amino ester intermediate). The protecting group of choice was the Fukuyama-type nitrosulfonamide [35], readily cleavable but stable in the oxidative-cyclization reaction.
1.NO 2 C 4 H 4 SO 2 CI(88%) 2. Mel,K 2 CO 3 (91%) 3. AICI 3 , EtSH (92%) OMe
Scheme 19. Synthesis of phenol 45
The reductive amination between aldehyde 45 and methyl 0-methylL-tyrosine ester gave the amino phenol 41, which incorporates all carbon atoms of the synthetic target FR901483. The crucial transformation in the synthetic pathway was the following oxidative cyclization of aminophenol 41. After much experimentation, it was found that exposure of a solution of 41 in hexafluoro-2-propanol to iodobenzene diacetate at room temperature resulted in the formation of azaspiro[4.5]decadienone 42 in 51% yield (based on 70% consumed 41). The outcome of this ring closure is noteworthy because it constitutes the first example of this type of oxidative cyclization starting from an amino compound (Scheme 20). After changing the protecting group of the exocyclic nitrogen atom, the dienone moiety of 42 was reduced to an epimeric mixture of alcohols 46 using Raney nickel under a hydrogen atmosphere. A reduction of the methyl ester with LiAlH4 in 46 produced a mixture of diols that could subsequently be converted by Swern oxidation into a single ketoaldehyde 43 in which the a-amino aldehyde unit is stereochemically stable [36]. For the closure of tricyclic skeleton of 1 by an aldol cyclization, Sorensen was mindful of the work of Snider and was able to convert 43 into the desired compound 44 in 34% yield using NaOMe in MeOH. When 43 in THF was treated with pyrrolidine and AcOH, the equatorial C(7) epimer of 44 was formed in 51% as the main product.
21
Owing to its rigid and concave architecture, hydroxyketone 44 was converted into diol 47 in a completely diastereoselective fashion by hydrogenation. To achieve a synthesis of 1 from 47, inversion of stereochemistry at C(9) and production of a C(9) monophosphate ester were required. It was possible to accomplish both objectives in a single step by a Mitsunobu reaction between 47 and dibenzyl hydrogen phosphate [37]. Despite its modest yield, this reaction was achieved in the presence of a free C(7) hydroxyl group, and directly produces the natural stereochemistry and the phosphate ester moiety at C(9). Hydrogenolysis of the dibenzylphosphate 48 followed by cleavage of the Boc group afforded FR901483 (1) as its hydrochloride salt.
CO2Me
(51 %)
LUAIH4
Ar
CO2Me
2. Swern (90 %)
H2, Raney Ni (92 %)
(BnO) 2 PO 2 H, (C6H4CI)3P, HO,,,
HO,,, HO.
DIAD, Et3N (37%)
K JVIe Bo c
OP(O)(OBn)2 47
Scheme 20. Sorensen's synthesis of (-)-FR901483
48
1.H 2 , Pd/C 2. HCI (81
FR901483.HCI
(D
22
In summary, Sorensen completed a concise synthesis of FR901483 (16 steps from methyl ester of O-methyltyrosine, 1.5% overall yield) by a strategy that for the first time featured an oxidative cyclization of a phenolic secondary amine (obtained by a reductive coupling of two tyrosine units). The other notable aspect of his synthesis is the one-step conversion of a hydroxyl group to a phosphate unit with an inversion of configuration that shortens the global sequence in comparison with other routes. 3.2.3 Ciufolini synthesis via oxidative cyclization of an oxazolinetethered phenol The synthesis of Ciufolini, published in 2001 [6], is based on the strategy he had previously developed to assemble the spirobicyclic skeleton through an oxidative cyclization of phenolic oxazolines (see 3.2.1). The strategic plan for the synthesis of FR901483 (1) is outlined in Scheme 21 and starts with the same disconnection C(7)-C(8) as used by Snider and Sorensen, involving a retroaldol process which leads to aldehyde 6. This advanced intermediate could be prepared from the spirodienone 49 available from an adequate oxazoline such as 50, which comes from two tyrosine units. IVIH(PG)
50
Scheme 21. Ciufolini's retrosynthetic analysis of FR901483
The construction of the suitable oxazoline 50 requires compounds 51 and 52, both of which may be made from L-tyrosine. For the coupling of amino alcohol 52 and carboxylic acid 51, the Vorbriiggen protocol [38] was the method of choice, since it leads to the desired heterocycle in one step and tolerates an unprotected phenolic function in 51 (Scheme 22). In the key step of this synthesis, oxazoline 50 underwent DIB oxidation/acetylation to 49 through a novel process, which has been mentioned in section 3.2.1. The nature of the protecting group applied to the lateral group in 50 is crucial for the success of the cyclization step. In particular, the Boc blocking group is unsatisfactory because it competes effectively with the oxazoline for the electrophilic intermediate produced
23
through DIB activation of the phenol. The choice of a tosylamide form instead of the nitrosulfonamide for the protection of the pendant amino group allows the iV-protecting group to be retained until the spirolactam reduction step. Although acetylation of the primary product of oxidative cyclization also results in 7V-acylation of the tosylamide, it is unimportant, because both acetyl groups are removed simultaneously at a later stage of the synthesis. Conversion of dienone 49 to the corresponding cyclohexanone was carried out by hydrogenation in the presence of PtC>2, while other hydrogenation catalysts (Pd or Rh) also provided variable quantities of a rearomatized product. A straightforward series of reactions advanced intermediate 53 to keto aldehyde 6, which constitutes the substrate for the crucial aldol cyclization leading to the intermediate 55. Ciufolini optimized the regio- and diastereoselectivity for this stage using sodium methoxide in 90% aqueous methanol, bridged compound 55 being obtained in 44%. It should be noted that aldehyde 6 is fairly resistant to epimerization at C(6), minimizing the probability of formation of aldol isomers possessing the undesired C(6) (.^-configuration. This stereochemical stability, first recorded by Snider, is consistent with observations by Myers and Garner concerning the configurational stability of amino acid-derived aldehydes [36]. The final sequence that produced the first Ciufolini approach to FR901483 commenced with reduction of the ketone in the acetyl derivative of 55 to the corresponding diol 56. The shape of the molecule disfavors the approach of reducing agents from the top face of the ketone, so that the desired C(9) axial carbinol is not directly available. The reduction was achieved by the use of L-selectride, in the hope of obtaining at least some of the correct carbinol diastereomer, but the reaction occurred with complete diastereocontrol in favor of the equatorial alcohol 56. Inversion of the C(9) configuration was achieved by Snider's method, via />-nitrobenzenesulfonate ester, and the resulting acetate 57 was obtained in a satisfactory 73% yield, the elimination product also being isolated (12%). Global deprotection/reduction of 57 was achieved in high yield by vigorous LiAlH4 treatment, and the emerging secondary amino group was protected as a benzyl carbamate prior to selective phosphorylation of the C(9) carbinol, without protection of the rather hindered C(7) alcohol. Final hydrogenolysis of all benzyl groups in 58 in the presence of aqueous HC1 provided the bis-hydrochloride of 1. Sorensen's synthesis of 1 demonstrated the feasibility of a Mitsunobutype inversion of the configuration of the C(9) carbinol in substrates similar to 59. This allowed Ciufolini to simplify his own synthesis as shown in Scheme 22 (on the right). Thus, LiAlH4 reduction of compound 55 produced a 6:1 mixture of equatorial, 59, and axial amino alcohols.
24
1.Phl(0Ac)2 CF3CH2OH 2.Ac2O, Py (41 %)
lyie
TPAP, NMO
first generation
<^N
^
(77%)
'
1. Ac2O, Py 2. L- Selectride
NaOMe, 90%aq MeOH
~ UMU
Y
(44%) O 6 (PG=Ts) second generation 1. LiAIH4
(53 %)
59
2. (BnO)2PO2H, (C6H4CI)3P, DIAD, then Et3N 3. CbzCI
(70'
OMe
/-Pr2NP(OBn)2, then f-BuOOH Cbz
<26%) H2, Pd/C HCI
Hfl
"
FR901483.HCI
•l<
Cbz
(29 %) OPO(OBn)2
Scheme 22. Ciufolini's synthesis of (-)-FR901483
58
(94%)
25
This somewhat surprising stereochemical result is attributable to the greater reactivity, and hence the lower selectivity, of LAH relative to other reducing agents. Working with alcohol 59 and its epimer at C(9), the Mitsunobu process leads to phosphate 58, as the major product, after conversion of the resulting extremely polar compounds to their iV-Cbz derivatives. So, although the Mitsunobu step may be conducted in the presence of an unprotected methylamino substituent, technical difficulties rule in favor of protection of the secondary amine prior to inversion at C(9), exactly as Sorensen described. In summary, the total synthesis of (-)-FR901483 was accomplished by an oxidative cyclization of a phenolic oxazoline to a spirolactam in the key step. The longest linear sequence leading to FR901483 through a Snider-type inversion encompasses 20 steps from commercial L-tyrosine and it proceeds with an overall yield of 1%. The alternative synthesis involving a Sorensen-type Mitsunobu inversion is shorter (17 steps), and it affords identical overall yield (1.3%).
3.3 Funk synthesis of (±)-FR901483 via an amidoacrolein cycloaddition In 2001, Funk reported a synthesis of (±)-FR901483 using an approach very different from those described previously, taking advantage of his amidoacrolein cycloaddition methodology to prepare 1-alkyl-laminocyclohexane derivatives, starting the synthesis from acyclic compounds. Moreover, Funk neither uses tyrosine derivatives nor needs an inversion at C(9) to install the phosphate unit. In Funk's retrosynthetic analysis, shown in Scheme 23, he envisaged that lactam 60 possessed the necessary functionality for the introduction of the C(6) /?-methoxybenzyl and C(3) methylamino substituents via enolate alkylation and amination reactions, respectively.
(±)-FR901483(1)
-CHO 63
Scheme 23. Funk's retrosynthetic analysis of FR901483
26
A number of scenarios, albeit multistep, are conceivable for the transposition of the P-hydroxy ketone functionality present in lactam 61 to the isomeric version in lactam 60. Lactam 61 could be constructed by an aldol ring closure of 62, which in turn could be fashioned by another aldol-type cyclization within aldehyde 63. Finally, a Diels-Alder cycloaddition of silyloxydiene 65 with the 2-amidoacrolein 64 was expected to furnish 63. It should be noted that this intermolecular cycloaddition was expected to install not only the central 1-alkyl-laminocyclohexane substructure of FR901483 but also the electrophilic and nucleophilic components, appropriately tuned, for the pending sequential aldol cyclizations. The precursor to amidoacrolein 64, 1,3-dioxin 66, was prepared as follows [39]: the imine derived from the condensation of 2,2-dimethyll,3-dioxan-5-one with aminoacetaldehyde dimethyl acetal was acetylated with acetic anhydride/triethylamine to afford dioxin 66 in 83% yield (Scheme 24). Retro Diels-Alder of dioxin 66 in warm benzonitrile (120 C, 16 h) generated the amidoacrolein 64, which was trapped in situ with the silyloxydiene 65 to afford the desired cycloadduct 63 (64%). An aldol cyclization between the acetamide and neighboring aldehyde functionalities within 63 proceeded smoothly (2 equiv. of KO/-Bu, 10 equiv. of EtOAc, THF, 0 °C, 40 min) and directly afforded the corresponding conjugated lactam. This product was of sufficient purity for the second aldol reaction, which was best accomplished under acidic conditions, presumably proceeding through the achiral keto aldehyde intermediate 62 enroute to the desired, but racemic, P-hydroxy ketone 61 obtained in 79% yield after the two consecutive ring closures. The next phase of the synthesis involved the transposition of aldol adduct 61 to the protected "aldol" adduct 60. p-Hydroxyketone 61 was subjected to conditions (NaBH4, AcOH) which effected a direct reduction of the carbonyl moiety of 61 and thereby introduced the axial C(9) hydroxyl functionality of 67 with complete stereocontrol through an intramolecular delivery of hydride within an alkoxide intermediate at C(7). After diprotection of both hydroxyl groups of 67, chemoselective deprotection of hydroxyl at C(7) and Swern oxidation, ketone 60 was isolated. The enolate derivative of 60 could be stereoselectively pmethoxybenzylated, and the resulting ketone was reduced to the wrong equatorial alcohol 68. The C(7) stereogenic center was inverted by treatment of the nosy late derivative of 68 with rubidium acetate to afford the desired acetate 69 accompanied by the syn elimination product (15%). The final stage of the synthesis from lactam 69 required three major operations: introduction of the nitrogen atom at C(3), reduction of the C(4) carbonyl, and formation of the C(9) phosphate moiety. The first of these objectives was accomplished by saponification of the acetate group
27
-f
(65) CH(0Me)2
CH(OMe)2
CXX>
CH(OMe)2 (64 %) OTIPS
66
63
1. KOf-Bu 2. TFA, H2' (79 %)
1.K0t-Bu, p-MeOBnBr 2. Red-AI OMe
(72 %) OTBS 69 OMe
OMe 1.KOH 2. TfOSiEt3
1.LiAIH4
Et3SiQ,
I.NaH, Mel (64%)
HO..
N 3 2. CICbz
3. H2, PtO2 4. LDA, TrisN3
2. HF(92%)
(71%) OTBS
70
.OMe
HO,, A ^ T N"\
k^°
OH
*sMe Cbz
/-Pr2NP(OBn)2, thenm-CPBA
HO,,,,
e
H2, Pd/C
Cbz
(97o/o)
FR901483
(73 %) 3PO(OBn)2
72
rac-58
Scheme 24. Funk's synthesis of (±)-FR901483
of 69, protection of the resulting hydroxyl group as the corresponding triethylsilyl ether, reduction of the unsaturated lactam, and azidification
28
of the enolate derived from the saturated lactam following Evan's protocol [40] to afford a diastereomeric mixture of azides. X-Ray crystallographic analysis of the major isomer (59% yield) showed that it possessed the adequate relative stereochemistry at C(3). Simultaneous reduction of the lactam and azide functional groups with concomitant removal of the triethylsilyl protecting group was accomplished by subjecting lactam 70 to LiAlHj to afford a diamino alcohol whose primary amine functionality was selectively protected with benzyl chloroformate to deliver carbamate 71 (71% yield for two steps). The remaining carbon atom of the natural product was introduced by methylation of carbamate 71, and then deprotection of the TBS ether gave diol 72. The selective phosphorylation of the less encumbered hydroxyl was capricious when tetrabenzyl pyrophosphate was employed but was reproducible if diol 72 was first converted to the corresponding phosphite ester and then oxidized with m-CVBA in the presence of triethylamine to produce the dibenzyl phosphate rac-5H. Finally, hydrogenolysis of the dibenzyl phosphate and benzyl carbamate functionalities provides (±)-FR901483 which was characterized as the hydrochloride salt. In conclusion, Funk completed a synthesis of (±)-FR901483 in 22 steps and 2.4 % overall yield from the starting dioxanone, demonstrating that the easily accessible trifunctional arrays of 2-amidoacroleins can be exploited in the rapid assembly of tricyclic ring systems. Attention should also be drawn to the /7-methoxybenzyl substituent introduced at C(6) by alkylation of a tricyclic lactam in an advanced stage of the synthesis. 3.4 Brummond's synthesis through the tandem cationic aza-Cope rearrangement-Mannich cyclization reaction In 2001, Brummond published an approach to the core structure of FR901483 [12] and two years later communicated a formal synthesis of enantiopure FR901483 [8]. The key strategic element in both synthetic processes is the tandem cationic aza-Cope/Mannich cyclization reaction [41], via a bridgehead iminium ion. This sequential reorganization constitutes a powerful method for preparing nitrogen heterocycles that has been extensively developed by Overman and successfully applied to the synthesis of a number of structurally complex alkaloids [42]. The 3acylpyrrolidine unit is the basic structure formed by the aza-Cope rearrangement-Mannich cyclization, which could be carried out under mild conditions and with enhanced selectivity. The high efficiency of this tandem reaction is the result of two factors: i) the [3,3] sigmatropic rearrangement is highly favored, because of the presence of a charged atom in the molecular array involved in the process, and ii) after the azaCope rearrangement, the resulting iminium ion is trapped by the
29 OMe
OMe
Mannich
(HO) 2 P-O V '2
OPG
FR901483 (1)
QPQ. A ^ r O M e
OPG
0 P G
OPG
Scheme 25. Brummond's retrosynthetic analysis of FR901483
proximate enol to give the 3-acylpyrrolidine system, shifting the otherwise reversible process. Brummond's approach to FR901483 involves compound V, which can be accessed in a very efficient manner using a tandem cationic aza-Cope rearrangement/Mannich cyclization strategy. The complete retrosynthetic analysis leads to three relatively simple starting materials (Scheme 25). In an initial work to quickly establish the feasibility of the proposed route, Brummond targeted cyclohexanone 77, which possesses only the functionality necessary for the tandem rearrangement-cyclization process (Scheme 26). The synthesis of the cyclization precursor 77 was initiated by addition of the lithium anion of trimethylsilylacetonitrile to cyclohexenone to give the protodesilylated 1,4-addition product [43], which was protected to afford acetal 73. The choice of butanediol as the reagent for protection is based on the facile removal of the resulting dioxepane acetal (see 5.2). Reduction of nitrile 73 gave the primary amine 74. On the basis of the Petasis coupling method involving amines, aldehydes and organoborates [44], amine 74 was treated with paraformaldehyde and excess allylboronate 75 [45] to give 76, which after hydrolysis furnished the desired amino ketone 77. Treatment of 77 with TsOH in refluxing benzene afforded the amino aldehyde 78 (dr, 2/1) via the tandem cationic aza-Cope rearrangement- Mannich cyclization
30
1. TMSCHLiCN
<X/0
2. HO(CH2)4OH, PPTS
j X .
(75 %)
Kj^
ri.
VN
(80 %)
(CH2O)n, EtOH, H2O
TsOH
Scheme 26. Synthesis of the skeleton of FR901483 by Brummond
sequence. The resulting aldehyde was then protected as acetal 79 for the purpose of isolation (72% for this two-step process). Having accomplished the construction of the bridged framework of FR901483, Brummond directed her efforts to the total synthesis of the natural product. The approach was undertaken as depicted in Scheme 27 . In this synthesis the introduction of the side chain upon the carbocyclic ring was carried out by an aldol condensation between the aldehyde 80 and the monoacetal of 1,4-cyclohexanedione. Treatment of the latter with LDA, followed by addition of 80 resulted in the formation of aldol 81 as the major diastereoisomer (42%). Reduction of [3-hydroxy ketone 81 with NaBH4 stereoselectively afforded diol 82, which was diprotected with benzyl bromide. The Boc group was then chemoselectively removed using a two-step sequence to give 83. A number of reactions were attempted to effect the formation of the secondary amine 85. Even the three component coupling with amine 83, paraformaldehyde and allylboronate 75 did not afford the secondary amine. However, on treatment with EDCI and 2-{tertbutyldimethylsilyloxy)but-3-enoic acid, amine 83 readily underwent acylation to afford amide 84. Protection of the a-hydoxy group as the methyl ether followed by amide reduction using LiAlH4 in toluene, and deprotection of the acetal group provided aminoketone 85. Treatment of
31
this compound with 1.2 equiv. of TsOH in refluxing benzene affords amino-aledhyde 86 via the tandem aza-Cope/Mannich reaction. Since the epimeric mixture of amino aldehydes was unstable, the diastereomeric ratio was determined from the corresponding alcohols 87 (2:1 dr). As the OMe
/
\ NHBoc Ar
LDA
NaBH4
(80 %)
/
/~\NH
\ NHBoc Ar
CO2H
1. BnBr, Ag2O, TBAI
OTBS
2. TBDMSOTf 3. TBAF
OBn 83
.O '
I.Mel, Ag2O
TsOH
2 .LiAIH4 3. HCI
OBn
OBn
OBn 84
85 ,Ar
NaBH 4
[ OH
T
)
~
CH2OH
1. Jones ox 2. CICO2Et; then BnO,,,, 3. 90 °C, PhMe; then MeOH
""Cfy
4. LiAIK. 87
,Ar
(47%, four steps)
OH
88
^Ar 1. (Boc) 2 O 2. H 2 , Pd(OH)2
BnO,,, 3>-N(Boc)Me
3. (BnO)2PN(/Pr)2 4. f-BuOOH OP(OXOBn)2
89 (3R)-isomer
48 (3S)-isomer
Scheme 27. Brummond's formal synthesis of FR901483
^
FR901483
mMe
32
configuration of a C(3) was not established, the synthesis was continued with both isomers of 87. At this point, to complete the synthesis of advanced intermediate 48 and hence FR901483 itself, the elaboration of the methylamino group at C(3) remained. Alcohol 87 was oxidized by Jones' conditions and the resulting amino acid was converted in the corresponding acyl azide, which underwent thermal rearrangement. Methanolysis of the isocyanate intermediate followed by reduction of the resulting methylcarbamate afforded the diamine 88, whose secondary amine was protected with Boc. The externally directed hydroxyl group of the debenzylated diol was selectively phosphorylated under a two step procedure to give phosphates 89 and 48, the latter being the minor isomer and, in turn, identical to that previously synthesized by Sorensen. In summary, Brummond has reported in a preliminary form a formal synthesis of FR901483, the pivotal reaction being the powerful tandem cationic aza-Cope rearrangement-Mannich cyclization process, which allows a highly efficient synthesis of FR901483's bridged ring fragment, incorporating the functionality needed to achieve the target. Although the overall process has a similar length to that of the previous syntheses due to the necessary ending steps for the functionzalization adjustment, it is worth of mention the straightforward framework assembling of this FR901483 synthesis. 4 OTHER SYNTHETIC APPROACHES TO THE FR901483 SKELETON 4.1 Wardrop's formal synthesis of (±)-desmethylamino FR901483 The synthesis of racemic desmethylamino FR901483 was reported by Snider in 1998 [9] (3.1.1). Three years later, using the jV-alkoxy-iVacylnitrenium ion methodology for the azaspirocyclization step (3.2.1), Wardrop published a formal synthesis of desmethylamino FR901483 [10] having reached the advanced intermediate 15 previously reported in Snider's pioneering work (Scheme 8). The retro synthetic pathway is outlined in Scheme 28 and starts by considering that 15 could be prepared from tricycle 90 through a sequence involving alkylation of the corresponding C(7) ketone. Wardrop opted to prepare the bridged skeleton from alkyne 91 via a 6exo-trig radical cyclization, mediated by the addition of a stannyl radical [46] and using a silyl enol ether as radical acceptor [47]. The cyclization precursor 91 could be obtained from dienone 36 which, in turn, would be accessible through the spirocyclization of the iV-acyl-JVmethoxynitrenium ion VI (3.2.1).
33 OMe ,Ar HO.
ref 9
OPO(OBn)2
90
15
(±)-Desmethylamino FR901483 (19) OMe Me3Sii OMe
91
VI
Scheme 28. Wardrop proposal for the synthesis of (±)-desmethylamino FR901483
The synthesis commences with the preparation of methoxyamide 35 starting from commercially available 92 followed by treatment of 35 with PIFA (15 seconds) resulting in rapid spirocyclization to furnish 36 (Scheme 29). Dienone hydrogenation, protection of the resulting ketone, and reductive cleavage [48] of the 7V-methoxyl amide provides spirolactam 93. N-alkylation with propargyl bromide and acetal hydrolysis furnished 94, which was converted to the corresponding silyl enol ether 91 using TMSI and hexamethyldisilazane [49]. For the C(7)-C(8) bond formation and ring closure Wardrop used a radical procedure (Bu 3 SnH, AIBN, benzene) and after protodestannylation of the crude reaction mixture, the desired product 90 together with tricycle 95 were isolated. The latter arises from a translocation of the stannylvinyl radical intermediate (1,5-hydrogen transfer [50] of the adjacent allylic hydrogen atom) followed by cyclization of the allylic radical formed with the pendant vinyl stannane [51]. Probably, the competing radical pathways leading to 90 and 95 are correlated with the two conformations of the azaspirodecane ring. Having established a protocol for the formation of the 2azabicyclo[3.3.1]nonane core of the target, Wardrop proceeded to install the C(6) side chain. Protection of the hydroxyl group of 90 as the benzyl ether and oxidative cleavage of the exo-olefin gave ketone 96. Lewis acid-mediated a-alkylation of triethylsilylenol ether of 96 with pmethoxybenzyl bromide and ZnCl2 stereoselectively gave 97. Reduction of the C(7) ketone with samarium iodide cleanly generated the desired exo-alcohol 98. Reduction of lactam 98 with LiAlHj gave the corresponding amine (28%) and, rather unexpectedly, diol 15 (39%), the
34
product of benzyl cleavage [52]. Catalytic hydrogenolysis of the remaining benzyl ether allows the total conversion in 66% overall yield from 98. .0
Phl(OCOCF3)2 CH 2 CI 2 ; NHOMe then NaHCO3
COOH MeONH2.HCI, DCC ( 92 %)
OMe
(72 %)
I.NaH,
1.H 2 , Pd/C 2. (CH2OH)2, PPTS
(TMS)2NH, TMSI 2. HCI, acetone
3. Na, NH 3
(86 %)
(81 %)
0
From 90 LBnBr, NaH 2. OsO4; NalO4 (85 %)
1.KHMDS;Et3SiCI 2. p-MeOBnBr, ZnCI2 (68 %) 96
98
97
OMe
,Ar 6 steps according the Snider protocol
1. LiAIH4 2. H 2 , Pd(OH)2
HQ
See Scheme 9 (38 %)
(66 %) 15
OPO(OH)2 (±)-Desmethylamino FR901483 (19)
Scheme 29. Wardrop's alternative route to azatricyclic compound 15.
35
In summary, Wardrop developed a synthetic route to azatricycle 15 (16 steps), which Snider had previously transformed to 19. Accordingly, a formal synthesis of racemic desmethylamino FR901483 was achieved, the key steps being: (i) formation of the azaspirodecane ring by an Nmethoxy-iV-acylnitrenium ion-induced spirocyclization; (ii) construction of the bridge framework by a 6-exo-trig radical cyclization, and (iii) installation of the C(6) /»-methoxybenzyl side chain by Lewis acidmediated alkylation of a silyl enol ether. 4.2. Kibayashi's approach from bridgehead azabicyclic iminium ions In 1997 Kibayashi reported the first approach to the tricyclic framework of FR901483 [11] based on the synthesis of l-alkyl-2-azabicyclo[3.3.1]nonanes (e.g. IX), as depicted in Scheme 30, through a nucleophilic bridgehead alkylation on an anti-Bredt iminium ion (e.g. VIII), which is available by means of the Lewis acid-induced cleavage of tricyclic N,Oacetals (e.g. VII).
RM
Lewis acid
VII
VIII
IX
Scheme 30. Lewis acid-induced nucleophilic alkylation of /V,<9-acetals in the approach by Kibayashi of the tricyclic core
The requisite tricyclic N,O-aceta\ 101 was prepared by initial condensation of 3-(2-bromoethyl)cyclohexanone ethylene acetal 99 [53] with 2-(aminomethyl)benzyl alcohol. Deacetalization of 100 followed by heating in refluxing CHC13 resulted in the formation of tricyclic N,Oacetal 101 with construction of the 2-azabicyclo[3.3.1]nonane skeleton (Scheme 31). Compound 101 reacted with [2-(l,3-dioxolan-2yl)ethyl]magnesium bromide in the presence of Et2AlCl, which acts as a Lewis acid to weaken the C-0 bond in 101, promoting the formation of the reactive bridgehead iminium ion pair and thus allowing the subsequent nucleophilic attack by the Grignard reagent leading to the 1 alkylmorphan 102 in 75% yield. Hydrogenation of 102 over Pd-C was initially carried out in MeOH to cleave the 2-hydroxybenzyl group and continued after addition of 3 N HC1, resulting in deacetalization and subsequent in situ iminium ion cyclization and reduction to afford 103.
36
1.HCI-MeOH 2. CHCI3, rfx
99
100
101
H2, Pd/C, MeOH then in HCI/MeOH
MgBr
(71 %)
Et2AICI (76 %)
102
103
Scheme 31. Kibayashi's synthesis of the FR901483 skeleton
Kibayashi later reported [13] a modified version of the aforementioned approach. Ketalization of 104 and oxidative cleavage of the olefin moiety gave aldehyde 105, which was converted through a two-step reductive
1.(CH2OH)2, TsOH 2. OsO4, NalO4
HO
(83 %)
(90 %) 104
1.H2N(CH2)2OH NaBH4
2.
106
105
1.HCI-MeOH 2. CHCI 3 ,rfx
Et2AICH=CH2 (93 %)
(80 %) 108
107
H 2 , Pd/C, MeOH
CI2(Cy3P)2RuCHPh
(67 %) (62 %)
(68 %) 109
110
Scheme 32. Kibayashi's alternate synthesis of the tricyclic skeleton
103
37
amination to the secondary amine 106 (Scheme 32). After removal of the acetal group of 106, the resulting keto amino alcohol was converted to the tricyclic oxazolidine 107 in 80% yield. Using (CH2=CH)3A1 as the nucleophile, which at the same time acts as the Lewis acid, the tricyclic oxazolidine 107 reacts to give the vinylated product 108 in 93 % yield. Compound 108 was subjected to Swern oxidation and the resulting aldehyde was converted to the amino diene hydrochloride salt 109 by Wittig olefination followed by acid treatment. Five-membered ring construction was efficiently achieved by application of ring-closing olefin metathesis [54] to the hydrochloride salt 109 with 20 mol% of Grubbs' catalyst [55] at room temperature to furnish 110. Hydrogenation converted 110 to 103, thus achieving an alternative route to the azatricyclic core skeleton of FR901483 (10 steps, 18% overall yield from 104) based on vinylation at the bridgehead position of the 2-azabicyclo[3.3.1]nonane ring via the anti-Bredt iminium ion using the trivinylaluminium reagent. 4.3 Bonjoch's approach via palladium-catalyzed cyclization of aminotethered vinyl bromides and ketone enolates In 2003 we published a new synthetic entry to the tricyclic skeleton of FR901483 [14], involving the formation of the C(7)-C(8) bond through a Pd-mediated intramolecular coupling of an amino-tethered vinyl halide and ketone enolate. This type of ring closure was first reported in the carbocyclic area by Piers in the early 1990's [56] and ten years later it was introduced in the synthesis of nitrogen-containing compounds by Cook [57] and Bonjoch and Sole [58]. The potential usefulness of compounds 111 lies in the feasibility to transform these 7-methylene derivatives into compounds embodying the functionalization of FR901483 in the piperidine ring. This aspect was brought to the fore by Funk and Wardrop in their respective approaches to FR901483 using a three-step sequence consisting of oxidative cleavage of the exocyclic methylene [10], followed by a-benzylation and reduction of the resulting ketone at C(7) [7,10]. As outlined in Scheme 33, to access the tricyclic skeleton of FR901483 through the proposed methodology we required a cyclization precursor embodying a l-azaspiro[4.5]decan-8-one framework. We prepared two series of compounds and used different methodologies in each series to build the azaspiranic nucleus. For the synthesis of the simplest compound Ilia, which lacks the methylamino group at C(3), we decided to assemble the azaspiranic system of 113a by using a protocol inspired by the classical procedure for preparing spirolactams from nitrocyclohexanes, based on the a-alkylation of a nitrocycloalkane followed by the reduction and subsequent lactamization of the nitro ester
38
114
113
111
Series b
NHBn
115
Series a: R = H Series b: R = N(CO2Me)Me
Scheme 33. Pd-Mediated cyclization of tethered vinyl halides and ketone enolates in the approach by Bonjoch and co-workers.
obtained [59]. Meanwhile, the azaspirocyclic system present in 113b was prepared following our procedure developed in the synthesis of a seco derivative of FR901483, in which the pyrrolidine ring is formed by iodoaminocyclization of a homoallylamine [60]. The synthesis of I l i a starts from the unknown 4-nitrocyclohexanone, which was achieved by a Diels-Alder reaction between nitroethylene and 2-(trimethylsilyloxy)-l,3-butadiene, and following the sequence depicted in Scheme 34, vinyl bromide 112a was obtained. This amino-tethered ketone vinyl halide was treated with 0.2 equiv of Pd(PPh3)4 and 1.5 equiv of KO^-Bu at reflux temperature (THF) for 30 min to give the azatricyclic compound I l i a in 54% yield. This promising result prompted us to begin the synthesis of compound 111b, which embodies the amino group at C(3) present in FR901483 and CO2Me
NO, 1. benzene, rfx 2. (CH2OH)2 OTMS
(51%)
1.Pd/C, HCO2NH4
CH2=CHCO2Me
2. NaBH4, AcOH
TMG
(51%)
(71%) 114
N-H 1.BrCH2CHBr=CH2
Pd(PPh3)4
2. HCM0%, THF (30%)
KOf-Bu
113a
Scheme 34. Bonjoch's FR901483 skeleton synthesis
(54%) 111a
39
thus could be considered as an advanced intermediate for the synthesis of this fungal metabolite. The synthesis of compound 111b was carried out as depicted in Scheme 35. Reaction of the monoethylene acetal of 1,4cyclohexanedione and benzylamine followed by the addition of allylmagnesium bromide upon the initially formed imine gave 115. Treatment of 115 with iodine provided iodide 117, which was converted into the corresponding methylamino derivative and the resulting secondary amine reacted with methyl chloroformate to furnish the 3methylamino protected azabicyclic compound 118. Debenzylation of the latter rendered the amine 113b, which, after deprotection of the acetal, was alkylated with 2,3-dibromopropene to give the vinyl halide 112b. Treatment of 112b with 0.2 equiv of Pd(PPh3)4 and 1.5 equiv of KO/Bu in refluxing THF gave 113b in 48% yield as a nearly equimolecular mixture of diastereoisomers. It became clear from this result that the substituent at C(3) does not influence the regiocontrol in the formation of the enolate that reacts with the vinylpalladium intermediate species. BnNH2
.NHBn 2,
.N-Bn
NaHCO3
2
(95
C^jO
(70 %)
115
fO. 1.CH 3 NH 2 2. CICO2Me
1. HCI, THF-H2O
H2,Pd(OH)2/C
2.
(66 %)
(66 %) 113b
p°2Me
<po2Me Pd(PPh3)4, KOf-Bu , rfx, 30 min Bf 48%
112b
Scheme 35. Bonjoch's second FR901483 skeleton synthesis
H
111b
40
In summary, our team developed a new approach to the tricyclic core of FR901483, the key step being the closure of the bridged piperidine ring from an azaspirodecanone using a Pd-catalyzed cross coupling of vinyl bromides with ketone enolates. Additionally, a novel synthetic entry to l-azaspiro[4.5]decan-8-ones based on an iodine promoted cyclization of 1-allyl-l-aminocyclohexane derivatives is reported. 5. SYNTHESES OF TAN1251 ALKALOIDS Since the isolation of the four TAN1251 alkaloids (A-D, 2-5) in 1991, several total syntheses of members of this series have been reported.
Kawahara-Nagumo (1,3) Wardrop (2) Honda (4)
Snider (5) Ciufolini (6)
OH
.OTBDMS
pO 2 Me NH D HN-OMe
O ^—I
OMe
£>PG 3oc-N K j ) Oh
O
Scheme 36. An overview of TAN1251 alkaloid syntheses.
41
In a landmark paper that established the absolute configuration of TAN1251C and D, Snider reported the enantioselective synthesis of the four TAN 1251 alkaloids [61]. Other enantioselective syntheses of TAN 1251A have been reported by Kawahara and Nagumo [62], who published the first total synthesis of this compound in his racemic form [63], Wardrop [64] and Honda [33], while Ciufolini reported the enantioselective synthesis of TAN 1251C [6b]. Tyrosine is the chiral starting material in all the enantioselective syntheses, except in the Kawahara-Nagumo approach where a proline derivative was used. As depicted in Scheme 36, Snider and Ciufolini (entries 5,6) generate the tricyclic framework by formation of an imine from an azaspiranic intermediate incorporating the benzyl group. In contrast, KawaharaNagumo, Wardop and Honda (entries 1-4) assemble the diazatricyclic TAN 1251 skeleton by a lactamization process to give an intermediate to which the benzyl substituent should be introduced in the last stages of the synthesis. To access the azaspirodecane fragment Ciufolini, Wardrop, and Honda used a hypervalent iodine oxidation of a phenol derivative (3.2.1). Snider employed an oxazoline intermediate formed through a dipolar cycloaddition of a nitrone derivative and ethyl acrylate. For the elaboration of the azaspiranic ring system, Kawahara and Nagumo reported two very different approaches: in the racemic series an intramolecular iV-alkylation process was carried out to close the nitrogen five-memberd ring, while in the enantiopure series the carbocyclic ring was built by an aldol process from a 2,2,4-trisubstitued pyrrolidine. 5.1 The Kawahara-Nagumo synthesis of (±)-TAN1251A In 1998, Kawahara and Nagumo reported the first total synthesis of a member of the TAN1251 series [63] and five years later both authors revisited the TAN 1251A alkaloid by means of a new enantioselective synthesis (see Section 5.6). The retrosynthetic analysis of TAN1251A is outlined in Scheme 37. The target compound could be obtained by aldol reaction of tricyclic lactam 119, whose disconnection at the amide bond led to the bicyclic amino acid 120, which could be prepared from azaspirocyclic compound 121 by means of alkylation of the secondary amine and Mitsunobu-type chemistry. Azabicycle 121 may be prepared by an intramolecular alkylation of 122, which in turn could be available from allyl derivative 123. The latter can be prepared from carboxylic acid 124 by alkylation and subsequent Curtius rearrangement. The TAN1251A synthesis starts from acid 124, which was prepared from />-hydroxybenzoic acid according to the reported procedure [65]. aAlkylation of 124 (Scheme 38) with allyl bromide followed by a Curtius rearrangement [66], using diphenylphosphoryl azide (DPPA) [67] and trapping the isocyanate formed with benzylalcohol, resulted in carbamate
42 OTBDMS
TAN1251A (2)
122
123
124
Scheme 37. The Kawahara-Nagumo retrosynthetic analysis of TAN 1251A
123. Dihydroxylation of 123, selective protection of the primary alcohol as tosylate and silylation of the other secondary hydroxyl group gave silyl ether 125. Catalytic hydrogenolysis of 125 and subsequent treatment with DBU gave azaspirocycle 121. Construction of the piperazine ring commences with alkylation of the secondary amine using ethyl bromoacetate, desilylation of the protected alcohol and conversion of the hydroxyl into the azido group [68] to provide 126. Hydrogenolysis of the azide and hydrolysis of the ester led to amino acid 120. Treatment of the latter with DPPA [69] afforded the desired piperazine, which was methylated to yield 119. Aldol reaction of the enolate of lactam 119 and 4-(3-methyl-but-2enyloxy)-benzaldehyde [70] provided a mixture of two separable alcohols 127. Subsequent elimination of the mesylate of both aldol adducts afforded 128. Finally, reduction of the amide using A1H3 [71] and acid hydrolysis of the acetal provides racemic TAN 1251 A. In summary, Kawahara and Nagumo reported the first synthesis of (±)2 in 20 steps and 1.4% overall yield, the prenylated arylidene functionality being installed through an aldol process after the elaboration of the diazatricyclic skeleton of the target. The two nitrogen-containing ring were formed from a carbocyclic precursor by the succesive intramolecular processes of amino alkylation (pyrrolidine ring) and amino acid coupling (piperazine ring).
43
POOH
1-LDA(2q) ally! bromide
1. OsO4, NMO 2. TsCI, DMAP
2-DPPA,Et3N 3-BnOH
3. TBDMSCI
P*^
1 • Pd/C, H2 2. DBU
(73%)
(51%)
124
,OTBS
3
EtO
HO:
1.BrCH2CO2Et
1. Pd/C, H2
2. TBAF 3. DPPA, DEAD, Ph3P
2. LiOH (75%)
(68%)
1.DPPA, Et3N, DMF (50%)
1.MsCI,Et3N LDA.THF -78 "C 2. f-BuOK, THF
ArCHO 2. Mel, NaH, DMF (56%)
(85%, the two epimers)
°^
y3 127
1. LiAIH4, AICI3 2. HCI
Ar
128 TAN1251A (2)
Scheme 38. The Kawahara and Nagumo synthesis of (±)-TAN1251A
64-82%
44
5.2 Snider's syntheses of the four TAN1251 alkaloids (A, B, C, and D) In 2000, Snider published the total synthesis of the four TAN1251 derivatives (2-5) in their enantiopure form. The whole series was generated from the same advanced precursor 129, which in turn was obtained from aldehyde 130, a compound prepared using the synthetic methodology developed by Snider himself to synthesize aldehyde 6 in his approach to FR901483 (3.1). Aldehydes 130 and 6 only differ in the alkyl group of the phenolic ether, the size of the acetal ring, and the protecting group on the secondary amine. As shown in Scheme 2, this type of aldehyde is the point of divergence to access FR901483 or the TAN1251 derivatives, the former involving an aldol reaction and the latter a dienamine formation, which provides TAN1251C and access to the other members of this family, as depicted in Scheme 39.
of the double bond 130
TAN1251A
129 TAN1251C acetal I reduction
TAN1251D
hydroxylation
TAN1251B
EtO2C,
+ I 1,3-dipolar o"^O cycloaddition / A
R = prenyl; Ar = p-OBnC6H4 CO2Et
Scheme 39. Snider's proposal for synthesis of TAN1251C and its relationship to the TAN 1251 alkaloids
45
Synthesis of(+)-TAN1251C BO 2 C. NHOH
1. BnBr 2. TFA
O
3. p-OMeC6H4CHO 4. m-CPBA 5. NH2OH.HCI
132
(68%)
(53%)
Ar = p-OBnC6H4
2. TsCI, DMAP, Et3N 3. NaN3, DMF (79%)
2. ACOH (68%)
O"3 Ar = p-OHC6H4
y,^Y—-Ar [ J CO2Me
O
^^ / 134 Ar = p-OprenylC6H4
Me" LiAIH4 2. HCO2Ac 3. LiAIH4 (58%)
TFAA, Et3N,
POCF,
CH2OH
o
136
135
K2CO3, MeOH/H2O
(60% from 135)
f
129
(+)-TAN1251C(4)
Scheme 40. Snider's synthesis of (+)-TAN125lC
The synthesis starts from the N-Boc-tyrosine methyl ester, which was Obenzylated, since the prenyl ether required in the target compound is not compatible with the conditions required for the preparation of the hydoxylamine or the hydrogenation of the isoxazolidine intermediate (Scheme 40). Following cleavage of the iV-Boc, the resulting primary amine was treated with anisaldehyde, and after oxidation with m-CPBA
46
and reduction of the oxaziridine with hydroxylamine hydrochloride (Grundke's protocol [18]) 131 was obtained. Condensation of hydroxylamine 131 with the monodioxepane acetal of 1,4cyclohexanedione [72] gave the corresponding nitrone, which was treated with ethyl acrylate to yield an inseparable 6:1 mixture of the desired isoxazolidine 132 and its diastereoisomer. Hydrogenolysis of the latter mixture followed by treatment with a catalytic amount of HOAc in CH2CI2 afforded a 6:1 mixture of the desired lactam 133 and its diastereoisomer, in which the phenol group has been deprotected. The prenyl group was then introduced by alkylation of the phenolic hydroxyl group with l-bromo-3-methyl-2-butene. The two diastereoisomers were tosylated and then treated with sodium azide to provide after their separation 79% of pure azide 134 and 12% of the diastereomer. Simultaneous reduction of the azide, lactam, and ester of 134 was accomplished with LiAlH4. The resulting primary alcohol and amine were formylated [73] and reduction of the crude formamide provided secondary amine 135. Swern oxidation of amino alcohol 135 afforded the trifluoroacetamido aldehyde 136, which on basic treatment underwent deprotection and enamine formation. Hydrolysis with 0.1 N HC1 [74] of the TAN1251C acetal (129), which is the common intermediate for the synthesis of all members of this series, gives (+)-TAN1251C (4), confirming that this natural product has the S configuration. It is worth noting that when the dioxolane ring was used to protect the ketone group instead of the dioxepane ring, the product did not withstand the hydrolysis conditions and decomposed. Synthesis ofTAN1251A NaCNBH3, MeOH, AcOH
138
Scheme 41. Snider's synthesis of (-)-TAN1251A
(-)-TAN1251A (2)
47
Preparation of TAN 1251A (Scheme 41) proceeds by oxidation of TAN1251C acetal (129) with DDQ and subsequent reduction with NaCNBH 3 of the eniminium salt formed 137 to provide TAN1251A acetal (138), which after hydrolysis with HC1 (0.1 N) gave TAN 1251A (2). If in the initial oxidation step, DDQ was added too fast, an inseparable 7:1 mixture of 138 and the £-isomer was formed. This Snider synthesis of (-)-TAN1251A constituted the first enantiocontrolled synthesis of this natural product. Synthesis ofTAN1251D TAN 125 ID (5) is probably biosynthesized by reduction of the iminium salt formed by protonation of TAN1251C and should be available chemically by a similar protocol (Scheme 42). Snider studied this process using TAN1251C acetal (129). Its reduction with NaBH(OAc)3 in AcOH afforded 90% of a 1:9 mixture of acetals 139 and 140. Acid hydrolysis of the major acetal gave e/w-TAN1251D. Reduction of 129 with NaCNBH3 a stronger reducting agent in MeOH (pH 4) afforded a 2:1 mixture of 139 and 140. Changing the solvent to CF3CH2OH and then to the even more polar (CF3)2CHOH increased the diastereomeric ratio to 6:1 and then to 25:1 with 90% overall yield in the latter case. The stereochemical control in the reduction of enamine 129 occurs in the protonation step. If reduction of the iminium salt is slow and protonation is reversible, amine 140 is the major product, while using stronger reducing agent and increasing the polarity of the solvent, the ionic iminium salt is stabilized and the resulting product is 139 arising from the kinetic protonation from the less hindered axial face and further reduction. e
NaCNBH,,
140
TAN1251C acetal (129) R = prenyl
(+)-TAN1251D(5)
Scheme 42. Snider's synthesis of (+)-TAN1251D
48
In summary, Snider achieved the diastereoselective reduction of TAN1251C acetal and after hydrolysis (+)-TAN1251D was efficiently synthesized, confirming, in turn, that its absolute configuration is also S. Synthesis ofTAN1251B The first and so far only synthesis of TAN1251B (3) was achieved by Snider by oxidation of the TAN1251A (2) enolate (Scheme 43). This is a very challenging transformation since four a-hydroxy ketones can be formed from 2 and the choice of oxidants is limited by the presence of readily oxidizable amines and double bonds. Thus, for example, treatment of 2 with NaHMDS followed by oxidation with (15")-10(camphorsulfonyl)oxaziridine [75] gave 50% of a 2:3 mixture of undesired 141 and 142. Hydroxylation with the enantiomer of Davis' reagent also hydroxylated the wrong face of the enolates of 2. When hydroxylation was carried out using OsO4 upon the trimethylsilyl enol ethers of 2, a 2:1:8:4 mixture of 141, 142, 3, and 143 (39% overall yield) was obtained. After laborious purification, the major products were separated by HPLC on a Chiralpark AD column. In summary, with the synthesis of (+)-TAN1251B (3), Snider concludes the syntheses of the four TAN1251 alkaloids, all of them in their enantiopure form.
1. LDA; then TMSCI 2. OsO4 (20%), NMO, f-BuOH TAN1251A
141
Scheme 43. Snider's synthesis of (+)-TAN1251B
49
5.3 Wardop's synthesis of (-)-TAN1251A In his enantioselective synthesis of TAN1251A (2) Wardrop chose diazatricycle 119 as the synthetic precursor, whose conversion into 2 had been previously reported by Kawahara and Nagumo in their synthesis of (±)-2 (5.1). Thus, in his retrosynthetic analysis (Scheme 44) Wardrop postponed the construction of the Z-configurated benzylidene double bond of compound 2 to an advanced stage of the synthesis, achieving it by means of an aldol reaction followed by a stereoselective elimination process. The S configuration of the only stereogenic center of 2 was ensured using L-tyrosine as the starting material, which is also the precursor of the cyclohexanone ring. The key strategic bond was to be formed in the construction of the l-azaspiro[4.5]decane skeleton by an Nmethoxy-Af-acylnitrenium ion-induced spirocyclization (144 -> 145).
TAN1251A (2)
119
Scheme 44. Wardrop's retrosynthetic analysis of (-)-TAN1251A
The synthesis starts from L-tyrosine, which was converted to the corresponding iV-methyl carbamate derivative under Schotten-Baumann conditions. Methylation of the remaining phenol and carboxylic acid using dimethyl sulfate then gave 14. This ester was saponified and then the resulting acid subjected to coupling with methoxylamine to furnish 7V-methoxyamide 144. The latter was submitted to an azaspirocyclization (section 3.2.1) using bis(trifluoroacetoxy)iodo-benzene in CH2CI2 and MeOH to provide dienone 145 together with the corresponding dimethylacetal, which presumably arises from the trapping of the intermediate oxonium cation by methanol. Azaspirocompound 145 could be obtained in 69% overall yield, if water is added to the reaction mixture after cyclization. Hydrogenolysis of the dienone and subsequent reaction with ethylene glycol afforded acetal 147 (Scheme 45). Treatment of 147 with LiAlLLt allowed reduction of the amide as well as deprotection of the secondary amine leaving the tertiary methoxyamine intact [76]. The iV-methyl group was then protected as a Cbz derivative and the methoxy group of the pyrrolidine removed by treatment with Zn [77]. After alkylation with benzyl bromoacetate, amino
50
ester 148 was isolated. The Cbz and benzyl protecting groups were then simultaneously removed by hydrogenolysis and the resulting amino acid was submitted to DPP A coupling to provide 119. Having prepared the l,4-diazabicyclo[3.2.1]octanone core of TAN1251A from L-tyrosine in 13 steps and with 8% yield, Wardrop proceeded to install the benzyl side chain following the Kawahara-Nagumo protocol. Thus, generation of the enolate of 119 and its trapping with />-prenyloxybenzaldehyde [70] gave a mixture of aldol products, which were treated with MsCl. The resulting unseparated mesylates were submitted to an elimination process to steroselectively give the Z isomer. Reduction of the lactam with A1H3 gave an improved yield of the corresponding enamine, which finally was deprotected to provide (-)-TAN1251A. In summary, the enantioselective total synthesis of (-)-TAN1251A was accomplished by Wardrop in 17 steps and with 3% overall yield, the key step being the N - m e t h o x y -iV-acylnitrenium ion-induced spirocyclization. H
MeO,c \ HN-OMe
1.CIC0 2 Me 2. NaOH
LNaOH
3. Me2SO4
2. MeONH2.HCI, DCC HOBT, NMM
(76%) O MeOPhl(OCOCF3)2, CH2CI2, MeOH;
"
then H2O
2. (CH2OH)2
(69%)
0 M e
1.LiAIH4 2. CICO2Bn 3. Zn, AcOH 4. BrCH2CO2Bn
(86%) (43%)
lyie /H Cbz
BnQ >=O
1. LDA, ArCHO (80%) 2. MsCl; then Kf-BuO (75%)
1. H2, Pd/C 2. DPPA
3. AIH3 4. HCI
(57%)
148
119
Scheme 45. Wardrop's synthesis of (-)-TAN1251A
(61%) (-J-TAN1251A (2)
51
5.4 Ciufolini's synthesis of (+)-TAN1251C Ciufolini published in 2001 [6b] the synthesis of enantiopure TAN1251C (4) using an advanced intermediate of his FR901483 synthesis (3.2.3). The point of divergence of both synthesis is the keto lactam 54 (Scheme 22). The synthesis of 4 involves ten initial steps to achieve alcohol 54 from tyrosine (16% overall yield) and pursues with the change of the alkyl side chain on the phenolic hydroxyl group (Scheme 46). Thus, Odemethylation of 54 followed by prenylation and treatment of the resulting lactam with LiAlH4 gaves azaspiranic compound 149. Protection of the secondary amine and oxidation of both alcohols using TPAP/NMO lead to the keto aldehyde 150. Treatment of the latter with Cd/Pb couple [78] allows the deprotection of the secondary amine as well as the cyclization process to provide (+)-TAN1251C (4).
(10 steps, 16% overall yield)
see Scheme 22
Me-"1
OMe
1. BBr3 2. Prenyl bromide 3. LiAIH4 OH 149
CHO 1. TrocCI (51%, from 54)
Troc ,NI\/|e
RQ-
2. TPAP, NMO (63%)
Cd/Pb couple NH 4 OAc (79%)
150
(+)-TAN1251C (4)
Scheme 46. Ciufolini's synthesis of (+)-TAN1251C
In summary, the second enantiocontrolled synthesis of (+)-TAN1251C requires a maximum of 16 linear steps from L-tyrosine (4% overall
52
yield). The process not only uses the same methodology developed in the Ciufolini's FR901483 synthesis but even utilizes a common synthetic intermediate, which is a polyfunctionalized azaspirodecane (i.e. 54) that is obtained by aromatic oxidation of an oxazoline phenolic derivative to achieve the spirolactam unit. 5.5 Honda's Approach to (-)-TAN1251A In 2002, Honda developed a fruitful new synthetic entry to the known azatricyclic compound 119, which has led to a synthesis of (-)TAN1251A [33]. The retrosynthetic pathway is shown in Scheme 47, the synthetic target being the intermediate 119, which had previously been converted into TAN1251A by Kawahara and Nagumo [63] and Wardrop [64]. The strategy employed by Honda to form the spirocenter involved a hypervalent iodide-promoted phenol oxidation reaction, as in the Wardrop and Ciufolini syntheses, but on the more advanced intermediate 151, with the piperazine ring already incorporated. Compound 151 could be prepared by a coupling of L-tyrosine and glycine derivatives, the former acting as the chiral source to construct the TAN 1251A platform, followed by a lactamization process. Q
JVIe
ref63, 64
L-Tyrosine
TAN1251A
Glycine
119
152
151
Scheme 47. Honda's approach to the synthesis of (-)-TAN1251A
The starting material for the synthesis (Scheme 48) was the tyrosine aldehyde 154, which was readily prepared from the methyl ester of Ltyrosine. Reaction of the latter with ethyl chloroformate followed by Obenzylation of the phenol gave ester 153, which was reduced, reprotected at the nitrogen atom, and treated with the Dess-Martin reagent to give aldehyde 154. The construction of the piperazine ring was carried out through a reductive amination of 154 with glycine methyl ester, followed by deprotection of the nitrogen and lactamization of diamino ester 155. Hydrogenolysis of the benzyl ether group afforded the amino phenol 151 required for the next crucial transformation.
53
At this point, Honda focused on the critical step of the synthesis, the oxidative transannular cyclization to close the pyrrolidine ring, which involves the simultaneous construction of the quaternary center of the target. The oxidative cyclization of 151 in 2,2,2-trifluoroethanol with bis(acetoxy)iodobenzene (DIB) gave the desired spirocompound 152 in 43% yield, which was increased up to 69% by using hexafluoroisopropanol as the solvent. The successful cyclization leading to 152, featuring an unprotected aminophenol derivative, is the second time Sorensen's reaction (2.2) has been used. To achieve the Wardrop intermediate 119 and hence the formal synthesis of (-)-TAN1251A, the reduction of dienone 152 and an acetalization process were required. Although difficulties were initially encountered in obtaining the reduced compound by catalytic hydrogenation, Honda found that the dienone could be converted to the ketone using triethylsilane in the presence of CuCl and DPPF (20% mol of each) [79]. Finally, acetalization afforded the known intermediate 119. In summary, Honda reported a formal synthesis of (-)-TAN1251A in which a hypervalent iodine-promoted cyclization of a tethered secondary amine and phenol was used in the key step for the elaboration of the tricyclic skeleton.
NH2CH2CO2Me, NaCNBH3 2. BnBr (77%)
OBn
2. Boc2O 3. Dess Martin
OBn
2. TFA
153
(73%)
154
(83%)
H
Phl(OAc)2, (CF3)2CHOH (69%)
r^HVIe
1.Et3SiH, CuCI(60%)
TAN 1251A (2)
2. (CH2OH)2 (66%)
119
Scheme 48. Honda's formal synthesis of (-)-TAN125lA
54
5.6 The Kawahara-Nagumo approach to (-)-TAN1251A from a proline derivative In 2002, four years after publishing the first paper describing the synthesis of racemic TAN 1251 A, Kawahara and Nagumo reported a formal synthesis of the same antimuscarinic compound but in its enantiopure form [62]. Unlike the previous enantioselective syntheses where tyrosine was the chiral source, Kawahara and Nagumo chose a proline derivative as the starting material. The retrosynthetic pathway is outlined in Scheme 49 and starts with the simplification of the TAN 1251A again to the known intermediate 119, whose first conversion into the alkaloid had been reported by the same authors [63]. The target compound 119, in turn, can be prepared from 126 also following their known protocol. Compound 126 should be obtained from 156 via installation of an azide group and N-alkylation. Disassembly of the cyclohexanone ring from 156 in a retrosynthetic sense leads to keto aldehyde 155, which may be prepared by alkylation of proline 154 followed by some functional group interconversion steps.
Boo-
119
126
156
155
154
Scheme 49. Proline-based approach to (-)-TAN1251A by Kawahara and Nagumo
The synthesis starts with the alkylation of the trans-4-hydroxy-Lproline derivative 154 with 4-iodo-l-butene using 2.5 eq of LDA to afford 157 (67%) and its epimer (15%). Each one is useful for the synthesis of 156 since after hydrogenation of 159 the stereogenicity at spirocenter is lost. The sequence 157 -> 156 was carried out separately in both epimeric mixtures obtaining similar yields. The synthetic sequence, which is depicted in Scheme 50 for the major epimer 157, involves conversion of the latter to aldehyde 158 through a reduction-oxidation process, followed by Wacker oxidation of the terminal alkene to give the keto aldehyde 155. Aldol condensation and succesive elimination process in the ketol intermediate gave cyclohexenone 159, whose hydrogenolysis followed by reaction with ethylene glycol and a desilylation step afforded hydroxy acetal 156. Alcohol 156 was converted to the corresponding azide through a Mitsunobu-type process, and after
55
removal of the Boc group and alkylation of the secondary amine, polyfunctionalized azaspiranic compound (+)-126 was achieved. The synthesis was then pursued using the same methodology already described by these authors in the racemic series. Compound 126 was converted into tricyclic amide (+)-119 by the sequence of catalytic hydrogenation, saponification of the ester group, lactam ring formation and methylation. Since Wardrop had reported the conversion of (+)-119 into (-)TAN 1251A using the procedure already described by Kawahara and Nagumo in the racemic series, the enantiocontrolled synthesis of (+)-119 achieved by these authors constitutes a new formal synthesis of 2. This approach is noteworthy for its use of a proline derivative as the starting material and the elaboration of the carbocyclic ring by an aldol reaction. TBDPSQ
LDA, HMPA, 4-iodo-1-butene
^ N
TBDPSQ 1.DIBAH 2. TPAP, NMO
(67%)
(80%)
Boc 154
.OTBDPS
.OTBDPS TBDPSQ C H 0
Boc
Wacker oxidation
Boc—I 0H,
(92%)
158
1.H 2 , Pd(OH)2
1. DPPA, DEAD 2. CF3CO2H
Bo<
2. (CH2O)2 3. TBAF
3. BrCH2CO2Et (76%)
(85%)
(35%) 156
126
119
Scheme 50. Second synthesis of the tricyclic intermediate 119 by Kawahara and Nagumo
6. Concluding Remarks The isolation in the last decade of natural products incorporating the 1 azaspiro[4.5]decane ring system that have been shown to display
56
important biological activity has prompted the development of efficient strategies and synthetic routes towards these compounds. The synthetic studies summarized here cover the partially developed and fully completed programs to build FR901483 and TAN 1251 alkaloids, which together with the marine alkaloids cylindricines [80] and lepadiformine [81 ] are all the tricyclic natural products embodying the 1 azaspiro[4.5]decane ring to be isolated so far. ABBREVATIONS Ac AIBN Ar Bn Boc Cbz Cy DBU DCC DEAD DIBAL DMAP DMF DDQ DEAD DIAD DIB DIBAH DMAP DMSO DPPA EDCI ED50 HMPA HOBT HTIB KHMDS LDA w-CPBA Ms NMO NMM Ns
acetyl 2,2'-azobis(isobutyronitrile) aryl benzyl tert-butoxycarbonyl benzyloxycarbonyl cyclohexyl l,8-diazabicyclo[5.4.0]undec-7-ene A^-dicyclohexylcarbodiimide diethyl azodicarboxylate diisobutylaluminium hydride 4- (dimethy lamino )py ridine dimethylformamide 2,3 -dichloro-5,6-dicyano-1,4-benzoquinone diethyl azodicarboxylate diisopropyl azodicarboxylate iodobenzene diacetate diisobutylaluminium hydride 4-dimethyaminopyridine dimethyl sulfoxide diphenylphosphoryl azide 1-(3-dimethy laminopropyl)-3-ethylcarbodiimide hydrochloride median effective dose hexamethylphosphoramide 1 -hydroxybenzotriazole [hydroxy(tosyloxy)iodo]benzene potassium bis(trimethy 1 silyl)amide lithium diisopropylamide w-chloroperbenzoic acid methanesulfonyl TV-methylmorpholine iV-oxide JV-methylmorpholine /?-nitrobenzenesulfonyl
57
PG Ph PIFA PPTS pyr L-selectride TBAF TBAI TBDMS TBDPS TES Tf TFA TFAA TFEA TMG TMS TPAP Troc Ts
protecting group phenyl phenyliodosyl bis(trifluoroacetate) pyridinium/>toluene sulfonate pyridine tri-seobutylborohydride tetrabutylammonium fluoride tetrabutylammonium iodide tert-butyldimethylsilyl (also TBS) ter/-butyldiphenylsilyl triethylsilyl trifluoromethanesulfonyl trifluoroacetic acid trifluoroacetic anhydride 2,2,2-trifluoroethanol 1,1,3,3-tetramethylguanidine trimethylsilyl tetrapropylammonium perruthenate trichloroethyloxycarbonyl o-toluenesulfonyl
ACKNOWLEDGEMENTS Work in the authors' laboratory was supported by the MCYT (Spain) and DURSI (Catalonia).
7. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
Sakamoto, K.; Tsujii, E.; Abe, F.; Nakanishi, T.; Yamashita, M.; Shigematsu, N.; Izumi, S.; Okuhara, M. J. Antibiot. 1996, 49, 31-A4. Snider, B. B.; Lin, H. J. Am. Chem. Soc. 1999, 121, 7778-7786. Shirafuji, H.; Tsubotani, S.; Ishimaru, T.; Harada, S. PCT Int. Appl. 1991, WO 91 13,887; Chem.Abstr. 1992, 116, 39780t. Hida, T.; Takeda Chemical Industries, Ltd., May 6, 1999. Personal communication to Prof. Snider. Scheffler, G.; Seike, H.; Sorensen, E. J. Angew. Chem. Int. Ed. 2000, 39, 45934596. (a) Ousmer, M.; Braun, N. A.; Ciufolini, M. A. Org. Lett. 2001, 3, 765-767. (b) Ousmer, M.; Braun, N. A.; Bavoux, C ; Perrin, M.; Ciufolini, M. A. J. Am. Chem. Soc. 2001, 123, 7534-7538. Maeng, J.-H.; Funk, R. L. Org. Lett. 2001, 3, 1125-1128. Brummond, K. A.; Hong, S.-P. 226th ACS National Meeting, New York, 2003. Snider, B. B.; Lin, H.; Foxman, B. M. J. Org. Chem. 1998, 63, 6442-6443. Wardrop, D. J.; Zhang, W. Org. Lett. 2001, 3, 2353-2356.
58 [11] [12] [13] [14]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
61
BIOACTIVE NATURAL PRODUCTS FROM SOUTHERN AFRICAN MARINE INVERTEBRATES* MICHAEL T. DAVIES-COLEMAN Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa ABSTRACT: The coastline of southern Africa is divided into three bio-geographical zones, with each zone sustaining a unique diversity of marine fauna and flora. Associated with this marine biodiversity are relatively high levels of species endemism, often a useful pre-requisite for the discovery of novel, bioactive marine natural products. This review is the first comprehensive presentation of the structures of 143 marine natural products isolated over the last two decades from 30 species representing five phyla of southern African inter-tidal and sub-tidal marine invertebrates.
INTRODUCTION The approximately 3000km long southern African coastline, stretching from Namibia in the west to southern Mozambique in the east, is broadly subdivided into three bio-geographical zones: the cool temperate west coast, the warm temperate south east coast, and the subtropical east coast, Fig. (1) [1]. Each of the southern African bio-geographical zones sustains distinctive populations of marine flora and fauna and of the over 10 000 species of marine organisms recorded off the southern African coast, a large proportion are reported to be endemic [2-5]. The bioactive natural products reviewed here are grouped together according to the phylum to which their source invertebrate belongs. The phylogenetic sequence {i.e. Hemichordata before Porifera) reflects the chronological order of investigations of five phyla of southern African marine invertebrates by natural products chemists. The bioactive natural products described within each of the phyla are in turn grouped according to the bio-geographical region where the source organism was collected. The bio-geographical distribution of bioactive metabolites is presented in a sequence starting with the west coast cool temperate zone and ending This review is dedicated to the memory of the late John "Jack" Elsworth, a pioneer of marine natural products chemistry research in South Africa.
62
with the east coast subtropical zone. To date there are no records of the isolation of bioactive natural products from southern African marine algae or micro-organisms and this review accordingly records only those southern African marine invertebrate species that have yielded one or more biologically active marine natural products. Other metabolites, cooccurring with the bioactive natural products and whose bioactivity (if any) has not been established, are also included to provide a more holistic view of the natural product diversity produced by southern African marine organisms. Where applicable, reference is made to syntheses of bioactive metabolites capable of addressing the supply problem often associated with development of Pharmaceuticals from marine natural product lead compounds. Bio-geography of the Southern African Coastline The bio-geography of the southern African coastline is largely the product of the prevailing winds and the two major ocean currents that follow the edge of the continental shelf (ca. -400m) on either side of the subcontinent, Fig. (1) [1,2]. The Agulhas current is a warm, fast flowing current that dominates the oceanography of the east and south east coasts of southern Africa. Following the continental shelf, the main body of the massive Agulhas current is deflected away from South Africa along the southern Transkei coast where the continental shelf broadens (at the junction of the sub-tropical and warm temperate bio-geographical zones). Closer in-shore, a variable, cooler counter-current flows in an opposite direction to the Agulhas current. This cooler counter-current, together with eddies of warm water moving inshore off the Agulhas current, significantly influences the biodiversity of inter-tidal and benthic marine coastal communities in the warm temperate bio-geographic zone [1,2]. Conversely, the cold Benguela current moves in a northerly direction up the west coast of southern Africa. A combination of strong southerly and south-easterly winds, together with Coriolis forces, result in the deflection of surface waters of the Benguela current away from the coast [1]. The concomitant upwelling of deep, nutrient-rich, cold water supports substantial populations of phytoplankton, and leads to the proliferation of kelp forests in this region. Surprisingly, despite the higher primary productivity of the west coast, this cool temperate bio-geographic region has a lower biodiversity of marine invertebrates than the east coast
63
where the occurrence of large numbers of circumtropical Indo-Pacific species contributes significantly to overall species diversity [1,2]. —pS
\
\
V
' V
South Coast: warm temperate
j ^ ^
West Coast: cold temperate
\
\^\
NT
=L)
East Coast: subtropical
•^^
••
Main current
p.
Secondary currents
\
-Wi ^'. MJi
SOUTH
AFRICA
'•Tl 35-s
INDIAN OCEAN
1
i
^>
\ /
/
0
Continental shell
CON
J
Agnilms
1
Roium
!
r\
30 S
35'S
~^>
ATLANTIC OCEAN 0 21) "E
30'F;
500 KILOMETRES
Fig. (1) Map of southern Africa indicating the three bio-geographical zones, ocean currents and sites of major marine invertebrate collections (A = Cape Peninsular, B = Tsitsikamma Marine Reserve, C = Algoa Bay, D = Coffee Bay, E = Aliwal Shoal, F = Sodwana Bay, G = Ponto do Ouro)
Marine Natural Products Research in Southern Africa - A Brief Historical Perspective Regions of high species diversity and endemism can offer rich rewards for marine natural products chemists in search of bioactive compounds with possible medicinal properties. The potential for South Africa's marine resources to provide a source of new pharmaceuticals was first recognized over thirty years ago in a South African government report entitled "Drugs from the Sea" that concluded with the following statement, "Very little attention has yet been paid in South Africa to the recovery of drugs from the sea. This field offers exciting and rewarding challenges to South African scientists. Once research brings down the unit cost, the sea may offer a vast potential for the production of drugs for South Africa [6]." Prior to this report, South African contributions to the
64
chemistry of marine organisms were modest and comprised a series of research publications emanating from the National Chemistry Research Laboratory in Pretoria describing the characterization of highly unsaturated fatty acids and alcohols from fish oils [7-9] and the pioneering marine algal polysaccharide structural research of Nunn and co-workers at Rhodes University [10-12]. The first studies in South Africa of the chemistry of southern African marine invertebrates began at the University of Cape Town in 1977 under the direction of Elsworth and Cragg who focussed on the natural products chemistry of selected marine organisms collected around the Cape peninsular, the acknowledged dividing line between the cold temperate west coast and the warm temperate south east coast marine fauna and flora [5]. Their initial research involved a GC-MS study of the complex sterol profiles of two oceanic and three intertidal species of annelid worm [13] the ribbed mussel Aulacomya ater, sea stars Marthasterias glacialis and Henrica ornate, and the sea cucumber Cucumeria frauenfeldi [14, 15]. Interestingly, South African marine worms and coelenterates also attracted the attention of international researchers at this time and Mazzanti and Piccinelli compared the occurrence of indole and imidazole compounds in six species of southern African annelid worms and six species of coelenterates [16]. In contrast to the increased global interest in marine natural products as a source of potential pharmaceuticals in the 1980s, the natural products chemistry of southern Africa's marine resources was largely ignored for most of this decade. However, in the last fifteen years, this situation has been reversed. Four research groups have provided a significant insight into the chemistry of biologically active natural products from southern Africa's marine fauna and flora and the contributions of the research groups led by George Pettit, Yoel Kashman, the late John Faulkner and myself, predominate in the following review. BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE WORMS (PHYLUM HEMICHORDATA) Cephalostatins The most significant group of bioactive metabolites to be isolated from southern African marine worms have undoubtedly been the powerful cell growth inhibitors, cephalostatins 1-17 (1-17), isolated by Pettit et al. from
65
the marine tube worm Cephalodiscus gilchristi (Class Pterobranchia) initially collected off the south east of southern Africa in 1972 and again in 1981 [17-24]. The cephalostatms are closely related to the ntterazines isolated by Fusetani et al. from the Japanese ascidian Ritterela tokioka [25]. The trisdecacyclic pyrazine structure of cephalostatin 1 (1) was eventually solved by X-ray analysis many years after the initial collections of C. gilchristi [17]. The structures of the related cephalostatins 2-4 (2-4) essentially followed from comparison of the spectroscopic data with those of 1 [18]. All four compounds exhibited "7
Q
1
similar exceptional ED50 values in the range 10" -10" |J.gmL~ in the US National Cancer Institute's (NCI's) P-388 lymphocytic leukaemia cell line screen.
1 2 3
R1 = R = H R1 = OH, R2 = H R1 = OH, R2 = Me H
OH
66
Conversely, cephalostatins 5 and 6 (5, 6), in which ring C of the left hand steroid unit is aromatized, exhibited reduced activity in the PS system ( 1 0 - 1 0 ugmL") suggesting the importance of C - D' ring junction and possibly C-22' spiroketal structural integrity to the cytostatic properties of the cephalostatins [19]. OH
5 6
R = Me R =H
Further clues pertaining to the structure activity relationships within this intriguing group of metabolites were provided by the isolation of further compounds in this series from C. gilchristi. Cephalostatins 7-9 (79) and 1-4 displayed almost identical activity and selectivity (TI50 values of 0.1 -1 nM) against a series of cancer cell lines (including non-small cell lung HOP 62, small cell lung DMS-273, renal RXF-393, brain U-251 and SF-295, leukaemia CCRF-CEM, HL-60 and RPM1-8226) in the NCI's in vitro 60 cell line primary screen [20]. Not unexpectedly, concurrent screening of cephalostatins 5 and 6 revealed that these two compounds only exhibited modest activity against two cell lines, CNS U251 and renal SN12K1 [20]. The conclusion drawn from this profile of activity was the apparent importance of the pyridizine right hand side unit to the bioactivity of the cephalostatins, and that minor changes in the left hand side E' and F' rings does not significantly reduce this activity [20].
67
HO
A re-collection (450 kgs wet weight) of C. gilchristi in 1990, for possible pre-clinical development of 1, led to the isolation and identification of seven more cephalostatin analogues. The differential cyctotoxicity of cephalostatins 10 and 11 (10-11), in the NCI's anticancer screens mirrored that of 1 [21]. Further compounds isolated from this re-collection included the first symmetrical cephalostatin, cephalostatin 12 (12), and the C-l' hydroxylated derivative of 12, cephalostatin 13 (13) [22]. Interestingly, while 12 and 13 showed substantial growth inhibitory activity against many of the NCI's cancer cell lines, the GI50 values obtained from these assays were on average
68
orders of magnitude (400 and 1000 nM respectively) higher than those of 1 (1 nM). OH
R ! = H , R2 = OMe
OH
13 R = OH
Comparative evaluation of 1 and cephalostatins 14 and 15 (14, 15) in the NCI in vitro primary screen again revealed a reduced average GI50 cytotoxicity (100 nM and 68 nM respectively) for 14 and 15 compared to 1 [23]. The final pair of cephalostatins (16 and 17) isolated from C. gilchristi exhibited the same cyto toxic profile as the majority of the other cephalostatins with the panel averaged GI50 values of 16 (lnM) and 17 (4nM) comparable to that of 1 (lnM) [24].
69
14 R = H 15 R = Me
17
Cephalostatin 1 (repeatedly the most active in the series) has recently been shown to induce a novel pathway of receptor-independent apoptosis of leukaemia cells at nanomolar concentrations [26]. Given the unique profile of activity of 1 in the NCI 60 cell line screen, this compound was identified in 2000 as a Rapid Access to Intervention Development (RAID) project by the NCI [27]. Unfortunately, preclinical development
70
of 1 was hampered by the poor availability of this compound from natural sources (ca. lOOmg of 1 isolated from one ton of C. gilchristii) and although numerous cephalostatin analogues have been synthesized [28], there are relatively few syntheses reported for the naturally occurring cephalostatins [29]. Fuchs et a/.'s synthesis of 1 (from the abundant plant derived steroid, hecogenin acetate) provides possibly the best route for the production of multi-gram amounts of the cephalostatins to alleviate the supply problem [30]. BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE SPONGES (PHYLUM PORIFERA) Although, marine sponges are a prolific component of both inter-tidal and sub-tidal southern African marine ecosystems they are poorly described and the number of species occurring along the southern African coast is unknown [31]. It is noteworthy that our recent investigations of the natural products chemistry of southern African members of the sponge family Latrunculiidae have contributed to the discovery of five new species and the raising of two new genera [32,33]. Spongiostatins Pettit et al. initiated the discovery of potential anti-cancer compounds from southern African marine sponges with the isolation of the potent spongiostatins 4 - 9 (18 - 23)[34-36] from the bright orange "wallsponge" Spirastrella spinispirulifer (Family Spirastrellidae), a large, conspicuous sponge common on sub-tidal reefs along both the cool and warm temperate south west and south east coasts of southern Africa [31]. Spongiostatins 4-9 are related to the first three compounds isolated in this series, spongiostatin 1-3 (24-26)[37,38] isolated from a different species of sponge, Spongia sp. (Family Spongiidae) collected off the Republic of the Maldives, a group of islands in the northern Indian Ocean. The family of spongiostatin compounds can be broadly divided into two structural groups with the presence or absence of a tetrahydrofuran ring in the macrocycle defining the difference between these two groups. The members of the first group (18, 20, 24-26) without a tetrahydrofuran ring differ from each other in the substitution pattern at C-5, C-15 and C-50
71
while those in the latter group (19, 21-23) only differ in substitution at C5 and C-50.
OMe
OH
H I OH OH
^ C l , R2 = Ac, R 3 = H 18 20 R1 = R3 = H, R2 = Ac 24 R1 = Cl, R2 = R3 = Ac 25 R1 = H, R2 = R3 = Ac 26 R1 = Cl, R2 = H, R3 = Ac
The structures of 24 and 25 were found to be consistent with the structures of altohyrtin A and C respectively, isolated from an unrelated Hyrtios sponge, and reported by Kobayashi and Kitagawa [39, 40]. Initial discrepancies in the assignment of the absolute stereochemistry of the spongiostatins and altohyrtins were eventually resolved in 1997 by the first total synthesis of altohyrtin C/spongiostatin 2 by Evans et al. [41], followed by the total synthesis of altohyrtin A/spongiostatin 1 by the Kishi group [42]. The structures of compounds 18, 20, 24 - 26 presented here are drawn with the revised stereochemical assignments [43]. Although these revised assignments could probably be extrapolated to compounds 19, 21 - 23, the original stereochemistry proposed by Pettit et al. is retained here.
72
.0 OMe
H 1 OH OH
OH
19 R = C 1 , R = H 21 R1 = H, R2 = H 22 R1 = H, R2 = Ac 23 R1 = Cl, R2 = Ac The trace amounts of the spongiostatins (ca. 10"7% yield) present in S. spinispirulifer required increasingly excessive and environmentally controversial recollections of this sponge (e.g. 2409 kgs) during the 1980s for the investigation of their potent in vitro activity (Table 1) [34]. The antineoplastic mechanism of action of the spongiostatins is well established. Spongiostatin 1 blocks microtubule assembly through inhibition of the glutamate-induced polymerization of tubulin [44]. Microtubules maintain cell shape and are an integral part of the mitotic spindle necessary for cell division. The collapse of microtubule assembly therefore halts cell division and leads ultimately to cell death [45]. The competition provided by the plethora of tubulin inhibitors presently in clinical development [45] and a paucity of spongiostatin 1 has hampered the preclinical development of this compound [46]. Paterson et a/.'s synthesis of 24 (1% yield from 33 steps) presently provides the most economic solution to the supply problem [47].
73 Table 1 T h e isolated yields of spongiostatins 1 - 9 and their cytoxicity in the NCI's 60 cell line screen and as an inhibitor of tubulin polymerization [43].
Yield
Compound
Spongiostatin 1
3.5xlO' 6 % (13.8mg)
GIso NCI 60 cell line screen
ICso inhibition of tubulin polymerization
1.3xl0"10M
5.3xlO" 6 M
Spongiostatin 2
1 xlO" % (4.3mg)
8.5xl0-'°M
4.6xlO"<'M
Spongiostatin 3
6.7xlO' 7 % (2.7mg)
8.3xl0-'°M
1.3 x 10"5M
Spongiostatin 4
4.4x10-'% (10.7mg)
1.0xl0-'°M
5.1 xlO"6M
Spongiostatin 5
6
7
5.4 x 10" % (12.9mg)
lo
1.2xl0 M
3.5xl0" % (8.4mg)
1.1 xlO" M
4.4 x 10-6M
Spongiostatin 7
2.2xlO" 7 % (5.4mg)
1.0xl0- 9 M
SJxlO^M
8
9
aJxlO^M
Spongiostatin 6
7
Spongiostatin 8
7.5xl0- % (1.8mg)
2.3xl0-'°M
5.5 x 10"6M
Spongiostatin 9
2.2xlO- 7 % (5.4mg)
4.0xl0-"M
4.2xl0- 6 M
27 R^^R 28 R'=R 2 = H 29 R ! =OH,R 2 =
30 R1 = R2 = H 31 R1 = H, R2 = Me 36 R1 = OH, R2 = H 37 R1 = OH, R2 = Me
Dilemmaones There is only one other report of bioactive natural products from sponges of the cool temperate bio-geographic region of the southern African coast [48]. Bioassay (brine shrimp lethality assay) guided chromatography of a mixed collection of three orange sponges comprising 47% Ectynonanchora flabellate, 41% Crambe chelastra and 12% Antho sp. yielded three novel indole alkaloids, the dilemmaones A C (27-29) [48]. Although the names of these compounds reflect the initial
74
confusion over their original sponge source, a process of elimination was used to propose E. flabellate as the source of 27-29. The structures of the dilemmaones were readily resolved by standard spectroscopic techniques. Pyrroloiminoquinones Our investigations of the marine natural products chemistry of southern African marine invertebrates began in the Tsitsikamma Marine Reserve on the warm temperate south east coast of South Africa in 1993. This region of coastal southern Africa has recently been proposed as an area of high latrunculid sponge (Family Latrunculiidae) biodiversity [33]. An initial study of a new species of the latrunculid sponge, Tsitsikamma favus, collected in the Tsitsikamma Marine Reserve in 1994, yielded two bis-pyrroloiminoquinone alkaloids, tsitsikammamines A and B (30, 31) and the pyrroloiminoquinone 14-bromo-discorhabdin C (32) together with its dihydro analogue (33) [49]. A recent re-examination of extracts of this sponge also yielded 3-dihydro-7,8-dehydrodiscorhabdin C (34), 14bromo-1-hydroxydiscorhabdin V (35) and the novel N-18 oxime analogues of tsitsikammamine A and B (36 and 37) [50]. A second deep-water species of the genus Tsitsikamma, T pedunculata, from Algoa Bay (200kms to the east of the Tsitsikamma Marine Reserve) in addition to compounds 32, 33 and 35, produced the known 3-dihydrodiscorhabdin C (38) [51] and two new minor metabolites 14-bromo-3-dihydro-7,8-dehydrodiscorhabdin C (39) and discorhabdin V (40) [50]. Extracts of two other latrunculid sponges from Algoa Bay, a dark green, encrusting Latrunculia sponge, L. bellae, gave the new discorhabdins; 1-methoxydiscorhabdin D (41) and 1-aminodiscorhabdin D (42) and five known metabolites; damirone B (43) [52], makaluvic acid A (44) [53] makaluvamine C (45) [54] and discorhabdins G* and N (46 and 47) [55]. Discorhabdins A (48) [56], D (49) [57], H (50) [55] and 32 were the major pyrroloiminoquinone metabolites in polar extracts of he shallow-water brown sponge Strongylodesma algoaensis [50]. The structures of 30 - 50 were all elucidated from 2D-NMR spectroscopic data despite the relatively few proton resonances in the 'H NMR spectra of some of these compounds leading ultimately to a paucity of heteronuclear multibond coherence (HMBC) correlations. The cohort of twenty pyrroliminoquinone metabolites isolated from the four species of Southern African latrunculid sponges provided a unique opportunity for a comparative analysis of their cytotoxicity against human colon
75
tumour cells (Table 2). Interestingly, the ubiquitous discorhabdin A (48) was the most bioactive compound in this series [50].
16
32
33 34 38 39
R1 + R2 = O, R3 = Br R1 = H, R2 = OH, R3 Br R1 = H, R2 = OH, R3 = H, A7 R1 = H, R2 = OH, R3 = H R ! = H, R2 = OH, R3 = Br, A7
35 R = O H , R =Br 40 R1 = R2 = H
43
41 R = OMe 42 R = NH2 47 R = ^ . . . .
45
44
46
76
Br
O
48
49 R = H 50 R =
COOH
CH 3 Table 2.
Human colon tumor (HCT-116) cytotoxicity data for compounds 30 - 49 [ 50] Compound
HCT-116 (ICsoj*M)
Compound
HCT-116 (ICsojtM)
30
1.414
40
1.266
31
2.382
41
0.232
32
0.077
42
0.119
33
0.645
43
3.102
34
0.197
44
28.399
35
12.496
45
1.089
36
128.213
46
0.327
37
16.541
47
2.249
38
0.323
48
0.007
39
0.222
49
0.595
Halistanol disulphate B Approximately 700 kms to the north east of Algoa Bay, on the warm subtropical coast of South Africa off southern Kwazulu-Natal, lies an extensive offshore reef system (the Aliwal Shoal) rich in marine invertebrate life. A three-year collaborative research program (19941996) between Scripps Institution of Oceanography (SIO), Rhodes University (RU) and SmithKline Beecham Pharmaceuticals (SKB) was initiated with a collection of approximately ninety marine invertebrates
77
from the Aliwal shoal. As part of a high-throughput screening programme to find potential anti-hypertensive agents, an extract of the Aliwal Shoal sponge Pachastrella sp. (Family Pachistrellidae) was found to inhibit the endothelin converting enzyme (ECE) [58]. ECE is a membrane bound metalloprotease enzyme responsible for hydrolysing an inactive polypeptide big-endothelin (B-ET) to the smaller, bioactive polypeptide endothelin-1 (ET-1). Overproduction of ET-1 is implicated in hypertension and renal failure and compounds that inhibit ET-1 production may therefore be useful in treating these two conditions. The ECE inhibition of the Pachastrella extract was found to reside in a sulfated sterol, halistanol-disulfate B (51) [58]. The structure and relative stereochemistry of 51 was readily delineated from standard 2D NMR and nuclear Overhauser effect (nOe) data. Halistanol disulphate B is the desulfated analogue of halistanol sulphate B (52) a thrombin receptor antagonist isolated from the Japanese marine sponge, Halichondria cf. moorei [59]. Although halistanol disulfate B inhibited ECE at low concentrations (IC50 2.1p.M), the poor membrane solubility and broadspectrum general bioactivity (e.g. anti-microbial and haemolytic [60]) of marine sterol sulfates against a wide range of drug targets (e.g. feline leukaemia and HIV-1 [61]) counted against any further development of this polar compound [58]. The hydrolysis product of 51, diol 53, was inactive in the ECE assay [58].
R 1 O'
51 R 1 =SO 3 H,R 2 = H 52 R1 = SO3H, R2 = OSO3H 53 R ' = R 2 =
78
Sodwanones A productive ongoing collaboration between marine biologist Michael Schleyer at the Oceanographic Research Institute in Durban, South Africa and Yoel Kashman and co-workers at Tel Aviv University has thus far led to the discovery of twenty-one bioactive metabolites from five species of marine sponges collected from the Sodwana Bay region along the warm tropical northern Kwazulu-Natal coast of South Africa. The first sponge metabolites to emerge from this collaboration were the appropriately named, rearranged triterpenes, sodwanones A-C (54-56), from the IndoPacific fan sponge Axinella weltneri (Family Axinellidae) [62]. The structure of 54 was acquired by X-ray analysis and provided the key to the structures of other members in this series, which later also included sodwanones D-F (57-59) [63] and sodwanones G-I (61-62) from a later recollection of A. welterneri [64].
o
54 R=OH 55 R=H o
o
56 o
58
79
The left hand hemispheres of the sodwanone structures are dominated by a fr-aws-perhydrobenzoxepine ring, a structural moiety common to other rearranged marine triterpenes isolated from the Mediterranean Axinellid sponge Raspaconia aculeata [65] and the unrelated Red Sea sponge Siphonochalina siphonella [66]. Direct comparison of the NMR data of the different sodwanones, supported by standard 2D NMR experiments, facilitated the structural elucidation of these compounds. Careful analysis of ' H - ' H coupling constants and nOe data was used to establish the relative stereochemistry of 55-57, 61 and 62, with X-ray diffraction analysis conclusively providing the structure and relative stereochemistry of 58-60 [64].
o
60 OH
61
62
Although no bioactivity data was originally reported for sodwanones A-F, sodwanones G-I were found to be cytotoxic to four cancer cell lines, P-388 (murine leukaemia), A-549 (human lung carcinoma), HT-29 (human colon carcinoma) and MEL-28 (human melanoma) (Table 3) [64]. The apparent selective toxicity of 57 and 58 in the A-549 cancer assay is of interest. The anti-tumor activity of sodwanones A, G, and H has been patented [67].
80 Table 3.
Cytotoxicity of Sodwanones G-I Against a Panel of Four Cancer Cell Lines |64|. Mean IC50 (nM) P-388
A-549
HT-29
MEL-28
Sodwanone G
2.0
0.2
2.0
2.0
Sodwanone H
10.5
0.02
10.5
10.5
Sodwanone 1
20
20
20
20
Cell line
Phorbazoles The second group of bioactive metabolites to emerge from Sodwana Bay sponges were the novel chlorinated phenylpyrroloxazoles, phorbazoles A-D (63-66) from the Indo-Pacific sponge, Phorbas aff. clathrata (Family Anchinoidae) [68]. X-ray diffraction analysis of the dimethyl ether derivative (67) of phorbazole A indirectly provided the structure of 63 and hence a model for the structure elucidation of the other compounds in this series, which differ only in the chlorine substitution pattern around the pyrrole and oxazole rings. No further details have been published describing the immunomodulatory activity of the phorbazoles originally alluded to by Kashman et al. [68]. Radspieler and Liebscher have provided the first total synthesis of phorbazole C and thus a potential solution to the supply problem possibly hampering investigations of the bioactivity of this group of compounds [69].
63 R1 = R2 = R6 = H, R3 - R4 = R5 - Cl 64 65 66 67
R ' = R 5 = R6 = H, R2 = R3 = R4 = C1 R*=R 2 R6 = R5 = H, R3 = R4 = Cl R1 = R2 = R3 = R5 = R6 = H, R4 = Cl R1 = R6 = Me, R2 = H, R3 = R4 = R5 = Cl
81
Cyclic Peroxides The Sodwana Bay sponge Plakortis aff. simplex (Family Plakinidae) yielded four unsaturated peroxy-carboxylic acid and ester metabolites (68-71) [70]. Cyclic-peroxides containing a characteristic tri-substituted six-membered 1,2-dioxene ring are common constituents of Plakortis sponges [71]. Carboxylic acids 68 and 70 proved to be very unstable and their structures were determined via methylation to give the more stable esters 69 and 71. The structures of 69 and 71 followed from analysis of their mass and NMR data, while the relative stereochemistry at C-3 and C-7 was unequivocally established from nOe data. Overlapping resonances in the lH NMR spectrum of 71 prevented the assignment of the relative stereochemistry of the second 1,2-dioxene ring in this compound. The possibility that 69 and 71 may be artefacts, arising from the use of methanol during the chromatography of the sponge extract, cannot be ruled out. Both 69 and 71 were reported to be moderately cytotoxic to P-388 murine leukaemia cells (IC50 <0.3(iM) [70].
68 R = H 69 R = Me o-o
70 R = H 71 R
Halitulin and Halichlorensin Kashman and co-workers' investigation of Sodwana Bay sponge chemistry continued with a bioassay guided fractionation of an extract of Haliclona tulearensis (Family Chalinidae) [72, 73]. The sponge genus Haliclona is a well-known source of bioactive nitrogen-containing heterocycles e.g. haliclonacyclamines [74], papuamine [75] and
82
manzamines [76]. The P-388 cytotoxicity observed for the initial sponge extract was attributed to a novel bisquinoline pyrrole, halitulin (72) [72]. Definitive mass spectral fragments, supported by NMR spectroscopic data, confirmed the incorporation of the azamacrocycle halichlorensin (73), isolated earlier from extracts of//, tulearensis [73], into the structure of 72. Further mass and NMR spectroscopic data, obtained from 72 and its unstable tetra-acetate (74), were used to delineate the structure of these two compounds. Halitulin was found to be cytotoxic to four cancer cell lines (Table 3) [72]. Rp
OR
RO
OR
72 R = H 74 R = Ac Table 3.
Cytotoxicity of Halitulin Against a Panel of Four Cancer Cell Lines [12]. Mean IC50 (\xM)
Cell line
P-388
A-S49
HT-29
MEL-28
Halitulin
0.04
0.02
0.02
0.04
Jaspamide, Hemiasterlin and Geodiamolide TA The Sodwana Bay sponge Hemiastrella minor (Family Hemiasterellidae) yielded three cytotoxic depsipeptides, the known ichthyotoxic, antihelminthic and antifungal jaspamide (75) [77] and the related hemiasterlin (76) and geodiamolide TA (77) [78]. Although the structures of 75 - 77 were elucidated from NMR and mass data, a paucity of material prevented degradation studies to establish the absolute
83
stereochemistry of 76 and 77. All three compounds exhibited toxicity against P-388 cancer cells at relatively low concentrations (ca. 15fiM). Kashman and co-workers proposed a symbiont origin for all three peptides from firstly, the distinct taxonomic differences between H. minor and other sponges that produce 75 and geodiamolide D (a close analogue of 77) and secondly, the occurrence of the unusual amino acid t-leucine (a metabolite previously reported from actinomycetes) in hemiasterlin [78].
77 Swinholide A and Theopalauamide The SIO-RU-SKB collaboration also involved the collection, in 1994 and 1995, of over 100 marine invertebrates from the Ponto do Ouro area of southern Mozambique, about 80kms north of Sodwana Bay Fig. (1). Two cyclic peptides, the known swinholide A (78) and the new bicyclic theopalauamide (79), were isolated from Mozambique specimens of the Indo-Pacific lithistid sponge Theonella swinhoei (Family Theonellidae) [79]. Interestingly, a concurrent investigation of T. swinhoei from Palau,
84
Micronesia revealed that the source of 78 and 79 was not the sponge but rather the symbiont unicellular bacteria and filamentous eubacteria resident in the interior of T. swinhoei [80]. Swinholide A was reported to be toxic to L1210 (IC50 = 21 nM) and KB (IC50 = 28 nM) cell lines [81]. Theopalauamide inhibited the growth of Candida albicans in the standard paper disk assay at a concentration of 10 p,g/disk.
OMe
85
OH
H2NOC
)
O
H
HO'
79
BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE ASCIDIANS (PHYLUM ASCIDIACEAE) The ascidian fauna of southern Africa are poorly known and the 145 species presently described are thought to represent only a small fraction of the overall ascidian biodiversity [3]. Not unexpectedly, common Pacific and Atlantic fouling species e.g. Cystodytes delechiajei, Clavellina lepadiformis, Didemnum listerianum and Ciona intestinalis frequent harbours in the southern African region [3]. Unsaturated Amino Alcohols and Amines There have been no reports of bioactive metabolites from the ascidians of the cool temperate west coast of southern Africa. Bioactivity guided fractionation of an ethyl acetate extract of the marine ascidian, Pseudodistoma sp., collected in the Tsitsikamma Marine Reserve on the warm temperate south-east coast of southern Africa, revealed that the anti-microbial activity in this extract resided in a group of detergent-like acyclic amino alcohols isolated as their peracetylated derivatives 80 - 83
86
[82]. The relative stereochemistry of the amino alcohol functionality in 80 and 81 was established from nOe studies performed on the oxazolidinone derivatives of these two compounds [82].
NHAc
OAc
80 A5, A 13 81 A5
AcHN' OAc
82 A4, A 12 83 A4
86 A recent investigation of a second Pseudodistoma species from Algoa Bay nearly 200 kms to the east of the Tsitsikamma Marine Reserve, collected as part of a large collaborative marine invertebrate and algal survey of the warm temperate coast of southern Africa by Rhodes University, the Coral Reef Research Foundation and the NCI, also yielded bioactive acyclic amines, pseudodistamine (84), two unsaturated amines (85 and 86) and the quaternary N-methylated P-carbolinium alkaloid (87) [83]. Standard spectroscopic data were used to elaborate the structures of these compounds with Mosher's method providing the absolute stereochemistry of the chiral alcohol functionality in 84. Oxidative
87
ozonolysis of 85 and 86 gave £>-alanine and thus established the (R)stereochemistry of the chiral amine in these two compounds. Of compounds 84-87, only 85 showed moderate activity (IC50 values of approximately 28 (J.M) against LOX (melanoma), A549 (non-small cell lung), SNB-19 (CNS) and OVCAR-3 (ovarian) human tumor cell lines. Lissoclin disulfoxide One of the invertebrates collected during the 1994 SIO-RU-SKB collaborative marine invertebrate collection from the Aliwal Shoal off southern Kwazulu-Natal, South Africa was the ascidian Lissoclinum sp.. Bioassay guided fractionation of a methanol extract of this ascidian yielded a novel interleukin-8 inhibitor, lissoclin disulfoxide (88) [84]. Interleukin-8 (IL-8), a promoter of neutrophil aggregation and activation, is implicated in a wide range of inflammatory disorders e.g. psoriasis and rheumatoid arthritis [85]. In addition to its potent inhibition of both IL-8 Ra and IL-8 R(3 (IC50 = 0.6 and 0.82 |jM respectively) 88 also exhibited activity against the protein kinase C enzyme (IC50 = 1-54 fiM) [84]. OMe MeO
OMe
MeS
SMe
88 Polycitone and Polycitrins As part of their ongoing investigation of bioactive natural products from tropical marine invertebrates collected in Sodwana Bay (situated in northern Kwazulu-Natal, South Africa), Kashman and co-workers isolated three novel alkaloids, polycitone A (89) and polycitrins A and B (90 and 91) from an ascidian Polycitor sp. [86]. The structure of 89 was established from X-ray data while the structures of 90 and its mono-0methyl ether analogue, 91, followed from analysis of the NMR data of these two compounds. Two possible biosynthetic precursors of 89-91,
88
prepolycitrin A and polycitone B (polycitone B is the de-phydoxyphenylethyl analogue of 89) were later isolated from the ascidian Polycitor africanus collected off Madagascar [87]. The ability of polycitone A to inhibit retroviral reverse transcriptase enzymes {i.e. of HIV-1, murine leukaemia virus and mouse mammary tumor) via interference with reverse transcriptase catalysed DNA primer extension and the formation of the reverse transcriptase-DNA complex [88], has prompted a total synthesis of this compound [89]. The penta-O-methyl derivative (92) of polycitone A was also found to inhibit the growth of SV40 transformed fibroblast cells at a concentration of 7.6 |uM [86].
Br
89 R = H 92 R = Me
90 R = H 91 R = Me
OR
BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE SOFT CORALS (PHYLUM COELENTERATA) Stoloniferous octocorals and soft corals form a major component of the invertebrate fauna that inhabit benthic ecosystems off the southern African coast and of the approximately 200 species known from this region as many as 60-70% are thought to be endemic with the remaining 30-40% made up of either cosmopolitan Indo-Pacific or Atlantic species [4]. Eleven families of stoloniferous octocorals and soft corals are recognized globally and of these, seven are represented off the southern African coast [4]. In contrast to most tropical Indo-Pacific coral reef systems, e.g. the Great Barrier Reef, the coral reefs of northern Kwazulu-
89
Natal and southern Mozambique (in the warm tropical bio-geographic zone of the southern African coast) are not dominated by stoloniferous corals but instead have up to 70% of their biomass composed of soft corals [90]. The benthic environment of the coast of southern Africa is very varied and soft corals are not confined to the coral reefs of the warm tropical regions. The adaptability of southern African soft corals to different environments is reflected by their occurrence in diverse habitats off all regions of the southern African coast ranging from the littoral zone to the edge of the African continental shelf at a depth of 468m [4]. Tsitsixenicins There have been no natural products investigations of soft corals from the cool temperate west coast of southern Africa. Soft corals are common along the south-east coast of South Africa and as part of our ongoing search for bioactive metabolites from the marine invertebrate fauna of the Tsitsikamma Marine Reserve we isolated four bioactive xenicane diterpenes, the tsitsixenicins A - D (93 - 96) from the endemic soft coral Capnella thyrsoidea (Family Nephtheidae) [91]. The structures of 93 96 were delineated from standard analysis of their NMR data and comparison of these data with those published for related compounds e.g. 9-deacetoxy-14,15-deepoxyxeniculin (diastereomic with 93) isolated from the Red Sea soft coral Xenia macrospiculata [92].
93
94
90
OAc
95
96
The production of reactive oxygen species e.g. superoxides is implicated in the biosynthesis of prostaglandins from arachidonic acid during tissue inflammation. Oxygen can be reduced to superoxide by plasma membrane bound NADPH-oxidase in rabbit and human cell neutrophils. The tsitsixenicins inhibited (> 80% at a concentration of approximately 30 fJVI) the production of superoxide in isolated rabbit neutrophils with only 94 retaining this level of activity on ten-fold dilution. The results observed with rabbit neutrophils cannot usually be directly extrapolated to human neutrophils and only 93 and 94 exhibited moderate activity when the assay was repeated using the latter neutrophils [91]. Rietones The endemic soft coral Alcyonium fauri (Family Alcyoniidae) is one of the most common and conspicuous shallow water octocorals occurring along the south-east coast of southern Africa [4] and specimens of this species yielded three new sesquiterpene hydroquinones, rietone (97), 8'acetoxyrietone (98) and 8'-desoxyrietone (99) [93]. The stereochemistry at C-8' was established by application of Mosher's method to the quinone (100) obtained from mild Ag2O oxidation of rietone. Rietone exhibited moderate activity (IC50 = 9.3 f^M) in the NCI's CEM-SS cell line screen; a general screen designed to identify metabolites acting at any stage in the HI virus reproductive cycle [93].
91
COOMe
97 98 99
R = OH R = OAc R =H
COOMe
OH
100 Valdivones The potential anti-inflammatory properties of southern African soft coral metabolites were further exemplified by the isolation of five diterpene esters; valdivones A (101), B (102), their corresponding methoxy ketals (103, 104) and dihydrovaldivone A (105) from Alcyonium valdivae (Family Alcyoniidae) collected off Coffee Bay on the warm temperate east coast of southern Africa (Figure 1) [94]. The structures of these compounds were determined from analysis of their 2D-NMR data with coupling constant analysis and nOe data providing the relative stereochemistry of 101 and 102. Valdivones A and B showed fairly strong inhibition of chemically-induced inflammation in the mouse ear assay (93% and 72% inhibition at 50 |ag/ear respectively) but did not 1 significantly inhibit bee venom phospholipase A2 (ca. 43% at 16 [94].
92
OR'
101 102 103 104 105
R ! =H,R 2 = a R'=H,R 2 = b R'=Me,R 2 = R ! =Me,R 2 = R ! = H , R2 = c
o
Zahavins and Eleuthosides An unusual intraspecific variation in the structures of the diterpene metabolites produced by the soft coral Eleutherobia aurea (formerly Alcyonium aurea) has been observed from several collections of this species made from the Aliwal Shoal and Sodwana Bay regions [95]. The Sodwana Bay specimens yielded: antheliatin (106), zahavins A and B (107,108) [96], sarcodictyin A (109) and eleuthosides A and B (110, 111) [95]. In addition to 107, other xenicane diterpenes including 9deacetoxy-14,15-deepoxyxeniculin (112), 7,8-epoxyzahavin A (113) and xeniolide C (114) were isolated from the Aliwal Shoal specimens of E. aurea [97]. Sarcodictyon A and 9-deacetoxy-14,15-deepoxyxeniculin were isolated previously from the stoloniferous octocoral Sarcodictyon roseum [98] and the soft coral Xenia obscuronata [99]. Standard analysis of spectroscopic data provided the structures of the xenicane diterpenes 107, 108 and 112 - 114 while the structure and stereochemistry of 106 were established via X-ray crystallography. The diacetoxypentose moiety in 110 and 111 was assigned as diacetoxyarabinose from 13C chemical shift data and coupling constant analysis. The total synthesis of 109 - 111 by Nicolaou et al. established that the ring junction stereochemistry for all three compounds was identical with that of eleutherobin (115) [100]. The structural similarity of these four compounds to the valdivones (101 - 105) is clearly evident. The total synthesis of 110 and 111 also confirmed the expected D-
93
configuration for the arabinose moiety. Eleutherobin was originally isolated from a western Australian soft coral (possibly Eleutherobia albiflora) by Fenical and co-workers [101].
-OAc OAc
OAc
106
107 108
R =H R = OH
OH COOMe
109
110 R ! =R 3 = H,R2 = Ac 111 R ! = R2 = H,R 3 = Ac 115 R ! = M e , R 2 = R 3 = H
While compounds 106-108 exhibited cytotoxicity (IC50 = 1.7-2.1 \xM) to P-388 mouse leukaemia, A-549 human lung carcinoma, MEL-28 human melanoma and HT-29 human colon carcinoma cell lines, only 106 showed enhanced cytotoxicity against the latter cell line (IC50 = 0.2
94
[96]. Eleutherobin exhibits potent cancer cell cytotoxicity (IC50 = 10-15 nM) against a diverse range of cancer cell lines with a hundred fold increased potency towards selected breast, renal, ovarian and lung cancer cell lines [101]. Although the mechanism of action of 109 and 115 mimics the microtubule stabilizing functions of paclitaxel (Taxol), these two marine metabolites appear to be slightly less potent [102]. Eleutherobin's expression of cross-resistance in cells that are P-gppositive has dampened enthusiasm for the development of this compound and its analogues into potential anti-cancer drugs [103]. Initial studies of the bioactivity of 107 and 108 have been hampered by the paucity of material isolated from E. aurea (ca. 2 mg of each compound) [95]. Access to larger amounts of synthetic material [100] should ease the supply problem if either of these compounds is considered as suitable candidates for drug development. Our interest in the anti-inflammatory properties of soft coral metabolites provided a vehicle to screen the inhibition of superoxide production in rabbit neutrophils by compounds 107, 112 and 113 [97]. The results obtained (> 90% inhibition at a concentration of 50 JJM) were comparable to those observed, vide supra, for the structurally related tsitsixenicins.
AcO
114 Tetraprenyltoluquinols Of the soft corals present on the coral reefs of Kwazulu-Natal and southern Mozambique, three genera, Sinularia, Sarcophyton and Lobophytum predominate [90]. Two soft corals, Sinularia dura (Family Alcyoniidae) and a Nephthea sp. (Family Nephtheidae) collected in Sodwana Bay yielded four tetraprenyl-toluquinols (116 - 119) [104]. The
95
structures of these compounds were established from spectroscopic data with nOe correlations providing the relative stereochemistry of the dioxabicyclooctane moiety in the side chain of sindurol (116). Acid hydrolysis of nephthoside (118) gave the aglycone, 117, and D-arabinose. The stereochemistry of the pentose was established from its optical rotation. Sindurol was twice as toxic to P-388 mouse leukaemia cells (IC50 - 2.1p,M) when compared with compounds 117-119 (IC50 = 5 |oM) [104]. AcO
OH
118 R = H 119 R = Ac
Sarcoglane and Flaccidoxides Two common in-house bioassays to screen for metabolites exhibiting general cytotoxicity, the fertilized sea urchin egg cytotoxicity assay and the brine shrimp lethality assay, were used to identify bioactive diterpene metabolites in two soft coral species collected from Sodwana Bay and Ponto do Ouro respectively. Sodwana Bay specimens of Sarcophyton
96
glaucum (Family Alcyoniidae) yielded the tricyclo-[7,5,010'14]-tetradecane diterpene sarcoglane, (120) [105] while Ponto do Ouro specimens of Cladiella kashmani (Family Alcyoniidae) gave the known flaccidoxide (121) [106], and the related cembrane diterpene (122) [107], in addition to flaccidoxide-13-acetate (123) [108]. The structures of all these diterpenes were solved by analysis of spectroscopic data. The relative stereochemistry of 120 followed from coupling constant analysis and nOe data while a combination of Mosher's method and molecular modelling studies established the absolute stereochemistry of 121.
OH
120
121
R1 = OH, R2 = OAc
122 R1 = H, R2 - OH 123 R ! =R 2 BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE MOLLUSCS (PHYLUM MOLLUSCA) In southern Africa, nearly three-quarters of the 381 known species of marine opisthobranch molluscs belong to the order Nudibranchia [5]. In contrast to shelled marine molluscs (prosobranchs), nudibranchs reflect an evolutionary trend towards the complete loss of the shell [109]. Forgoing the obvious protection against predation provided by a shell, nudibranchs are generally brightly coloured as a warning to potential predators that they are non-palatable. The non-palatability of nudibranchs is attributed to bioactive natural products, which they selectively sequester from their diet and store in glands lining their mantle tissue [109]. Accordingly, nudibranchs prey on a variety of organisms rarely fed upon by other animals (e.g. sponges and octocorals) because of the toxic metabolites produced by these organisms. Therefore natural products derived from nudibranchs generally have an implied bioactivity and the structures of 27
97
metabolites isolated from three nudibranch species collected off the coast of southern Africa are presented here. Variabilins and Nakafurans There are no reports of natural product investigations of nudibranchs collected along the west coast of southern Africa. The endemic nudibranch Hypselodoris capensis is a colourful member of the family Chromodorididae and the linear p-substituted sesterterpenes (18R)variabilin (124) [110], 22-deoxyvariabilin (125) [111] and 22-deoxy-23hydroxymethylvariabilin (126) in addition to the sesquiterpenes nakafurans 8 and 9 (127, 128) [112], were isolated from specimens of H. capensis collected in the Tsitsikamma Marine Reserve [113]. Compounds 124 -126 were also isolated from one of//, capensis' dietary sponges (Fasciospongia sp.) . A further investigation of the sponges preyed upon by H. capensis established a Dysidea sponge as the source of the nakafurans sequestered by the nudibranch [114]. Standard spectroscopic techniques established the structures of the compounds isolated from H. capensis. Variabilin is a common metabolite of Dictyoceratid sponges and possesses anti-microbial [115], anti-tumor [116], anti-inflammatory [117] and icthyotoxic [118] properties. Nakafurans 8 and 9 have also been shown to be icthyotoxic [112].
o
124
R1 = OH, R2 = Me, R3 = a-Me
125
R 1 = H , R 2 = R3 = Me
126
R1 = H, R2 = CH2OH, R3 = Me
127
128
98
Millecrones and Millecrols The endemic nudibranch Leminda millecra is the single representative of the family Lemindidae occurring off the southern African coast. There have been two investigations of the natural products sequestered by this species. The first investigation, by Pika and Faulkner, of L. millecra specimens collected from Coffee Bay yielded the sesquiterpenes millecrone A (129), millecrone B (130), millecrol A (131) and millecrol B (132) [119]. The structures of compounds 129 - 132 were delineated from NMR and mass data while their relative stereochemistry was delineated from nOe data. Of the four metabolites only millecrone A inhibited the growth of Candida albicans (50|ag/disk). Millecrone B was active against Staphylococcus aureus and Bacillus subtilus (50|j.g/disk) and millecrol B was only active against the latter bacterium at similar concentrations. A diversity of soft coral spicules (from Alcyonium foliatum, A. valdivae and Capnella ihyrsoidea) found in the digestive glands of the Coffee Bay specimens of L. millecra suggested a possible soft coral source for the sequestered metabolites 129-132 [119].
130
131
132
Sesquiterpenes, Triprenylquinones and Triprenylhydroquinones L. Melinda is particularly abundant in Algoa Bay and specimens collected from this region of the southern African coast yielded a plethora of diverse metabolites including 129, 130, isofuranodione (133)[120], (+)-8hydroxycalamenene (134)[121], algoafuran (135), cubebenone (136) and seven closely related triprenylquinones and hydroquinones (137 - 143) [122]. The structural elucidation of these compounds was accomplished from spectroscopic data. Gas chromatographic analysis of several octocorals preyed upon by L. millecra in Algoa Bay confirmed that the
99
sea fan Leptogorgia palma was the source of 130 and 136 and an unidentified Alcyonium soft coral was the origin of 129 [122]. OH
OAc
o 133
135
134
o
136
OH
138 139 R = OH
o 140
141
100
Hamiltonins, Lati unculins and Spongiane Diterpenes Four unusual chlorinated homo diterpenes, hamiltonins A-D (143 - 146), the sesterterpene hamiltonin E (147) and the toxins latrunculin A and B (148, 149) [123], were isolated from the brightly coloured dorid nudibranch Chromodoris hamiltoni (Family Chromodorididae) collected from the Aliwal shoal [124]. Since latrunculin A and B were initially shown to cause concentration dependent changes in cell shape and actin organization [125], they have now become the most widely accepted tools for exploring the inhibition of actin polymerization in molecular biology [126].
o o
147
101
149
148
Latrunculin B was also present in extracts of C. hamiltoni collected from the reefs off southern Mozambique. The latter specimens of C. hamiltoni also yielded two spongiane diterpene lactones (150, 151) [127]. No evidence of the hamiltonins was found in extracts of the Mozambique specimens of C. hamiltoni and possibly reflects geographical variation in the organisms that make up C. hamiltonVs diet in this region of the southern African coast. The structures of both spongiane diterpenes and the hamiltonins A -E were determined from spectroscopic data while the absolute stereochemistry at C-16 in 147 was established by comparison of its CD data with those of related compounds [127].
AcO,
12 H
OAc
OAc OAc
150 151 A 12
152 R = H 153 R = OAc
102
Diterpenes Finally, only one member of the marine pulmonate gastropod family Timusculidae, Trimusculus costatus, occurs along the intertidal zone in the warm temperate region of southern Africa. Two bioactive diterpene acetates 152 and 153 were isolated from this species and their structures and relative stereochemistry established from recourse to nOe and other spectroscopic data [128]. Both compounds were found to deter feeding of Pomadasys commersonni, a common, omnivorous intertidal and subtidal southern African fish at natural concentrations (ca. 2.5 mg/pellet) [128]. ABBREVIATIONS CD ECE HMBC NCI nOe SKB SIO RAID RU
= = = = = = = = =
circular dichroism endothelin converting enzyme heteronuclear multibond coherence National Cancer Institute nuclear Overhauser effect SmithKline Beecham Scripps Institution of Oceanography Rapid Access to Intervention Development Rhodes University
ACKNOWLEDGEMENTS I thank Dr Gordon Cragg of the Natural Products Branch at the NCI for helpful discussions and Professor Douglas Rivett and Dr Rob Keyzers for helpful editorial comments. Financial support from the South African National Research Foundation and the Department of Environmental Affairs and Tourism is gratefully acknowledged. REFERENCES [1] [2] [3] [4]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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BIOACTIVE MARINE SESTERTERPENOIDS SALVATORE DE ROSA1 AND MAYA MITOVA2 Istituto di Chimica Biomolecolare del C.N.R. Via Campi Flegrei, 34; 80078 Pozzuoli (NA), Italy. Institute of Organic Chemistry with Centre of Phytochemistry, B.A.N., 1113 Sofia, Bulgaria. ABSTRACT: Terpenoids are b y far t he 1 argest c lass of natural products. Within this class of compounds, the sesterterpenes form a rare group of isoprenoids, which occur in widely differing source. Marine organisms have provided a large number of sesterterpenoids, possessing novel carbon skeleton and a wide variety of biological activities. The more significant sesterterpenoids from marine organisms, which show biological activities, are reported and they are grouped in a biogenetic sequence. Natural products that do not contain 25 carbon atoms but are obviously sesterterpene derivatives are also included. The anti-inflammatory activity is the most relevant among the biological activities observed for marine sesterterpenoids. The high potential for some of these products suggested that they could be developed as drugs for the treatment of inflammation. The different directions that can be taken to obtain quantities of secondary metabolites are reported.
INTRODUCTION Natural products play a dominant role in the discovery of leads for the development of drugs for the treatment of human diseases. It should be realised that the bioactive compounds, which are synthesised in nature to protect a given organism, had been selected from a wide variety of possibilities and were under the pressure of evolution for several hundreds of million years to reach an optimal activity. The terpenoids (isoprenoids) are a class of secondary metabolites that may be formally considered to be constructed from the five-carbon isoprene unit [1]. The terpenes have been classified primarily on the basis of their number of isoprene units (monoterpenes Cio, sesquiterpenes C15, diterpenes C2o, sesterterpenes C25, triterpenes C30 and carotenoids C40) and then on their carbon skeleton. The monoterpenes, sesquiterpenes, diterpenes and sesterterpenes contain the isoprene units linked head to tail, while the triterpenes and carotenoids contain two C15 and C20 units, respectively 1 inked in the middle tail to tail. Several thousand terpenes
110 have been isolated and they are by far the largest class of natural products. Within this class of compounds, the sesterterpenes form a rare group of isoprenoids, which occur in widely differing source and have been isolated from terrestrial fungi [2], plants [3] and insects [4] as well as from marine organisms [5,6], mainly from sponges and nudibranches. Marine organisms have provided a large number of sesterterpenoids, possessing novel carbon skeletons different from those present in terrestrial species. Several sesterterpenoids isolated from marine organisms have shown a wide variety of biological activities. The aim of this contribution is to review the more significant sesterterpenoids from marine organisms, which show biological activities, emphasising those compounds with a potential industrial application. In this review the structures will be covered in a biogenetic sequence and also include natural products that do not contain 25 carbon atoms but are obviously sesterterpene derivatives, such as degraded sesterterpenes with 21-24 carbon atoms, and alkylated sesterterpenes with 26-27 carbon atoms. In addition, some of our own results on anti-inflammatory activity of marine metabolites have been reported. Likewise, some data on the different directions that can be taken to obtain secondary metabolites have been included in the final section to suggest alternative production of marine metabolites and to highlight the possible future importance of marine biotechnology in the production of large quantities of marine secondary metabolites. Previous specific reviews on sesterterpenoids have beenpublished [7-10]. Furthermore, several reviews on terpenoids [ 1113] and on marine organisms [5-6,14-16] all contain material on sesterterpenoids. LINEAR SESTERTERPENOIDS After the isolation of all-^ra«5-geranylnerolidol (1) and geranylfarnesol (2) from the phytopathogenic fungus Cochliobolus [17] and from the wax of the insect Ceroplastse albolineatus [18], a large number of acyclic (absence of formation of any additional carbon-carbon bonds compared with a linear combination of isoprene units) sesterterpenoids were isolated from marine organisms. Furanosesterterpenes are a prominent class of metabolites mainly isolated from marine sponges of the family Thorectidae. The less elaborate component of this interesting group is furospinosulin-1 (3), first isolated from Ircinia spinosula [ 19] and later
Ill
from several Dictyoceratida species, including the South African Fasciospongia sp. [20], the Australian Thorecta sp. [21] and Californian Spongia idia [22] that contains also the oxidized derivative, idiadione (4). OH
,CH 2 OH
O
5a A 12 ' 13 R = 5b A 12 ' 13 R = SO3H; 6b A 1 3 1 5 R = SO3H Both compounds 3 and 4 showed activity atlO |a.g/ml [22] in the Anemia salina bioassay, which gives results that correlate well with cytotoxicity in cancer cell lines such as human epidermoid carcinoma KB, murine lymphoma P388 [23], mouse lymphoma L5178y and murine lymphoma L1210 [24]. Minale and co-workers [25] reported in 1972 the isolation from the sponge /. oros of ircinin-1 (5a) and ircinin-2 (6a), both containing two furan rings and a conjugated tetronic acid moiety. Before the isolation of ircinins, only four others sesterterpenoids were known. Subsequently, it was reported that the mixture of both ircinins inhibited potently mouse ear oedema after topical application, with an ID50 of 51
112 |j.g/ear. Ircinin was found to be a good inhibitor of human recombinant synovial phospholipase A2 (PLA2) (IC50 3.1 \iM) and 5-lipoxygenase (IC50 1.3 \xM), and it was not cytotoxic on human neutrophils at all concentrations tested (0.1-100 (J.M) [26]. These results demonstrate that ircinin is a novel marine inhibitor of PLA2 with a potent topical antiinflammatory profile and they suggest a potential role of ircinin as an inhibitor of inflammatory processes. Recently, the anti-inflammatory activity has been recorded in many sesterterpenes, with a mechanism of action different from those of nonsteroidal, anti-inflammatory drugs (NSAID). The inflammatory response has been shown to be involved in a diverse array of pathological conditions such as arthritis, gout, psoriasis, bee stings and many chemically induced oedemas. The inflammatory response is mediated by the b iosynthesis o f eicosanoids from arachidonic acid (arachidonic a cid cascade), as well as other autacoids released locally in response to an irritant. Arachidonic acid is primarily stored in the sn-2 position of membrane phospholipids. The hydrolysis of the ester at this position is specifically catalysed by PLA2. Lipoxygenase, cycloxygenase and cytochrome P-450 are enzymes from arachidonic acid cascade [27]. The most commonly used non-steroidal, anti-inflammatory agents, indomethacine and the salicylate, inhibit the cycloxygenase pathway but the use of these inhibitors is associated with an increased risk of gastrointestinal bleeding and renal complications. Thus, the inhibition of release of arachidonic acid by PLA2 has become an attractive target for investigation. The development of marine specific inhibitors of PLA2 offers the prospect for a new generation of anti-inflammatory drugs without side effects, derived from non-selective inhibition of constitutive enzymes. From the sponge /. variabilis was isolated variabilin (7a), a furanosesterterpene containing a tetronic acid moiety, which showed antimicrobial activity against Gram-positive bacteria Staphylococcus aureus [28] and Sarcina lutea [29]. After the preliminary observation of Tiberio in 1895 [30] and Fleming in 1929 [31] that a metabolic product of the mould Penicillium notatum inhibited the growth of a staphylococcal culture, and the introduction of penicillin in the treatment of bacterial infections several antimicrobial drugs were produced. The introduction of antimicrobial drugs for the control of infection is the biggest achievement in the history of medicine.
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Unfortunately, many bacteria acquire resistance to one or more of the antibiotics to which they were formerly susceptible. Since most antibacterial agents interact with a specific protein or cellular component, modification of the target is a common means by which resistance can be conferred. Pharmaceutical companies are actually developing new antimicrobial agents against resistant bacteria.
7 a R = OH; 7 b R = OSO3H; 7cR = H; Later was reported that variabilin (7a) is a good inhibitor of human secretory and cytosolic PLA2 with anti-inflammatory activity [32] and shows in vitro antiviral activity against Herpes simplex (HSV) and Polio vaccine (PV1) viruses [33]. The high cytotoxicity against the BSC cell line exhibited by variabilin severely limits its potential usefulness as antiviral agent [33].
Thereafter, several compounds closely related to ircinins and variabilin have been isolated [5,6]. Fusetani and co-workers [34] reported the isolation of two compounds, dehydroderivative of ircinin (8) and an isomer of variabilin (9) from the Japanese sponge Cacospongia scalaris. Both compounds inhibited the cell division of fertilised starfish eggs at a concentration of 1.0 |j.g/ml. This assay is a variation on the test with sea urchin embryos, which can detect DNA and RNA synthesis inhibitors, microtubule assembly and protein synthesis inhibitors, the common leads
114
for the development of anticancer drugs [35]. From the Australian sponge, Thorecta sp. was isolated the rare 22-deoxy-variabilin (7c) that inhibited the growth of S. aureus at 100 |j.g/disk, Bacillus subtilis at 50 u.g/disk and Candida albicans at 100 )ug/disk in a standard agar plate assay [21]. From sponges of the genus Ircinia, collected in the Northern Adriatic Sea, together with the previously described ircinin-1 (5a), ircinin-2 (6a) and variabilin (7a), the corresponding sulphates 5b-7b were isolated. The sulphated derivatives 5b-7b showed greater activity in the A. salina bioassay (LC50: 1.72 and 1.22 |^g/ml, ircinins and variabilin sulphated, respectively), than the corresponding non-sulphated compounds 5a-7a (LC50: 2.38, 2.73 and 2.10 \iglm\), being less toxic in the fish {Gambusia affinis) lethality test (LC50: 5b-6b 5.09, 7b 9.50, 5a 3.35, 6a 3.03 and 7a 3.15 fxg/ml) [36]. Less common are those examples of this class of compound in which the tetronic acid moiety is nonconjugated. Palinurin (10a) was the first example of this class of compound, isolated from /. variabilis [37]. Subsequently, from an Australian Ircinia sp. was isolated a hydro derivative (11) of palinurin, which inhibits the growth of B. subtilis [38]. OR
10a R = H; 10bR=SO 3 H;
O
Additionally, three metabolites of this class of compounds, spongionellin (12), dehydrospongionellin (13) [39] and okinonellin B (14) [40], were isolated from a Japanese sponge of genus Spongionella and were shown to inhibit the cell division of fertilised starfish eggs at 2.0-5.0 u.g/ml. From an Australian Dysidea sp. was isolated isopalinurin (15) that possessed inhibitory properties against the protein phosphatase enzyme in chicken forebrain [41].
115
OH
Palinurin (10b) and fasciculatin (16b) sulphates, together with the previously described palinurin (10a) and fasciculatin (16a), were isolated from the Tyrrhenian sponges /. variabilis and /. fasciculata, respectively. Yet again the sulphated derivatives were more active in A. salina bioassay (LC50: 10b 3.04, 16b 1.44,10a 7.56,16a 2.03 fig/ml) and less toxic in the fish lethality test (LC50: 10b 2.30, 16b 2.20, 10a 1.67, 16a 1.04 ng/ml) [42].
o 16a R = H; 16bR = SO3H Several compounds closely related to palinurin and spongionellin have been isolated [5,6] that showed moderate antimicrobial and cytotoxic activities.
116
Sponges of the genus Sarcotragus are a rich source of sesterterpenes with both conjugated and nonconjugated tetronic acid moieties [43,44]. Sarcotins G (17) and H (18) are closely related to ircinin-1 (5a) and - 2 (6a), except that a furan ring is replaced by a 5-methoxy-2(5H)-furanone moiety. These compounds showed cytotoxic activity with IC50 values 5.010.0 |a.g/ml against five human tumour cell lines (lung carcinoma A549, ovarian carcinoma SK-OV-3, skin carcinoma SK-MEL-2, central nervous system carcinoma XF498 and colon carcinoma HCT15) [44], while sarcotin F (19), an oxidised derivative of palinurin (10a) showed less cytotoxic activity (IC50 7.6-24.1 fig/ml) in the same panel of cell lines [44].
OCH,
12
17 A 1 2 ' 1 3 ; 18 A 1 3 ' 1 5 H3CO
OH
Unusual trifuranosesterterpene acids, hippospongins A-C, were isolated from the Australian Hippospongia sp., which are speculated to be biosynthetically related to the C25 tetronic acids and the C21 furanoterpenes. Only hippospongin A (20) showed a mild antimicrobial activity, inhibiting the growth of S. aureus at a concentration of 200 ug/disk [45]. An a,(3-unsaturated. y-lactone linear sesterterpene (21) was isolated from the Caribbean sponge Thorecta horridus that exhibited inflammatory activity both in vivo inducing paw oedema and in vitro inducing release of histamine [46].
117
From a sample of the sponge Fasciospongia cavernosa, collected in the bay of Naples (Italy), were isolated two linear sesterterpenes, cacospongionolide D (22) with a p-hydroxybutenolide moiety, and luffarin-V (23) with two y-butenolide functionalities in the molecule. Cacospongionolide D showed a potent activity (LC50 0.1 (J.g/ml) [47] in the A. salina bioassay, and a moderate ichthyotoxicity to G. affinis (LC50 2.54 ng/ml) in the fish lethality assay. Luffarin-V was less active (LC50 1.72 |ag/ml) in the A. salina bioassay.
O
24 An unusual sesterterpenoid acid (24) with a tetrahydropyran ring was isolated from the Indonesian sponge Hippospongia sp. that inhibited the human Ras-converting enzyme (hRCE), with an IC50 value of 10 |J.g/ml [48]. The Ras signalling pathway has emerged as an important target for the development of anticancer drugs. Ras is a membrane bound G protein that functions as a molecular switch in a network of signalling pathways, controlling cell differentiation and proliferation. Mutated Ras genes, encoding activated Ras proteins, have been identified in approximately 30% of all human cancers. The approaches to therapeutic intervention in
118
the Ras signalling have focussed on the development of inhibitors that block the lipid modification needed for proper Ras membrane localisation (farnesyl transferase inhibitors) or to finding inhibitors of proteolytic processing of Ras (RCE protease inhibitors) [49]. Linear, closely related difuranoterpenes containing 21 carbon atoms have been found to occur in large amounts in the sponge of the genus Spongia. All of them possess the same carbon skeleton, and oxidation in the central chain accounts for all their differences. The first two C21 compounds, ninetin (25) and dihydroninetin (26) isolated from S. nitens [50] possess a y-lactone ring in the central part of the chain. S. officinalis and Hippospongia communis contain several C21 fiiranoterpenes in large amounts [51,52]. From a specimen of S. officinalis, collected in the Northern Adriatic Sea, together with furospongin-2 (27), previously reported from the same sponge collected in the Tyrrhenian Sea [51], its three isomers (28-30) were isolated. These C21 fiiranoterpenes (27-30) showed high activity (LC50 0.09-1.60 |u.g/ml) in the A. salina bioassay [53].
The most widely distributed component of this group, furospongin-1 (31), which possesses interesting biological activities, was first isolated from the Mediterranean S. officinalis, and the closely related H. communis [51], few years later, was isolated from two Australian sponges, Phyllospongia radiata and P. foliescens [54], Furospongin-1 showed analgesic [55] and antispasmodic activities [56]. Furthermore, furospongin-1 reduced, at yM concentration, the tissue levels of ATP and had no significant effect on ATP breakdown in bovine mitochondria, showing that its antispasmodic action has a mechanism different from that of the mitochondrial ATP synthase inhibitor, oligomycin [56].
119
O
Linear C21 furanoterpenes commonly occur in Spongiidae or Thorectidae sponges with structurally-related sesterterpenic tetronic acids from which they are biosvnthesised by the loss of a four-carbon fragment [14]. This hypothesis has raised considerable interest [15] and has received some experimental support from the degradation of linear conjugated furanosesterterpenic tetronic acids to C21 furanoterpenic carboxylic acids by oxidation in basic aqueous solution [57]; and from the isolation of C21 furanoterpenic carboxylic acids, ircinin-3 (32) and ircinin4 (33), related to ircinin-1 and ircinin-2, from the sponge /. Oros [58]; the acid 34 related to variabilin (7a) isolated from sponge of genus Sarcotragus [59]; and the acid 35 together with the C20 aldehyde 36 both related to fasciculatin (16a) [57]. The C21 furanoterpene acid 34 showed antiviral (HSV and PVl) and cytotoxic activities comparable with that of variabilin [33]. Further evidences of the hypothesis of Minale were obtained by the isolation of three chlorinated C24 norsesterterpenes (3739), closely related to ircinin-1 (5a) and ircinin-2 (6a), from the sponge /. oros, collected in the Northern Adriatic Sea [60]. In fact, we may suppose
120 that 37 and 39 are the first stage of degradation of the tetronic acid, through introduction of a chlorine atom by action of a chloroperoxidase, then hydrolysis of the lactone ring and subsequent decarboxylation to produce keto-chlorohydrins, which can easily be degraded giving the C21 terpenes. The norsesterterpenes, 37-39, showed less cytotoxic activity (LC50 8.2-8.6 |J.g/ml) than ircinin-1 (5a) and ircinin-2 (6a) (LC50 2.4 and 2.7 ng/ml), in the A. salina bioassay [60].
COOH 32 A 12 ' 13 ; 33 A 13 ' 15 COOH
COOH
O -,-
» 10,11 r,
37 A
TT
->o A 10,11
n
Cl
A
R = H;11 12 38 A R = Ac; 39 A ' R = H
Untenospongins A (40) and B (41), isolated from an Okinawan species of Hippospongia exhibited potent coronary vasodilating activity, markedly inhibiting KCl induced contraction of rabbit coronary artery with IC50 values of 10"6 and 2 x 10~6 M, respectively [61]. The norsesterterpenoids, sarcotins N (42), O (43) and e«?-kurospongin (44), isolated from Sarcotragus sp., showed moderate cytotoxicity, with
121 IC50 values 3.0-30.0 |a.g/ml, against a panel of five human tumour cell lines (A549, SK-OV-3, SK-MEL-2, XF498 and HCT15) [62]. OH OH
40
•o
OCH3
OH
O
O
Rhopaloic acids A-C (45-47), three unusual norsesterterpenes isolated from a Japanese species of Rhopaloeides, selectively inhibited the gastrulation of fertilised eggs of the Starfish Asterina pectinifera at jaM level. The minimum inhibitory concentrations of 45-47 were 0.5, 0.4 and 0.2 fjJVI, respectively [63]. Rhopaloic acid A, which was synthesised [64], exhibited also potent cytotoxicity against human myeloid K562 cells (IC50 0.04 fimol/1), human leukaemia MOLT4 cells (IC50 0.05 (imol/1), and L1210 cells (IC50 0.10 jxmol/1) [65]. Furthermore, rhopaloic acids A-C together with rhopaloic acids D-G (48-51) were isolated from an Indonesian Hippospongia sp.. Rhopaloic acids A-E showed a RCE protease inhibitory activity with IC50 values of ~ 10 |J.g/ml [48]. Compounds 45-49 were more active in the cell-based assay against colon tumour L0V0 cells (IC50 ~ 1 Mg/ml) than in the enzyme assays, suggesting
122
that the cytotoxic effect of the compounds might result from hitting more than one molecular target [48].
OH
51
123
Muqubilone (or aikupikoxide A) (52), a norsesterterpene peroxide acid, isolated from the Red Sea sponge Diacarnus erythraeanus showed in vitro antiviral activity against herpes simplex virus type 1 (HSV-1) with IC50 of 30.0 |J.g/ml [66], and cytotoxic activity with an IC50 > 1 Mg/ml, against three type of cancer cells, including P388, A549 and human colon carcinoma HT29 [67].
MONOCARBOCYCLIC SESTERTERPENOIDS Marine sponges of genus Luffariella (Thorectidae; Dictyoceratida) are a rich source of monocarbocyclic sesterterpenoids and most of them possess interesting bioactivities. In 1980 and 1981, Scheuer and coworkers [68,69] reported the isolation of manoalide (53), seco-manoalide (54), (6E)- (55) and (6Z)-neomanoalide (56a) from the Palauan sponge L. variabilis, which showed interesting antimicrobial activity against Gram positive bacteria Streptomyces pyogenes, S. aureus and B. subtilis [68,69]. Later, Kobayashi and co-workers [70] reported, from the Okinawan sponge Luffariella sp., the isolation of manoalide (53), (6E)- (55) and (6Z)-neomanoalide (56a) that showed cytotoxic activity against L1210 cells (IC50 0.032, 9.8 and 5.6 |ig/ml for 53, 55 and 56a, respectively), and only manoalide was active against KB cells with an IC50 value of 0.3 |ag/ml [70,71]. Manoalide is the first compound of this group to be reported, characterised by cyclisation that is reminiscent to those of the carotenoids and one or two potentially reactive rings, yhydroxybutenolide ring and a 8-lactol ring (a-hydroxy-dihydropyran ring) or its derivative. Subsequently, it was found that manoalide showed molluscicidal activity towards Biomphalaria glabrata at 1.5 ppm [72], analgesic activity at 50 mg/kg in the phenylquinone test, and antiinflammatory activity in the induced inflammation of the mouse ear, with a potency greater than that of indomethacine and less than that of hydrocortisone [73]. The most important finding has been that manoalide is the inhibitor of various secreted forms of PLA2 a t n M concentration
124 [74-77]. It was suggested that the binding of manoalide to PLA2 is irreversible and involves initial formation of a Schiff base (imine) between a lysine residue on PLA2 and the aldehyde group of y-hydroxybutenolide, than a second lysine reacts with the aldehyde group of cthydroxy-dihydropyran ring to produce an adduct in which the manoalide is irreversibly bound to PLA2 [75,78]. Over 140 citations concerning manoalide recorded in MEDLINE show the high interest pointed to this compound. Eight total syntheses have been reported [79-86]. Secomanoalide (54), which is the geometrical isomer of manoalide, has similar potency and efficacy in the inhibition of bee venom PLA2, suggesting that the inhibition reaction is not dependent on a rigid geometrical relationship between the aldehyde group and the second lysine residue [75].
56a A
6>7
55 A 6J £, R = OH Z,R = OH;56b A 6 ' 7 Z,R; OAc
From the Western Pacific sponge L. variabilis was isolated dehydromanoalide (57) that showed a marked decrease in inhibition of bee venom PLA2 (IC50 0.28 \xM) [76, 87].
125 In 1992, Konig and c o-workers [72] reported the isolation of Z-2,3 dihydro-neomanoalide (or luffariolide C) [88] (61a), its 24-acetyl derivative (61b), 6Z-24-acetoxy-neomanoalide (56b) and Eneomanoalide-24-al (58), from an Australian sponge of genus Luffariella. All these compounds showed antibacterial activity against Escherichia coli, B. subtilis and Micrococcus luteus, in a TLC bioautographic test [72].
.0
59 HO
61a R = OH; 61b R = OAc Kobayashi and co-workers [70] reported, from the Okinawan sponge Luffariella sp., the isolation of several sesterterpenoids related to manoalide, named luffariolides A-J (59-67). All luffariolides showed cytotoxic activity against L1210 cells (IC50 1.1-4.5 ng/ml) and only luffariolides F (64) and G (65) exhibited weak activity also against KB
126
cells [70,71,88]. Luffariolides H (66) and J (67) showed antimicrobial activity against S. aureus, with minimum inhibitory concentrations (MIC) of 16.7 and 33.3 ng/ml, respectively, B. subtilis (MIC, both 8.4 |ig/ml) andM luteus (MIC, both 8.4 ng/ml) [88].
62 R = H, OH; 63 R = O
HO CHO
67
127
Faulkner and co-workers reported the isolation of luffariellolide (68) from a Palauan sponge Luffariella sp., which was a potent antagonist of topical induced inflammation in the mouse ear, but it was less potent than manoalide (53) inhibitor of bee venom PLA2 with an IC50 value of 1.6 x 10~7 M. Luffariellolide is a partially reversible inhibitor of bee venom PLA2, because it lacks one of the two masked aldehyde groups that appears to be responsible for the irreversible reaction of manoalide with lysine residue of PLA2 [89].
From the Fijian sponge Fascaplysinopis reticulata were isolated two sesterterpenoids related to luffariellolide, zso-dehydro-luffariellolide (69) and dehydro-luffariellolide diacid (70). Zso-dehydro-luffariellolide inhibited at 1 mg/ml 81% of the HIV-1 reverse transcriptase activity [90] and reduced the activity of p56lck tyrosine kinase at 0.5 mM to 45% in ELISA based assays [91]. Hyrtiolide (71) was isolated from the Fijian sponge Hyrtios erecta together with its correlated /so-dehydro-
128
luffariellolide. Hyrtiolide showed weak antifungal activity towards Ustilago violaceae [91]. Muqubilm [92] (or prianicin A) [93] (72), a norsesterterpene peroxide acid, isolated from the Red Sea sponges, Prianos sp. [92-94] and Diacarnus erythraeanus [66] showed antimicrobial activity against Streptococcus beta haemolytic (MIC 2.5 |j.g/ml), S. aureus (MIC 12.0 |u.g/ml) and Corynebacterium diphteriae (MIC 3.0 |ug/ml) [93], and it displayed potent in vitro activity against Toxoplasma gondii at a concentration of 0.1 foM without significant toxicity [66]. Furthermore, muqubilin totally inhibited the cell division of fertilised sea urchin eggs at 16 )J.g/ml [94]. .EnZ-muqubilin (72), 2-e/n-muqubilin (73) and deoxydiacarnoate B (121) (see bicyclic section) were isolated from the New Caledonian sponge Diacarnus levii [95]. The mixture of all three compounds showed cytotoxicity against both chloroquine sensitive and resistant strains of Plasmodium falciparum, the human parasite responsible for the most severe cases of malaria [95].
-O 2 XOOH
72;
73 2-epi
COOH
COOH
COOH
The finding of new antimalarial drugs, particularly those against multiresistant P. falciparum, is extremely important, because in the last
129
years the malaria has regained its status as an extremely important threat to the human health. It is estimated that, in regions where malaria is endemic, each year about 1.5 million of people die from this disease. Tasnemoxides A-C (74-76), closely related to muqubilin, were isolated from the Red Sea D. erythraeanus, and showed moderate cytotoxicity (IC50 > 1 ng/ml) against three cancer cell lines including P388, A549 and HT29 [96]. In order to provide sufficient manoalide for continued pharmacological evaluation, F aulkner and coworkers m ade an e xtensive c ollection of L. variabilis, from different locations in Palau. From a small number of specimens of L. variabilis were isolated two new metabolites, luffariellin A (77) and Luffariella B (78) in place of manoalide and seco-manoalide [97]. Despite the different carbon skeleton, the functional groups in luffariellins A and B are identical with those in manoalide and secomanoalide, respectively, and they showed almost identical antiinflammatory properties. Both luffariellins were potent antagonists of topical induced inflammation in the mouse ear, and inhibitors of bee venom PLA2. with an IC50 value of 5.6 x 10'8 M and 6.2 x 10'8 M, for luffariellins A and B, respectively [97].
HO" CHO
78 Hippospongin (79), isolated from the Okinawan sponge Hippospongia sp., is an unusual sesterterpene containing an isolated cyclohexenofuran ring and a tetronic acid moiety, which showed antispasmodic activity (5 x 10" M), abolishing the contractile responses to carbachol and histamine on the guinea-pig ileum [98]. Further sesterterpenes (80 and 81) and two
130
norsesterterpene (82 and 83), related to hippospongin, were isolated from the Okinawan sponge Ircinia sp.. The norsesterterpenes 82 and 83 were more cytotoxic (IC50 < 1 fig/ml) than the sesterterpenes 80 and 81 (IC50 > 1 fig/ml) against KB cells [99]. An additional norsesterterpene, untenic acid (84) was isolated from an Okinawan sponge Spongia sp., which activates sarcoplasmic reticulum Ca2+-ATPase [100].
HO.
79 HO. 12 -
O"
80 A 1 2 ' 1 3 £; 81 A 12 ' 13 Z O
R
OH
82 R = H; 83 R = C1
O
COOH
84 From the Caribbean sponge Cacospongia linteiformis were isolated cyclolintemone (85) [ 101] and its 3-deoxy derivative (86) [ 102] with a novel rearranged monocarbocyclic skeleton, l-alkyl-l,2,6-trimethyl-2cyclohexene ring system. Both compounds were ichthyotoxic at 10 ppm to G. affinis, and showed antifeedant activity at a concentration of 30 |a.g per cm of food pellets against the fish Carassius aurantus [101,102]. Furthermore, cyclolinteinone showed anti-inflammatory activity, inhibiting the nuclear transcription factor-KB binding activity, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) enzymes,
131
and it was capable of controlling the excessive production of both prostaglandin (PGE2) and nitric oxide (NO) [103].
85 R = O; 86 R = H2
OSO3Na
Halisulphate 2 (87), a sulphated sesterterpene with a monocarbocyclic skeleton related to cyclolinteinone, was isolated from the Californian sponge Halichondria sp. [104]. Halisulphate 2 showed anti-microbial activity against S. aureus, C. albicans and B. subtilis at 20 jag/disk, it inhibited mouse ear oedema after topical application and was an inhibitor of PLA2 [104]. BICARBOCYCLIC SESTERTERPENOIDS Sesterterpenoids with a bicarbocyclic skeleton in many instances show structures reminiscent of the clerodane and labdane diterpenoids. Palauolide (88), isolated from an unidentified Palauan sponge, is structurally a classical example of clerodane type [105]. From the Palauan sponge Fascaplysinopsis sp. was isolated palauolol (89) that maybe a biosynthetic precursor of palauolide [106]. Palauolide (88) and palauolol (89), both containing a functional group y-hydroxybutenolide ring, inactivate bee venom PLA2 with 85% and 82% inhibition for 88 and 89, respectively, at 0.8 |u.g/ml [27,106], and showed anti-microbial activity against B. subtilis and S. aureus at 10 u.g/disc [105,106]. From the Palauan sponge Thorectandra sp. were isolated palauolide (88), palauolol (89) together with their derivatives, named thorectandrols A-E (90-94). Compounds 88-94 were tested for antiproliferative and cytotoxic activities against 12 human tumour cell lines originated from breast, CNS,
132
colon, lung, ovarian and renal carcinoma, leukaemia and melanoma. Palauolol (89) was active in all the cell lines with IC50 in the range 0.5-7.0 (J.g/ml, while palauolide (88) showed a decrease in activity in all the cell lines with IC50 7.7-53 jxg/ml. Thorectandrols A-D were weakly active with IC50 over 30 fj.g/ml, whereas thorectandrol E was not cytotoxic to any of the cell lines at the maximum dose tested [107,108].
O
92 R = OAc; 93 R = H 94 R = OH
90R = H;91 R = OAc AcO
95 Luffalactone (95) from the Pacific Luffariella variabilis is a sesterterpene with a labdane type skeleton, related to manoalide (53). Luffalactone showed 52% inhibition of oedema in the mouse ear assay at 50 |ng/ear [87], In order to find compounds related to cacospongionolide (155) (see tricyclic section) [109], we have investigated other Mediterranean horny sponges belonging to the family Thorectidae. From a specimen of Fasciospongia cavernosa, we isolated, in good yields, an isomer of cacospongionolide, named after for uniformity cacospongionolide B (96)
133
[110]. Structural differences between the two compounds are due to the absence of the cyclopropane ring and the presence of an exomethylene group. There are two varieties of F. cavernosa, one is massive, and the second is encrusted. The massive form is very common along the Adriatic coasts and Aegean Sea, while the encrusted form is distributed in all the Mediterranean Sea. Normally, from specimen of the massive form were isolated only one or two correlated metabolites, while from specimen of the encrusted form were isolated a complex mixture of cacospongionolides: cacospongionolides D (22) [47], E (97) [111] and F (98) [112] that was recently synthesised [113], and related metabolites, such as 25-deoxycacospongionolide B (99) [114] and cavernosolide (151) [115].
The isolation of several related constituents from individual specimens of F. cavernosa confirms the peculiarity of the sponges belonging to the family Thorectidae. In fact, similar variation of related metabolites was observed for the sponges Luffariella variabilis, L. geometric and Thorectandra excavatus [5]. The structures of cacospongionolides are similar to that of manoalide (53) (see monocyclic section). The differences between the two compounds, apart the non-polar region, are due to the lack of the hydroxyl group at C-24 in cacospongionolide. This lack renders the cacospongionolides more stable than manoalide. Despite the absence of the C-24 hemiacetal function, cacospongionolides showed potent inhibitory activity on recombinant human synovial PLA2 similar to that of manoalide, while a lower inhibitory activity was shown on other secretory PLA2S [116].
134
As cacospongionolide (155), cacospongionolide B (96) showed a high cytotoxicity (LC50 0.25 M-g/ml), in the A. salina bioassay. It was moderately ichthyotoxic to G. affinis (LC50 1.05 (J.g/ml) and showed a high antibacterial activity against the Gram-positive bacteria B. subtilis and M. luteus, with an MIC value of 0.78 fig/ml, comparable with that of gentamycin [110]. Further pharmacological screening revealed that cacospongionolide B is a new inhibitor of PLA2; preferentially inhibiting the human synovial PLA2 (IC50 4.3 |aM), and pancreatic PLA2 (IC50 4.0 }iM), and its potency on the human synovial enzyme was comparable to that of the reference inhibitor, manoalide (IC50 3.9 |oM). This activity was confirmed in vivo on a model of chronic inflammation, the established adjuvant-induced arthritis. Cacospongionolide B was less active than indomethacine, an NSAID. Nevertheless, the stomachs of the animals treated with this NSAID showed redness and perforations, while these toxic effects were absent in the rats treated with cacospongionolide B [116]. Furthermore, it has been shown that cacospongionolide B inhibited nuclear factor-^B (NF-A:B)-DNA binding activity and nuclear translocation of this transcription factor. The NF-&B pathway has emerged as an important target for the development of drugs against chronic inflammatory disorders and cancer. Moreover, cacospongionolide B is able to downregulate the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), resulting in decreased production of the two important mediators of inflammation process detected in high levels in rheumatoid synovial tissues, nitric oxide (NO) and prostaglandin E2 (PGE2). Cacospongionolide B also reduced the mRNA expression of the major factor in the development of chronic inflammatory conditions, tumour necrosis factor- a (TNF-a) [117]. The use of cacospongionolide B as inhibitor of the PLA2 is covered by patents [118]. Recently, Snapper and co-workers reported the total synthesis of cacospongionolide B and its enantiomer [119]. The examination of SPLA2 inhibition with synthetic variants of cacospongionolide B revealed that the inhibition is enantioselective, i.e. the natural product is a more potent inhibitor of bee venom SPLA2 (IC50 49 \iM) than the unnatural enantiomer (IC50 106 |i.M). Moreover, the inhibition is notable for synthetic precursor possessing the furan group (IC50 76 |uM) in place of y-hydroxybutenolide moiety. These results suggest that the y-hydroxybutenolide moiety is not the sole structural feature of the natural product involved in SPLA2 inhibition [119].
135
All cacospongionolides isolated showed more or less similar biological activities. In particular, as anti-inflammatory agents, they preferentially inhibited bee venom and human synovial PLA2 in the |uM range (Table 1). Cacospongionolide E (97), however, was the most potent inhibitor towards human synovial PLA2, showing higher potency than the referenced compound monoalide [111]. Our results confirmed the suggestion [76] that the pyranofuranone part interacts with PLA2 enzymes, but that the hydrophobic region of the molecule, which can be partially linear (manoalide) or cyclic (cacospongionolides), may facilitate this interaction. These results demonstrate that cacospongionolides are a novel class of marine metabolite inhibitors of PLA2 with a potent topical anti-inflammatory profile and a high antimicrobial activity and this suggests a potential role of cacospongionolides as drugs. Table 1.
Effect of Different Cacospongionolides on a Panel of Secretory PLA 2 a [lll]
PLA2 Enzymes
Cacospongionolide (155)
N. naja venom %I(10nM)
3.5
Pancreas %I (10 uM) IC50 (uM)
Human synovial %I(10uM) IC50 (uM)
RAP+zymosan %I(10nM) IC50 (uM)
Bee venom %I (10 uM) IC50 (uM)
14.1 N.D.
90.7 3.0
21.8 N.D.
96.3 2.3
Cacospongionoiide B (96)
0.0
64.2 4.0
86.7 4.3
36.9 7.8
35.4 N.D.
Cacospongionolide E (97)
0.0
5.3 N.D.
96.7 1.4
65.1 N.D.
94.8 2.8
Manoalide (53)
17.0
32.3 N.D.
93.2 3.9
38.4 N.D.
62.5 7.5
a IC» values were determined for those compounds that reach 50% inhibition at 10 uM; N.D. = not determined.
A number of carbobicyclic sesterterpenoid sulphates were found, including halisulfate 1 (100), isolated from Halichondria sp. [104]; halisulfates 8-10 (101-103), isolated from the Australian sponge Darwinella australensis [120]; hipposulfates A (104) and B (105), isolated from the Okinawan Hippospongia metachromia [121] and sulfircin (106) that was isolated as its N,N-dimethylguanidinium salt, from a deep-sea member of the genus Ircinia [122]. Halisulfate 1 (100) is an mhibitor of human 12-lipoxygenase (12-HLO) (IC50 1.0 \xM) and 15HLO (IC50 0.9 uM) [123]. 12-HLO is involved in the development of
136
psoriasis and controlling cancer cell proliferation, while 15-HLO in the development of atherosclerosis and tumourigenesis. Halisulfates 9 (102) and 10 (103) inhibited cell division of the fertilised eggs of the sea urchin Strongylocentrotus intermedius (IC50 50 and 35 |J.g/ml for 102 and 103, respectively) [120]. Hipposulfates B (105) showed cytotoxic activity with an IC50 of 2.0 (ig/ml against four human tumour cell lines, A549, P388, melanoma MEL28 and HT29 [121]. ,OSO3Na
OSO 3 Na
106
105 Sulfircin (106) showed activity against the fungal pathogen C. albicans with a MIC of 25 ng/ml [122].
137
A new class of sesterterpenes in which the middle three units of a penta-isoprenoid chain cyclised into a bicyclic system, leaving the first and the last isoprenoids to substitute the decaline moiety, was isolated from sponges of genus Dysidea and Irdnia. From the Palauan Dysidea sp. was isolated dysideapalaunic acid (107) that inhibited the aldose reductase [124]. An inhibitor of aldose reductase is expected to prevent neuropathy or cataract as a complication of diabetes. These diseases are caused by the accumulation of sorbitol in the peripheral nerve or the crystalline lens, as a result of enzymatic reduction of glucose by the aldose reductase in the sorbitol cycle [125,126]. The absolute stereochemistry of dysideapalaunic acid was established by its total synthesis [127,128]. COOH
COOH
OH COOH
Kohamaic acids A (108) and B (109) were isolated from the Okinawan Ircinia sp.. They exhibited cytotoxicity against P388 cells, with IC50 values of >10 (32%) and 2.8 |a.g/ml, respectively. Kohamaic acids are closely related to dysideapalaunic acid (107), but they have different stereochemistry at C-15 [129]. Dysidiolide (110), isolated from the Caribbean sponge D. etheria, is a potent inhibitor of the human cdc25A protein phosphatase (IC50 9.4 \xM), a potential target for anticancer therapy. Moreover, dysidiolide inhibited growth of the A549 (IC50 4.7 \xM) and P388 (IC50 1.5 pM) cells [130]. The interesting biological activities and the rare structural features of dysidiolide prompted several
138
researchers to undertake its total synthesis [131-139]. From D. cinerea were isolated two new inseparable metabolites, bilosespens A (111) and B (112). The mixture of both bilosespens showed cytotoxic activity with an IC50 of 2.5 (J.g/ml against four human tumour cell lines (A549, P388, MEL28andHT29)[140]. Carbobicyclic norsesterterpenoids, containing cyclic peroxides were isolated from four sponge genera, Mycale, Latrunculia, Sigmosceptrella and Diacornis. From a Thai Mycale sp. were isolated two related norsesterterpenoids 1,2-dioxanes, mycaperoxides A (113) and B (114), which showed significant cytotoxicity (IC50 0.5-1.0 |J.g/ml) against the cell lines P388, A549 and HT29 and displayed antiviral activity (IC50 0.25-1.0 |u.g/mi) against vesicular stomatitis virus and herpes simplex virus type-1 [141]. XOOH
COOH
HOOC
117 Trunculins A-E are norsesterterpene peroxides isolated from Latrunculia brevis [142,143]. Only trunculins A (115), B (116) [142] and E (117) [143] inhibited the growth of S. aureus, B. subtilis and C. albicans when tested at 100 mg/disk in the standard disk assay. From Sigmosceptrella laevis were isolated sigmosceptrellins A-C (118-120) that w ere i chthyotoxic (LD 5 ng/ml) a gainst L ebistes reticulatus [144]. Together with eM/-muqubilin (72) and 2-e/?/-muqubilin (73) (see
139 monocyclic section), from the New Caledonian sponge Diacornus levii was isolated the antimalarial agent deoxy-diacarnoate B (121) [95]. From a specimen of/7, cavernosa collected in the Aegean Sea, together with cacospongionolides B (96) and F (98), was isolated a new C21 terpene £-lactone (122), closely related to the cacospongionolide B, by the loss of four C atoms, through an oxidative rupture of the y-hydroxybutenolide ring [145]. XOOH -^\x
w
v^-n^/
—*s^
COOH
11816S,17S 11916R, 17S 120 16S, 17R
/ ''*"
121
N
'
122
This new compound, named cavernolide (122), showed antiinflammatory activity and exhibited specific inhibition of human synovial PLA2 in a concentration-dependent manner with an IC50 value of 8.8 |oM. Cavernolide was less potent in this assay than the referenced inhibitor manoalide (IC50 3.9 |j.M). In addition, this compound reduces TNF-a production, iNOS and COX-2 expressions [146]. TRICARBOCYCLIC SESTERTERPENOIDS Marine sponges are a rich source of tricarbocyclic sesterterpenoids with a cheilanthane skeleton, which seems to be derived from geranylfarnesol by a cyclisation initiated at the isopropylidene group that is typical of triterpenes. Luffolide (123), an anti-inflammatory compound, is a classic example of this class of compounds. The hydrolysis of phosphatidyl choline by bee venom PLA2 is completely inhibited by luffolide at a concentration of 3.5 |iM [147]. Further bioactive metabolites with cheilanthane skeleton were isolated from sponges of genus Spongia, Cacospongia, Petrosaspongia, Fasciospongia, and Ircinia and from the nudibranch Chromodoris. Spongianolides A-F (124-129) possessing a y-hydroxybutenolide moiety, were isolated from a Spongia sp. [148]. The absolute
140 stereochemistry of spongionohde A was established by its total synthesis [149]. CHO OAc
OAc
HO
123 O
HO
OH
126 16R,R = OCOCH3 127 16S,R = OCOCH3 128 16R, R = OCOCH(OH)CH3 129 16S, R = OCOCH(OH)CH3 130 16R, R = H 133 16R,R = OH 134 16S, R = OH
131 16R 132 16S Spongianolides A-E inhibited protein kinase C (PKC) at IC50 20-30 \\M, moreover, compounds 124-127 potently inhibited (IC50 0.5-1.4 JJM) the proliferation of the mammary tumour cell line MCF7 [148]. Simultaneously, from the Caribbean sponge Cacospongia linteiformis were isolated the spongianolides C a n d D (126 and 127) designated as lintenolides A and B, which showed high antifeedant activity against the fish C. aurantus (30 \xg per cm2 of food pellets) and ichthyotoxicity to G. affinis (10 ppm) [150]. Further, lintenolides C-G (130-134) were isolated from the Caribbean sponge Cacospongia cf. linteiformis [ 151,152]. All lintenolides A-G inhibited the growth of murine fibrosarcoma WEHI 164, murine monocyte/macrophage J774, bovine endothelial GM7373 and P388 cell lines (Table 2) [152].
141 Table 2.
Cytotoxicity of Different Lintenolides Against a Panel of Tumour Cells [152)
Mean IC» (ng/ml) Cell line:
WEHI164
J774
P388
GM7373
Lintenolide A (126)
0.92
0.36
0.098
0.085
Lintenolide B (127)
3.1
0.71
0.30
0.34
Lintenolide C (130)
50.0
23.4
2.7
0.22
Lintenolide D (131)
46.5
10.9
19.0
25.0
Lintenolide E (132)
53.3
30.7
125.0
0.021
Lintenolide F (133)
8.8
0.94
0.90
1.6
Lintenolide G (134)
3.2
1.70
0.037
0.30
From the New Caledonian sponge Petrosaspongia nigra were isolated several tricarbocyclic sesterterpenoids petrosaspongiolides A-J (135-144) [153,154] and M-R (145-149) [155]. From a Vanuatu Sponge sp., a 21hydroxy derivative of petrosaspongiolide P (150) was isolated [156]. All these compounds are biogenetically derived from luffolide (123). Petrosaspongiolides A-J exhibited cytotoxicity (IC50 0.5-14.8 iag/ml) against human bronchopulmonary non-small-cell-lung carcinoma cell line (NSCLC-N6) [154]. Petrosaspongiolides M-R (145-149) inhibited different preparations of PLA2 by irreversibly blocking these enzymes, particularly human synovial and bee venom, with IC50 values in the micromolar range. These compound displayed a much lower activity (or no activity at all) towards porcine and Naja naja PLA2 enzymes. The most potent compound, petrosaspongiolide M (145) (IC50 1.6 and 0.6 LIM for human synovial and bee venom PLA2 enzymes), was slightly more active than manoalide (53) (IC50 3.9 and 7.5 |aM) under the same experimental conditions. Petrosaspongiolide P (147) was more selective, inhibiting human synovial PLA2 (IC50 3.8 \xM) to a greater extent that bee venom PLA2 (37.9% inhibition at 10 \M) [155]. Furthermore, petrosaspongiolide M was able to reduce in a dose-dependent fashion.
142 PGE2, TNFa, LTB4 levels [157], and it has shown to modulate the expressions of COX-2 and iNOS by interfering with NF-kB [158].
OAc
136 R = CH 3 143 R = CH2OAc
135 R] = CH3, R2 = OAc 137 R, = CH2OAc, R2 = OAc 138 R, = CH2OH, R2 = OAc 139 R, = CHO, R2 = OAc 140 R, = COOH, R2 = OAc 141 R, = CH2OH, R2 = OH 142 R, = COOH, R2 = OH
OAc CH 2 COOH
OAc
HO
O
145 24S,R = H 146 24S,R = OAc 151 24R,R = H
HO
147 R = H 148 R=OAc 150 R = OH
HO
O
149
Besides, petrosaspongiolide M was capable of reducing the morphine withdrawal at 10" M [159]. The 21-hydroxy derivative of petrosaspongiolide P (150) inhibited human synovial PLA2 at 10 |jM with a value of IC50 5.8 (JM, showing a slightly lower potency but higher selectivity towards this enzyme than the referenced inhibitor m anoalide [156]. Cavernosolide (151), isolated from the Tyrrhenian sponge Fasciospongia cavernosa, is the 24 epimer of petrosaspongiolide M (145)
143
and showed high cytotoxicity (LC50 0.37 |ag/ml) in the A. salina bioassay and a moderate ichthyotoxicity (LC50 0.75 (a.g/ml) to G. affinis [115].
o 152
154
Suvanine (152), isolated from the sponge Coscinoderma mathewsi, has a cheilanthane skeleton with different stereochemical features and contains both sulphate and furan rings [160-162]. Suvanine was found to facilitate neuromuscular transmission in the indirectly stimulated rat hemidiaphragm preparations. Suvanine was also an acetyl cholinesterase inhibitor, and similar properties were exhibited by the suvanine sodium salt [161]. Besides, the suvanine sodium salt showed antithrombin and antitrypsin activity with IC50 of 9 and 27 |ug/ml, respectively [162]. Furthermore, suvanine was ichthyotoxic towards goldfish at 10 (J-g/ml, and exhibited 90% inhibition of sea urchin egg cell division at 16 ng/ml [160]. Inorolide C (153) was isolated from the nudibranch Chromodoris inornata. It was shown to inhibit the proliferation of KB (IC50 6.4 (ag/ml) and L1210 (IC50 1.9 ng/ml) cells [163]. From the Okinawan sponge Hyrtios erectus was isolated hyrtiosal (154), possessing a novel rearranged tricarbocyclic skeleton (hyrtiosane) [164]. Its structure was confirmed by total synthesis [165]. This compound exhibited in vitro antiproliferative activity against KB cells with an IC50 of 3.0 ug/ml [164]. In 1988, we reported the isolation and structural elucidation of a new tricarbocyclic sesterterpene [109], bearing a y-hydroxybutenolide moiety, from the Dictyoceratide sponge, Fasciospongia cavernosa, erroneously classified a s Cacospongia mollior, collected in the North Adriatic Sea. We named this compound after cacospongionolide (155), on the basis of the erroneous classification of the sponge [110]. Cacospongionolide was reported as a potent inhibitor of human synovial and bee venom PLA2 (Table 1) [111]. Besides, cacospongionolide showed high cytotoxic
144 activity (LC50 0.1 fig/ml), in the A. salina bioassay, very high inhibition (75%) in the crown-gall potato disc assay, an antitumoural like test [109].
155 From the Caribbean sponge Cacospongia linteiformis was isolated lintenone (156) with a new tricarbocyclic skeleton, which contains fused cyclohexane, cyclopentane and cyclobutane rings. Lintenone exhibited high antifeedant activity against the fish C. aurantus (30 p,g per cm2 of food pellets), ichthyotoxicity to G. affinis (10 ppm) and moderate toxicity in A salina assay (LC50 109 ppm) [166]. CHO CHO
157 = CH2OH, A13.= 13 158 = CH 2 OH,A'° = 13 161 R = COOH, A = Z 162 R = C O O H , A13I J = £
R 159 160 163 164
R = CH 2 OH ,A 1 3 = Z R = CH 2 OH , A 13 = £ R = COOH, A 13 = Z IJ R = COOH, A13 =E
From the New Caledonian sponge Rhabdastrella globostellata were isolated two isomalabaricane sesterterpenes, aurorals 1 and 2 (157 and 158) and the corresponding trinor-sesterterpenes aurorals 3 and 4 (159 and 160) [167]. From the Okinawan sponges Rhabdastrella (Jaspis) stellifera were isolated the corresponding oxidised compounds jaspiferals C-F (161-164) [168]. Since jaspiferals C-F were isolated together with the related triterpenes stelliferins A-F [169] and nortriterpenes jaspiferals AB [ 168], we can suppose that also aurorals 1-4 and jaspiferals C-F are degraded triterpenoids. Aurorals, which differ from jaspiferals by the presence of a primary alcohol group at C-4 position, exhibited higher
145 cytotoxic activity on the KB cells. The mixtures of aurorals 1-2 (157 and 158) and jaspiferals C-D (161 and 162) showed ID50 values of 0.2 and 5.5 jj-g/ml, respectively. The mixtures of aurorals 3-4 (159 and 160) showed moderate activity on KB cells with an IC50 of 8.0 M-g/ml, while jaspiferals E-F (163 and 164) were inactive until 10 |o.g/ml [167]. Furthermore, the mixtures of jaspiferals C-D, and jaspiferals E-F exhibited cytotoxicity against L1210 cells with IC50 values of 4.3 and 3.1 fj.g/ml, respectively [168]. Besides, jaspiferals E-F showed antifungal activity against Trichophyton memtagrophytes (MIC 50 (ig/ml) [168]. Halorosellinic acid (165) possessing an ophiobolane skeleton was isolated from the cultural broth of the marine fungus Halorosellinia oceanica. Compound 165 showed moderate antimalarial activity with IC50 value 13 |u.g/ml and weak antimycobacterial activity with MIC 200 Hg/ml [170].
HOOC
COOH
CH2CH2COOH
NCH2COOH ~CH2CH2COOH
167 Rt = CH3, R2 = CH2COOH 168 R! = CH2OAc, R2 = CH2COOH 170 R[ = CH3, R2 = CH2CH2SO3H From the New Caledonian Petrospongia nigra, together with the previously reported petrosaspongiolides A-J (135-144) was isolated a pyridium alkaloid 23-norsesterterpene named petrosaspongiolide L (166) that showed cytotoxic activity against NSLC-N6 cells with IC50 value of 5.7 (j.g/ml. Petrosaspongiolide L could be considered a condensation product with ammonia of a 16-keto, 18-al precursor, derived from petrosaspongiolide K (209) (see tetracyclic section) [154]. Four
146 pyridinium alkaloids, spongidines A-D (167-170), related to petrosaspongiolide L, were isolated from the Vanuatu Spongia sp.. These compounds inhibited mainly the human synovial PLA2 at 10 |oM and they were devoid of significant cytotoxic effect on human neutrophils at concentration up to 10 pM [156]. TETRACARBOCYCLIC SESTERTERPENOIDS The main group of marine tetracarbocyclic sesterterpenoids is of those with a scalarane skeleton, which appears to be of the same origin as cheilanthane and is formed by closely biosynthetic process involving additional cyclisation. Metabolites of this class have been reported from marine sponges of the order Dictyoceratida and their predator nudibranches [5, 6]. The first example of this group was scalarin (171), isolated from the sponge Cacospongia scalaris bearing a yhydroxybutenolide moiety. [171].
171 R = a-OAc 172R=p-OAc 173 R = p-OH
174
175
A number of 19-deoxy, 20-deoxo, 12-O-deacetyl and 12-epimers were isolated [5,6]. From the Japanese Spongia sp. were isolated \2-episcalarin (172), 12-0-deacetyl-12-epi-scalarin (173), 12-e/?z-deoxoscalarin (174) and 12-0-deacetyl-19-deoxyscalarin (175) [172]. These compounds exhibited selective cytotoxicity against four tumour cell lines, being more active on L1210 cell line (IC50 13.2, 2.3, 2.1 and 1.6 jig/ml for 172-175, respectively) and less active on A549, KB and HeLa cell lines with an IC50 of the range 14.3-29.4 |ag/ml [172]. 12-0-deacetyl-19-deoxyscalarin (175), first isolated from the sponge Hyrtios erecta, showed also
147
cytotoxicity against P388 cells with IC50 of 2.9 |ig/ml [173]. Moreover, compound 175 showed antitumour activity in vivo on sarcoma-180implanted mice with an increase of lifespan (ISL) of 50.3% at 5 mg/kg intraperitoneal administrations. This activity is more potent than of a positive control, 5-fluorouracil (ISL: 32.9%) at the same dose [172]. 12£/>/-acetylscalarolide (176), isolated from the Spanish C. scalaris, showed significant cytotoxic activity towards a panel of four tumour cell lines (Table 3) [174]. 12-O-acetyl-16-O-methylhyrtiolide (177), with an additional methoxy group at C-16 exhibited cytotoxicity against L1210, A549, KB and HeLa cell lines with IC50 values of 2.2, 5.3, 15.6 and 5.3 fag/ml, respectively [172]. AcO
r
OAC
Heteronemin (178), first isolated from the sponge Heteronema erecta [175], was toxic to A. salina and gametes of the giant kelp Macrocystis pyrifera at 10 |J.g/ml and also immobilised the larvae of the red abalone Haliotis rufescens at 1 fig/ml [22]. Furthermore, heteronemin showed antituberculosis activity, inhibiting the growth of Mycobacterium tuberculosis with an MIC of 6.25 |ag/ml [176]. Salmahyrtisol B (179), isolated from the Red Sea Hyrtios erecta [177], is related to scalarafuran (180), isolated from Spongia idia, a compound toxic to A. salina at 10 Hg/ml, [22]. Salmahyrtisol B showed cytotoxic activity with an IC50 > 1 Hg/ml against P388, A549 and HT29 cells [177]. Generally, scalarane sesterterpenoids are not functionalised on A- and B-rings. A structure-activity study showed that an oxygen-bearing substituent at C-3 of scalaranes, together with the presence of hydroxyl groups at C-12 and C-19, leads to increase of antitumour activity [178]. Accordingly, salmahyrtiol C (3-oxo-12-O-deacetyl-12-epi-deoxyscalarin) (181), first isolated from the Japanese H. erecta [178] and subsequently from the Red Sea H. erecta [177], exhibited potent cytotoxicity against P388 (IC50 of 14.5 ng/ml) and human gastric carcinoma MNK-1 (IC50 of
148
57.7 ng/ml), MNK-7 (IC50 of 56.0 ng/ml) and MNK-74 (IC50 of 36.8 ng/ml) cells. Intraperitoneal administration of 181 (0.5-8.0 mg/kg) on mice with P388 leukaemia increased the mean survival time (10.7-15 days) and ISL (24.4-74.4%) dose-dependently [178]. 12-Deacetoxy-21acetoxyscalarin (182), isolated from the Japanese H. erecta, showed cytotoxic activity against P388 cells with IC50 value of 0.9 )J.g/ml [179]. OH
HO
HQ, '-r-0
= CH 3 , R2 = CH2OH = CH2OH, R2 = CH3 From the Maldivian H. erecta were isolated sesterstatins 1-3 (183-185) that showed cytotoxic activity against P388 cells with IC50 value of 0.46, 4.2 and 4.3 ng/ml, respectively [180]. Additional 3- (186 and 187) and 19-oxygenated scalaranes (188 and 189) were isolated from the nudibranch Chromodoris inornata that showed cytotoxic activities against L1210 (IC50 6.6, 0.95, 4.1 and 0.35 ng/ml for 186-189, respectively) and KB (IC50 22.8, 5.2, 21.0 and 3.1 ng/ml for 186-189, respectively) cell lines [163]. Scalaradial (190) and its 12-deacetoxy derivative (191) are two classical examples of compounds with a 1,4-dialdehyde moiety. Scalaradial (190) was isolated from two species of Cacospongia, C. mollior[ 181] and C. scalaris [174]; 1 2-deacetoxyscalaradial (191) was isolated from C. mollior [182]. The majority of terpenoids, containing an unsaturated 1,4-dialdehyde functionality, are intensely pungent [183] and
149 generally are very versatile repellents [184]. This activity was explained by their interaction with vanilloid receptors [185]. However, scalaradial (190) was tasteless and showed antifeedant activity at a concentration twice the sesquiterpene polygodial (192) [186]. The antifeedant activity of 12-deacetoxyscalaradial (191) was similar to that reported for 192, and moreover 12-deacetoxyscalaradial was hot to the taste. These results showed that the molecular size was not a restrictive factor in these activities and pointed out the specific importance of the substituent at C12 in 190 and 191, or in the equivalent C-l position of a supposed polygodial derivative [182]. CHO CHO
186 R = p-OAc 187 R = a-OAc
188R = CH2OH 189R = CH2OAc
AcO CHO
190 R = OAc 191 R = H
193R = OH 194 R = OAc, \8-epi
CH2OH CHO
CHO
192
195
196 Rx = OH, R2 = (3OH 197 R, = OAc, R2 = pOH 198 Ri = OH, R2 = aOAc
In 1991, de Carvalho & Jacobs [187] reported the potent activity of scalaradial (190) against bee venom PLA2 (IC50 0.07 |u.M). They observed that scalaradial completely inactivated the enzyme by a two-step mechanism, involving apparent non-covalent binding followed by covalent modification. Subsequently, we observed that scalaradial showed
150
a topical anti-inflammatory activity on ear oedema in mice, with an ID50 of 172 p,g/ear comparable with that of indomethacine. It is a potent inhibitor of several PLA2, with a high selectivity for human recombinant synovial PLA2 (IC50 0.5 |oM). Moreover, scalaradial showed cytotoxic effects on human neutrophils at concentrations of 5 ^M [26]. Many other scalaranes were screened in the bee venom PLA2 a ssay but a 11 showed less activity than scalaradial. From the Japanese C. scalaris was isolated deacetylscalaradial (193) that showed interesting cytotoxic activity against L1210 cells with an IC50 value of 0.58 |ug/ml [188]. Scalaradial (190) and deacetylscalaradial (193) were shown to act on both R- and Ctype vanilloid receptors [185]. From the C. scalaris, collected in the Southern Coast of Spain, were isolated 18-epz-scalaradial (194) and 19dihydroscalaradial (195). Both compounds showed significant cytotoxicity towards four tumour cell lines (Table 3) [174]. Table 3. Cytotoxicity of Compounds 176,194,195,199,206-208 Against a Panel of Tumour Cells [174]
Mean IC5o (ng/ml) Cell line:
P388
A549
HT29
MEL28
12-ep/-acetylscalarolide (176)
1.0
2.0
2.0
2.0
18-epi-scalaradial (194)
0.2
0.2
0.2
0.5
19-dihydroscalaradial (195)
2.0
2.0
2.0
2.5
16-acetylfuroscalarol (199)
2.5
5.0
2.5
10.0
norscalaral A (206)
1.0
1.0
1.0
2.0
norscalaral B (207)
2.0
2.0
2.0
2.0
norscalaral C (208)
1.2
2.5
5.0
2.5
From the Japanese H. erecta were isolated two sesterterpenoids (196 and 197) [179] related to scalarolbutenolide (198), isolated from the Mediterranean Spongia nitens [189]. Compounds 196 and 197 were cytotoxic against P388 cells with IC50 values of 0.4 and 2.1 p.g/ml, respectively [179]. These compounds cannot strictly be considered as
151
scalarane, because they show different arrangements of the carbons C-24 and C-25. 16-Acetylfuroscalarol (199), with moderate cytotoxicity (Table 3), isolated from the Spanish C. scalaris [174] and 12-0-acetyl-16-0deacetyl-12,16-episcalarolbutenolide (200), cytotoxic against L1210 (IC50 2.4 |J.g/ml) and KB (IC50 7.6 |ag/ml) cell lines, isolated from the nudibranch C. inornata [163], showed the same carbon skeleton of scalarolbutenolide. From the Indonesian Phyllospongia sp. were isolated two sesterterpenes (201 and 202), which exhibited cytotoxicity against KBcellsatl0ng/ml[190]. AcO
O
203 R = OAc 204 R = OH
205
206 R = 16(3-OH 207R=16a-OH
Tetracarbocyclic norsesterterpenoids a re extremely rare and are only isolated from sponge of subclass D ictyoceratida. Hyrtial (203), isolated from H. erecta, was the f irst 2 5-norscalarane to be reported. It showed anti-inflammatory activity at 50 |ig/ml close to the activity of indomethacine [191]. From the Okinawan sponge H. erecta were isolated 12-deacetylhyrtial (204) and its A17 isomer (205) that showed cytotoxic activity against KB cells with IC50 values of 10.0 and 2.82 fag/ml, respectively [192]. Norscalarals A-C (206-208) isolated from the Spanish C. scalaris showed cytotoxicity against four tumour cell lines (Table 3) [174]. Petrosaspongiolide K (209), isolated from the New Caledonian
152
Petrosaspongia nigra, was the first reported 23-norscalarane. Petrosaspongiolide K showed cytotoxic activity (IC50 1.3 p.g/ml) against NSCLC-N6 cells [154]. Scalarane sesterterpenoids also include alkylated derivatives, called homoscalaranes with methylations at C-20 or C-24 and bishomoscalaranes with methylations at C-20 and C-24 and rarely at C-23 and C-24 [193]. HOOC
AcO
209
208
AcO
CHO O
210
A series of 24-methylscalaranes were isolated from the Palauan sponges Dictyoceratida sp. and Halichondria sp. [194]. Only compound 210 was shown to have significant inhibitory activities (IC50 0.5 |i.g/ml) on the platelet aggregations caused by adenosine 5'-diphosphate, collagen, or arachidonic acid [194]. Another group of related compounds were isolated from the Australian sponge Lendenfeldia sp., as only the compound 211 was the inhibitor of platelet aggregation [195]. Further 24homoscalaranes were isolated from L. frondosa, and only the compound 212 exhibited moderate anti-inflammatory activity, inhibiting 35% of bee venom PLA2 at 8 JJM [196]. AcO AcO.
CHO O
AcO. 'OH
213 R = 214R = Ac
Four 24-homoscalaranes (213-216) that exhibited 30-95% inhibition of the growth of KB cells at 10 fj.g/ml were isolated from the Indonesian Phyllospongia sp. [190]. From the Pacific nudibranch Glossodoris sedna were isolated several scalarane and homoscalarane compounds, but only
153
compound 217 was ichthyotoxic at 0.1 ppm against G. affinis and inhibited mammalian cytosolic PLA2 (IC50 18.0 |u.M) [197]. Foliaspongin (218), a 20,24-dimethylscalarane derivative, isolated from the sponge Phyllospongia (Carteriospongia) foliascens, showed anti inflammatory activity [198,199]. HO
AcO AcO,
CHO O
HO
CHO O
A
215 Rj = OH, R2 = H 216 R, = OMe, R2 = OMe AcO
R
219 R = CHO 220 R = H AcO
222 R = 24a-Me 223 R = 240-Me
CHO O
OR
224 R = H 226 R = CH3CHOHCH2CO227 R = CH3CH2CHOHCH2CO228 R = CH3CH2CO229 R = CH3CO-
230 R = CH3CH2CH(OCOCH3)CH2CO231 R= CH3CH2CH(OCOCH2CH3)CH2CO232 R = CH3CH(OCOCH3)CH2CO-
Subsequently, several bishomosesterterpenoids were isolated from P. foliascens, collected in different seas. From the Neo Guinean sponge C. foliascens were isolated several bishomosesterterpenoids, but only
154
compounds 219-221 showed ichthyotoxic effects towards L. reticulatus at LD50 of 5, 20 and 40 mg/1, respectively [200]. Phyllactones A (222) and B (223), with moderate cytotoxicity against KB cells (IC50 20.0 fig/ml), were isolated from the Chinese P. foliascens [201]. From the Indonesian Phyllospongia sp. were isolated two 20,24-dihomoscalaranes (224 and 225) that showed cytotoxicity against KB cells at 10 |u.g/ml [190]. From the Australian Strepsichordaia lendenfeldi, together with the alcohol 224, were isolated four different acyl derivatives (226-229) and three esters with the same skeleton and different acyl groups (230-232). All these compounds exhibited potent cytotoxicity against both P388 and A549 cell lines (Table 4) [202]. Table 4.
Cytotoxicity of Compounds 224,226-232 Against a Panel of Tumour Cells [2021 Mean IC5o (ng/ml)
Cell line:
224
226
227
228
229
230
231
232
P338
0.1
0.23
0.5
0.67
0.91
0.12
0.12
0.2
A549
0.1
0.66
0.5
0.67
0.88
0.25
0.21
0.2
From the Red Sea Hyrtios erecta, together with hyrtiosal (154), previously reported [164], was isolated salmahyrtisol A (233), a furan sesterterpene with a new tetracarbocyclic skeleton. The coexistence of the unusual sesterterpenes 233 and 154 is noteworthy from the biosynthetic viewpoint and maybe hyrtiosal is the logical biosynthetic intermediate for salmahyrtisol A. Salmahyrtisol A showed cytotoxic activity with an with IC50 > 1 M-g/ml against three type of cancer cells including P388, A549 andHT29[177]. Suberitenones A (234) and B (235), isolated from the Antarctic sponge Suberites sp., are two sesterterpenoids with an unprecedented carbon skeleton. Suberitenone B inhibited (IC50 10 |umol/ml) the cholesteryl ester transfer protein (CETP), which mediates the transfer of cholesteryl ester and triglyceride between high-density lipoproteins and low-density lipoproteins. Many studies have found an inverse correlation between levels of high-density lipoproteins and incidence of atherosclerotic cardiovascular diseases. Therefore, CETP inhibition is considered to be a good target for the development of an effective agent against atherosclerotic diseases [203].
155
From the Japanese nudibranch Chromodoris inornata were isolated two sesterterpenes, inorolides A (236) and B (237) with a new carbon skeleton. Both compounds exhibited cytotoxic activity against L1210 (IC50 1.9 and 0.72 ^g/ml for 236 and 237, respectively) and KB (IC50 3.4 and 2.2 |J.g/ml for 236 and 237, respectively) cell lines [163].
AcQ
P
238R = C1 239 R = Br
OH
OH
241 R, = OH, R2 = H 242 R, = H, R2 = OH 244 R, = R2 = H
From the marine fungus Fusarium helerosporum were isolated two groups of sesterterpenes, neomangicols A-C (238-240) [204] and mangicols A-G (241-247) [205], both with unusual carbon skeleton that constitutes two new classes of rearranged sesterterpenes. Neomangicols A (238) and B (239) were found to be active against a variety of cancer cell lines. Neomangicol A was most active against MCF7 and human colon carcinoma CACO2 cell lines, displaying IC50 values of 4.9 and 5.7 |aM, respectively. Neomangicol B was less active having a mean IC50 value of
156
27 )j.M across the entire panel (versus 10 \xM for neomangicol A). Neomangicol B displayed antibacterial activity similar to that of gentamycin, against the Gram-positive bacterium B. subtilis [204]. Mangicols A-G (241-247) showed weak cytotoxicity with IC50 values ranging from 18 to 36 |a.M in the 60 cell lines panel. Mangicols A and C inhibited mouse ear oedema (81 and 57% reduction in oedema, respectively) at 50 (j.g per ear. These values are consistent with the potencies of the anti-inflammatory agent, indomethacine [205].
RO
243 R, = OH, R2 = H 245 R, = H, R2 = OH 246 R, = R2 = H
247
OR
248a R = H 248b R = Ac
Aspergilloxide (248a), a sesterterpene epoxide diol with a new carbon skeleton was isolated from the marine fungus of the genus Aspergillus. It showed little cytotoxicity towards HCT116, but its acetate derivative (248b) inhibited HCT116 cell line at 61 \xM [206]. PENTACARBOCYCLIC SESTERTERPENOIDS Although numerous marine sesterterpenoids have been found, only a few sesterterpenoids possessing a pentacarbocyclic skeleton have been isolated. Disidein (249a) and two halogenated related derivatives (250a, 251) were isolated from the Mediterranean sponge Dysidea pallescens [207-208]. The stereochemistry of disidein was determined by X-ray analysis of the acetyl derivatives of bromo-disidein (250b), which shows the same carbon skeleton of scalarane. The triacetyl disidein (249b) showed moderate analgesic activity [55]. From the Neo Guinean sponge Phyllospongia foliascens, together with bishomoscalarane derivatives (see tetracyclic section), was isolated a related compound (252) with an additional cyclobutane ring. This
157
compound showed ichthyotoxic effects towards L. reticulatus at the LD50 of5mg/l[200]. R1Q /^OFL
249a Rj = R 2 = H; 249b R{ = Ac, R 2 = H 250a Rj = H, R 2 = Br; 250b R, = Ac, R 2 = Br 251 Rj = H, R 2 = Cl AcO
252
253
Phyllofenone B (253), an additional bishomoscalarane derivative with a pentacarbocyclic skeleton was isolated from P. foliascens. It showed cytotoxicity against P388 cells with IC50 value of 5.0 |ig/ml [209]. PRODUCTION OF MARINE COMPOUNDS Although the marine environment is a plentiful source of interesting new products with pharmaceutical potential, only a few of these marine natural products have reached the stage of commercial production. Arabinofuranosyladenine (ara-A, isolated from the Gorgonian Eunicella cavolini) [210] is the unique marine secondary metabolite currently in clinical use and is one of most potent antiviral drugs [211]. The second one is avarol, a sesquiterpene hydroquinone isolated from the sponge
158
Dysidea avara [212,213] currently commercialised as a cream against skin disorder. Words such as "promising" and "potential" dominate the literature on marine natural products, while papers describing successful application of these products remain scarce. In fact, patent applications are less than 10% of the total number of papers published on marine natural products. The number of patent applications on marine natural products is very little when compared with those of terrestrial origin. The limited availability of larger quantities of a particular organism as starting material for extraction of the compounds is one of the major causes for the low attractiveness of such secondary metabolites for commercial utilisation. Furthermore, the isolation of large quantities of these compounds from animal tissues is unacceptable because of its devastating impact on the natural environment. Four different approaches can be undertaken to obtain bioactive marine secondary metabolites in bulk amounts: 1- Chemical synthesis 2- Aquaculture 3- Cultivation of marine organisms in bioreactor 4- Cell culture. Chemical Synthesis Generally, pharmaceutical companies need a strong patent position before starting the long and expensive path, of a drug development, and they prefer compounds that can be synthesised. This approach has successfully been undertaken specially for those compounds with a potential industrial application, but very often, for the high structural and stereochemical complexity of the metabolite, the synthesis includes many steps with low yield and it is not commercially realistic. Aquaculture The first attempt for i n situ a quaculture of c ommercial marine sponges (bath sponges) was made in Adriatic Sea in 1870, but no detailed statement of the methods employed was reported. Smith [214] first reported the description of cultivation of sponges in the late 19th century. Subsequently, Moore [215] described the procedures for the cultivation of sponges. The technique exploits the capacity of sponges to regenerate them and to form new colonies even only by small fragments. Then, the large-scale commercial sponge aquaculture was developed in several
159
countries [216-218]. Farming of sponges in a sustainable manner for the production of bioactive compounds has recently been started both in New Zealand [219] and Mediterranean Sea [220]. Cultivation of Marine Organisms in Bioreactor Aquaculture has the disadvantage that the growth rate of sponges is dependent on in situ conditions, which cannot be controlled. Therefore, some researchers have considered the possibility of producing sponge biomass under controlled condition. The main difficulties are the supply of an adequate food source and the accumulation of waste products. Recently, Osinga and co-workers [221] reported growth of the sponge Pseudosuberites andrewsi in a closed system, using the microalgae Chlorella sorokiniana and Rhodomonas sp. as food source. These two microalgae were selected, because it was microscopically observed, on fresh material, that these algae were ingested and digested by the sponge cells. The high growth rates observed for this sponge suggest a promising future for cultivation of sponges in closed systems. Cell Culture The high proliferation capacity of sponge cells suggests that it should be easily feasible to establish their cell cultures in vitro. Then, in analogy to the production of bioactive metabolites from fungi and bacteria, the production of secondary metabolites will be accomplished in a bioreactor using sponge cells in culture. In the last few years, there has been developed the production of axenic sponges cell culture, but until now, only the maintenance of sponge cells in vitro has been achieved [222224]. Primary cell cultures have been obtained from several sponges, with a low cell density in the cultures. This low proliferation can be explained in the culture condition utilised and/or in the experimental approach to establish the culture condition. The lack of in-depth knowledge of the nutritional requirements of marine sponges maybe one question to settle. Recently, we have reported that by optimising some physical parameters (pH, temperature, light) and supplementing the commercial medium with different compounds, such as cholesterol, fatty acids, glucose, it was possible to promote the sponge cell proliferation [225,226]. It has been observed that the single cells in suspension did not proliferate readily [223], because they loose telomerase activity and hence
160 their potency for cell division [227]. The formation of multicellular aggregates from dissociated single sponge cells regain telomerase activity, and with this their growth potential. These aggregates were termed primmorphs [228,229]. Another promising method is the fragmentation of intact sponges. Brummer and co-workers [230] reported the in vitro cultivation of sponge fragments without further dissociation and reaggregation. There are same limitations in the cultivation of sponge fragments. In fact, only species with high capability of wound healing can be used for fragmentation [230]. In all methods, cell culture, primmorphs and fragmentation, morphological changes indicate that the culture conditions may not be optimal. Further ecological parameters have to be involved in the optimisation of culture conditions and sponge bioreactor design. Recent studies have demonstrated the ability of sponge cell cultures to produce secondary metabolites [231,232]. If an appropriate growth medium and bioreactor system for primmorphs can be developed, this system may have promising biotechnological potential. ACKNOWLEDGMENTS One of the authors (M. Mitova) gratefully acknowledges a Marie Curie Research Training Grant of the European Community programme, "Quality of Life and Management of Living Resources" contract QLK5CT-2001-50974.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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ANTIMALARIAL LEAD COMPOUNDS FROM MARINE ORGANISMS ERNESTO FATTORUSSO AND ORAZIO TAGLIALATELA-SCAFATI Dipartimento di Chimica delle Sostanze Naturali, Universitd di Napoli "Federico II", Via D. Montesano, 49,1-80131, Naples, Italy ABSTRACT: A series of about 70 secondary metabolites produced by marine organisms have been grouped into three structural types and discussed in terms of their reported antimalarial activities. The major groups of metabolites include isonitrile derivatives, alkaloids and cycloperoxide derivatives. Structure-activity relationships and, when applicable, mechanisms of action of the isolated molecules, have been discussed. The following discussion evidences that antimalarial marine molecules can efficiently integrate the panel of lead compounds isolated from terrestrial sources with new chemical backbones and, sometimes, with "typically marine" functional groups (as isonitriles).
INTRODUCTION Malaria is an infectious disease caused by several protozoans belonging to the genus Plasmodium (P. falciparum, P. ovale, P. vivax, P. malariae), but P. falciparum is the parasite that causes most severe diseases and most fatal cases. The protozoan comes in contact with humans through the vector contribution of female mosquitoes of the genus Anopheles. The bite of infected mosquitoes injects protozoans in the sporozoite form, that invade selectively the parenchymal cells of the human liver. In this stage, the patient remains asymptomatic and, after an average incubation period of 5-7 days (in the case of P. falciparum), protozoans reach the merozoite stage and are released from the liver. The merozoites invade the erythrocytes and start feeding on the haemoglobin. After proliferation, the rupture of the erythrocyte membrane and the consequent liberation of other merozoites, that invade other erythrocytes, cause the massive infection and the symptoms. A small portion of merozoites develops into the sexual stage of gametocytes, a form that is able to re-start the life cycle of the malaria parasite when a mosquito takes a blood meal from an infected person [1].
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The clinical symptoms of malaria infections are exclusively attributable to parasites in the erythrocytic stage. The rupture of infected erythrocytes is associated with the release into the blood stream of cell debris responsible for the characteristic fever spike patterns. In the lethal cases, a specific protein produced by the protozoan is embedded into the cell membrane of the infected erythrocyte and, as a consequence of this modification, the erythrocyte sticks to the walls of capillaries causing obstruction of vessels. When this mechanism operates at the level of brain vessels, the loss of consciousness is the first symptom, but, if this form of cerebral malaria is not treated immediately, it is soon followed by death. The treatment of malaria infections holds a venerable place in the history of medicinal chemistry and of natural product chemistry. As commonly well-known, the first specific treatment for malaria dates back to the 17th century when the bark of Cinchona trees was used as the best tool to face infections of malaria, that was endemic in Africa, Asia but also in several parts of Europe and North America. Later, malaria was the first disease to be treated with an active principle isolated from a natural source, quinine [(1), Fig. (1)] isolated from the Cinchona bark in 1820, and, later again, the first human disease to be treated with a synthetic drug (methylene blue in 1891). In the course of 20th century, especially during World War II, a series of effective synthetic antimalarial drugs have been developed. Among them, chloroquine (2), mefloquine (3), and pyrimethamine (4), Fig. (1), became the drugs of choice in several programs and contributed to the almost complete eradication of malaria from Europe and North America. Unfortunately, in our days malaria still continues to be an extremely important threat to the health and economic prosperity of the human race, constituting a major cause of morbidity and mortality in tropical countries of Asia, Africa and South America. The reality is probably worse than that commonly conceived: a recent analysis estimates, at a minimum, between 700,000 and 2.7 million deaths each year from malaria (over 75% of them are African children) and between 400 and 900 million acute febrile episodes per year in African children under the age of 5 living in malaria-endemic regions [2]. Part of the reason for the failure to control malaria in these areas is the emergence and spread of resistance to
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first-line antimalarial drugs, cross-resistance between the members of the limited number of drug families available, and in some areas, multi-drug resistance [3]. In addition, the prevalent spreading of the disease to poor countries has suggested to many pharmaceutical companies to categorize malaria at low interest level. In this context, funds provided by public agencies, as European Community, are the good news of recent years. Their specific aim is to encourage the antimalarial research in spite of the poor economic interest.
Fig. (1) First-line antimalarial drugs: quinine (1); chloroquine (2); mefloquine (3); pyrimetamine (4).
Two major breakthroughs of the past few decades have renewed the assault of scientists to this infective disease. The first is the complete sequencing of the genome of Plasmodium falciparum [4] that is expected to provide useful information for the identification of new drug targets. The second is the discovery by Chinese researchers of artemisinin (qinghaosu), an endoperoxide sesquiterpene lactone, as the active principle of the sweet wormwood, Artemisia annua, an herbal remedy used in folk Chinese medicine for 2000 years [5]. This molecule and its
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oil soluble (e.g. artemether and arteether) and water soluble (e.g. artesunate and artelinate) semi-synthetic derivatives have shown excellent efficacy against chloroquine-resistant Plasmodium strains and are becoming increasingly used, especially in combination with traditional antimalarials (e.g. mefloquine). However, these important discoveries should be considered only as stimulating starting points in the continuing fight against malaria. As commonly believed, in spite of several efforts, an effective vaccine against malaria is still far to be introduced [6], and the complete removal of the vector of the transmission is practically impossible. Therefore, the need for rapid development and introduction of safe and affordable drugs against malaria continues to be urgent. THE MARINE POTENTIAL More than 60% of the earth's biosphere is made up by ocean, an unique environment that hosts a wealth of plants, animals and microorganisms. These, as a consequence of adaptation to their habitat, elaborate a wide variety of natural products often characterized by peculiar structures and promising bioactivities. The number of novel marine natural products until now discovered has been recently estimated to be about 15,000, and, when these molecules are divided according to the producer organisms, sponges play a dominating role as a source of new compounds (almost 40%), followed by coelenterates (21%) and micro-organisms (15%) [7,8]. Although the biogenetical origin of this plethora of marine secondary metabolites can be conceived in the realm of the biosynthetic pathways commonly proposed for their terrestrial counterparts, they often embed in their structures functional groups uniquely or predominantly marine. As an illustrative example we can cite the abundance of halogenated compounds, most likely as a consequence of the relative abundance of halogen atoms in the marine environment, but also some functional groups as isonitrile, thiocyanate (that we will see below in the structure of some antimalarial compounds), sulfamate and formamide that are much more abundant among marine metabolites than among the terrestrial ones.
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The incredible potential of even a single marine organism to produce a large array of secondary metabolites can be interpreted by considering the common features of the secondary metabolism in all the living organisms as well as some peculiar features of the marine environment. Secondary metabolites play an essential role for the adaptation of the producer organism to the environment, mainly, but not uniquely, in terms of defence; they are practically the sole tool in the hands of organisms at lower evolutionary levels or lacking of mechanical or morphological way for protecting themselves (this is the case of sessile organisms as plants, algae, and the marine invertebrates sponges, tunicates, and bryozoans). Since the production of secondary metabolites has been selected by these organisms as a strategy for their survival, and since a potent biological activity must be considered as a rare molecular property, the wider is the number of secondary metabolites produced by an organism, the more chances it has to be winning in the evolutionary competition. It is now generally accepted that metabolic pathways of the secondary metabolism are intrinsically different from those characterizing the primary metabolism [9]. Two main differences, both having as a consequence a wider chemodiversity at low cost, can be recognized: i) enzymes of secondary metabolism have generally a broader substrate tolerance and thus the same enzyme can be used to produce different products or, alternatively, the same product can be produced by more than one route; ii) biosyntheses of secondary metabolites are characterized by the possibility of producing more than one end product. This goal is reached at low cost by using enzymes that possess the ability to create and manage unstable intermediates (such as carbocations or radicals) that are able to result in the formation of a variety of stable products. A further variability is reached with the incorporation of non-enzymic reactions in the metabolic pathways. As far as marine invertebrates, in addition to these two points, the extremely rich secondary metabolism could also be interpreted in the light of the possible contribution of the symbiotic population to the metabolic work. Indeed, marine invertebrates harbor in their tissues, in the extra-
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and intra-cellular spaces, a series of microorganisms such as bacteria, cyanobacteria and fungi. In some cases, associated micro-organisms may constitute up to 40% of the biomass, this bacterial concentration exceeding that of the surrounding sea water by two or three orders of magnitude. For example, according to Hentschel, sponges can be regarded as "microbiological fermenters" containing novel speciesspecific marine microorganisms [10-12]. Although the real contribution of the microorganisms to the secondary metabolism of marine invertebrates has not yet fully understood and evaluated, essentially because of the difficulties encountered in culturing sponge-associated bacteria, it is generally accepted that these harbored microorganisms play a significant role in the biosynthesis of the natural products isolated from the invertebrate. For all the reasons above summarized, it is not surprising that a thorough chemical analysis of a single marine invertebrate, carried out with non-destructive modern spectroscopic techniques (allowing the stereostructure elucidation of molecules isolated in the low milligram range) can afford tens, when not hundreds, of secondary metabolites. These products provide a rich source of chemical diversity that can be fruitfully used as a "natural combinatorial library", frequently more rich and chemically diverse than the libraries obtained through the use of synthetic combinatorial chemistry. Ideally, this "natural library" can be screened in order to find lead compounds to be used as inspiration to design and develop new potentially useful therapeutic agents and to gain the first information about the structure-activity relationships. In spite of the difficulties associated with the limited availability of the compounds under investigation, which is strictly related to the limited supply of the biological material correctly protected for environmental concerns, some interesting results have been obtained. Through the combined efforts of marine natural product chemists and pharmacologists, an astounding array of promising compounds have been identified. Some of these molecules are either at advanced stages of clinical trials or have been selected as promising candidates for extended pre-clinical evaluation. The majority of these products fall within the area of antimicrobial and cancer therapies. Just to cite an example, ecteinascidin
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743 (ET-743), an anti-tumour compound especially effective against solid tumours, constitutes the most advanced compound among marine natural products under clinical investigation and it is expected to enter the drug market in Europe in the next year [13]. Luckily, two recent trends in marine science promise also to overcome the problem of compound supply: i) the increasing progresses in mariculture [14] and ii) the recent impressing advances in molecular genetics, currently allowing the identification of biosynthetic genes in the producing organisms and their cloning in bacteria suitable for large-scale fermentation [15]. If these techniques will be fully developed and utilized, the last obstacle to consider marine organisms as a potentially sustainable drug source would be overcome. Thus, hopefully, in the near future, the incredible chemical diversity of the secondary metabolites elaborated by marine invertebrates will be entirely utilized and the great marine potential will be turned into a brilliant reality of medicinal chemistry. The aim of this review is to highlight the contribution of marine chemistry in the field of antimalarial research. We will report on all the most important results obtained until the beginning of 2004, with particular emphasis to recent discoveries. Some interesting reviews have dealt with the broad topic of bioactive marine products, only skimming over the antimalarial agents [16, 17], while more targeted reviews are too old [18, 19] or not specifically "marine" [20-22]. By inspecting the literature, it appears evident that the number of potential antimalarial leads obtained from marine organisms is lower than that of the terrestrial counterparts and this is undoubtedly a consequence of the lower number of research groups working on the chemical investigation on marine organisms compared to those engaged in the chemical inspection of the terrestrial ones. However, some of the antimalarial "marine" molecules possess really innovative structures and they constitute a valuable contribution to the research in this field. We decided to include in this chapter all those compounds possessing a moderate to high antimalarial activity, cutting off very weak antimalarials or molecules for which the toxicity toward Plasmodium strains is not specific and/or is clearly due to a general cytotoxicity. In some cases the
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mechanism of action of the isolated molecules has been investigated and it will be discussed herein. Throughout this review marine antimalarials have been divided according to their chemical structures and they have been collected into three different classes: i) isonitriles and analogues; ii) alkaloids; iii) cycloperoxides. ISONITRILES AND ANALOGUES The first finding of an isonitrile-containing secondary metabolite from a marine organism dates back to 1973 with the isolation of axisonitrile-1 [5, Fig. (2)] from the marine sponge Axinella cannabina, where it cooccurred with the strictly related axisothiocyanate-1 (6) [23]. Remarkably, at that time, axisonitrile-1 constituted only the second example of a natural isonitrile derivative, after the antibiotic xanthocillin discovered in cultures of Penicillium notatum Westling [24]. Axisonitrile-1 was soon followed by other isonitrile-, isothiocyanate-, and formamide-containing sesquiterpenoids from the same source, namely axamide-1 (7), axisonitrile-2, (8) [25], axisothiocyanate-2 (9), axamide-2 (10) [26], axisonitrile-3 (11), axisothiocyanate-3 (12), and axamide-3 (13) [27] [Fig. (2)].
Fig. (2) Sesquiterpenoids isolated from the sponge Axinella cannabina
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In 1978 the complete synthesis of axisonitrile-3 confirmed its structure [28]. The co-occurrence in the same organism of the uncommon axisonitriles and of the corresponding formamide derivatives was considered to be evidence that the formamide is the biogenetic precursor of the isonitrile function through a dehydration reaction [26, 29]. However, subsequent biosynthetic experiments have unambiguously demonstrated that the isonitrile functionality present in sponge metabolites derives from direct incorporation of inorganic cyanide and, thus, most likely, formamide originates from the corresponding isonitriles through hydration reaction [30]. As frequently happened in the rising field of marine natural product chemistry, at the time of the isolation of isonitrile terpenoids and analogues from Axinella cannabina, and from other marine sources [31], Authors were intrigued by their unprecedented chemical structures and by their possible biogenetic origin, but they did not realize the pharmacological potential of the molecules in their hands. Almost twenty years later, axisonitrile-3 (11) was re-isolated from the sponge Acanthella klethra Pulitzer-Finali and found to possess a potent antimalarial activity both on chloroquine-sensitive (D6, 142 ng/mL) and chloroquine-resistant (W2, 17 ng/mL) P. falciparum strains, with an activity ten times higher on the chloroquine-resistant one [32]. The closely related axisothiocyanate-3 (12) was practically inactive, giving the first suggestion that the antiplasmodial activity should not (or, at least, not only) be ascribed to structural features of the carbon backbone but should be strictly dependent from the presence of the isonitrile functional group. Interestingly, axisonitrile-3 was found to be practically not cytotoxic toward KB cells. These remarkable findings stimulated an in-depth research activity aimed at isolating and testing isonitrile secondary metabolites from different marine sources, with particular regards to marine sponges of the families Axinellidae and Halicondridae that appeared to selectively elaborate this kind of metabolites. Significant advances were obtained with the chemical analysis of the sponge Cymbastela hooperi (Axinellidae) that afforded a series of diterpenes based on amphilectane, isocycloamphilectane, and neoamphilectane skeletons and bearing
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isonitrile, isothiocyanate, and the rare isocyanate functionalities [Fig.(3)] [33]. These molecules displayed a significant and selective in vitro antimalarial activity and the co-occurrence of several strictly related analogues gave also the possibility to elaborate some preliminary structure-activity relationships.
OCN
NCS
20
21
Fig. (3) Representative antimalarial diterpenoids isolated from the marine sponge Cymbastela hooperi
In general, these molecules showed an antiplasmodial activity in the low nanogram range, comparable or in some cases higher than that of axisonitrile-3, but, on the other hand, their selectivity between
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chloroquine-sensitive (D6) and chloroquine-resistant (W2) Plasmodium falciparum strains was poor, since the activity on D6 and on W2 resulted of the same order of magnitude. The cytotoxic activity of compounds 1421 is in the microgram range and, therefore, the average concentration for cytotoxicity can be estimated to be a thousand times higher than that required for antiplasmodial activity. Comparison among the activities exhibited by the closely related isocycloamphilectanes 14 (IC50 = about 4 ng/mL), 15 (IC50 = ab. 40 ng/mL), and 16 (IC50 = ab. 60 ng/mL) allowed the comparison among the relative potency of isonitrile, isothiocyanate and isocyanate groups, suggesting that the bioactivity is particularly associated to the presence of the isonitrile group. However, the location of functional groups also plays an important role, as suggested by the comparison between the activities of compounds 16 and 17, where the positions of isocyanate and isonitrile groups are inverted. Compound 17 is the most active compound obtained from this sponge (IC50 = about 3 ng/mL). The amphilectane derivatives 18-20 showed generally a lower activity, with IC50 values going from 100 to 800 ng/mL, the less active compound of the series being the isothiocyanate 20. This is in line with the lower activities of isothiocyanate derivatives, as observed also in other studies [34]. On the other hand, the activity of the neoamphilectane derivative 21 (IC50 = D6, 90 ng/mL; W2, 29.7 ng/mL) is considerably higher than that of compound 18, indicating that the carbon skeleton can modulate the antiplasmodial activity of isonitrile derivatives. For example, in some cases, unfavourable steric interactions may be important. Further isonitrile-containing antimalarial derivatives have been later isolated from the Japanese sponge Acanthella sp. [e.g. 22-25, Fig. (4)] [35]. These molecules belong to the class of the kalihinane diterpenoids, which comprises compounds known for their antifungal, anthelmintic and antifouling activities. The tested kalihinane diterpenes showed a potent antiplasmodial activity in the very low nanogram range. For example, the most active compound of the series, kalihinol A (25) exhibited in vitro activity against Plasmodium falciparum with IC50 = 0.4 ng/mL (data on chloroquine-resistant strains were not reported). Unfortunately, the cytotoxic activity of kalihinol A (tested against mouse mammary tumor
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cell line) was also quite remarkable (IC50 = 700 ng/mL), although it is more than a hundred times lower than the antimalarial one. Given this selectivity index, the activity of kalihinol A against Plasmodium falciparum should not be attributed to a general toxic effect and, consequently, this molecule is absolutely worthy of further investigation. A total synthesis of a kalihinol A analogue has been very recently reported [36].
NCS
NCS
Fig. (4) Representative kalihinane diterpenoids isolated from the marine sponge Ac ant he Ila sp.
A major advance in our knowledge about the antimalarial activity of marine isonitriles has been achieved with a recent pharmacological investigation that resulted in the elucidation of the mechanism of action of diterpene isonitriles isolated from Cymbastela hooperi [Fig. (3)] [37]. A hybrid modelling technique combining 3D-QSAR with quasi-atomistic receptor modelling methodologies was used to generate a pharmacophore hypothesis consistent with the experimental biological activities. Active
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isonitriles were demonstrated to interact with free heme by forming a coordination complex with the iron center. From the pseudoreceptor modelling study it was possible to conclude that the "pharmacophore" has an overall lipophilic rigid molecular core comprising at least a tricyclic framework carrying an isonitrile group in axial orientation and establishing further hydrophobic interactions above the ring plane. Equatorially oriented isonitrile groups would be much less active. Interaction of marine isonitriles derivatives with heme was shown to inhibit the transformation of heme into /?-hematin and then hemozoin, a polymer produced by Plasmodium in order to neutralize the toxic (detergent-like) free heme produced in the food vacuole. In addition, isonitriles were shown to prevent both the peroxidative and glutathionemediated destruction of heme under conditions that mimic the environment within the malaria parasite. In summary, isonitriles, similarly to quinoline antimalarials [38], exert their antiplasmodial activity by preventing heme detoxification. In principle, the sharing of the same mechanism of action of chloroquine could be a weak point for isonitrile antimalarials; indeed, in this case, cross-resistance phenomena could be stimulated. However, the remarkable selective activity of axisonitrile-3 on chloroquine-resistant strains (see before) encourages further studies. Marine isonitriles have recently inspired the synthesis of some analogues that showed good antimalarial activity in vitro [39, 40]. Among them, a series of easily accessible synthetic isonitriles [e.g. 26-29, Fig. (5)] have also been tested in vivo against the multi-drug resistant Plasmodium yoelii. Compound 29 showed a good activity, although its therapeutic index is poor.
26
27
Fig. (5) Synthetic isonitrile derivatives
28
29
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ALKALOIDS This is a heterogeneous group of marine metabolites that showed activity against Plasmodium falciparum. Metabolites included in this section have been isolated from different marine sources and possess extremely diverse chemical structures. Among them, we will give more emphasis to those molecules whose pharmacological profile is better defined. Manzamines are very complex polycyclic alkaloids first reported in 1986 from an Okinawan sponge belonging to the genus Haliclona [41]. These molecules are characterized by an intricate pentacyclic heterocyclic system attached to a (3-carboline moiety. Since the first report of manzamine A [30, Fig. (6)], at least 40 additional manzamine-type alkaloids have been reported from taxonomically unrelated sponges belonging to nine different genera (including Xestospongia, Ircinia, and Amphimedon) and to four different orders. These findings strengthen the hypothesis that manzamines are not true sponge metabolites but, more likely, they have a symbiotic origin. Microbial community analyses for one of the most common manzamine producing sponges resulted in the identification of Micronosphora sp. as the bacteria producing manzamines [42].
30
31
Fig. (6) Chemical structure of manzamine A (30) and 8-hydroxymanzamine A (31)
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Fourteen years after its first isolation, the research group of prof. Hamann discovered the antimalarial potential of manzamine A [43]. This molecule, and its 8-hydoxy derivative [31, Fig. (6)], were found to potently inhibit the growth of Plasmodium protozoans both in vitro and in vivo. Manzamine A showed in vitro activity against W2 and D6 strains of P.falciparum with IC50 = 8.0 and 4.5 ng/mL, respectively. As a result of in vivo experiments, a single intraperitoneal injection of manzamine A (50 umol/Kg) reduced the P. berghei parasitemia in mice by more than 90% compared to that in control for the first three days after treatment. Such suppression is of the same order of magnitude of that of artemisinin (see Table 1) at the same dose. Manzamine A resulted toxic to the mice at the concentration of 500 umol/Kg, a dose that is 10 times higher than the dose that suppress parasitemia and prolongs survival. Authors postulated that the antimalarial activity of manzamines is due to a stimulation of the immune response [44]. Interestingly, the same study revealed that a closely related derivative of manzamine A, manzamine F [32, Fig. (7)], is completely devoid of activity (IC50 > 1000 ng/mL). The lack of activity of manzamine F provided the first information on structure-activity relationships within this class of compounds, highlighting the key role of the eight membered ring, where the differences between the inactive manzamine F and the active manzamine A are confined. The reduction of the double bond and/or the insertion of a ketone group on the adjacent carbon is evidently deleterious for the antimalarial activity.
6
32
Fig. (7) Chemical structure of the inactive manzamine F (32).
184
Additional information on structure-activity relationships came with the isolation of «eo-kauluamine [33, Fig. (8)] a very complex molecule constituted by two manzamine units dimerized through ether linkages between the eight-membered rings [45]. Although also this molecule, like manzamine F (32), lacks the double bond in the eight-membered ring, it demonstrated to possess the same antimalarial activity of manzamine A.
OH
34
Fig. (8) Chemical structure of the active weo-kauluamine (33) and the inactive 12,34oxamanzamine A (34).
The lack of antimalarial activity for 12,34-oxamanzamine A [34, Fig. (8)] (IC50 = 5000 ng/mL) [42] indicates that the C-12 hydroxy, the C-34 methine or the conformation of the eight-membered ring are of key importance for the antimalarial activity. Authors proposed that these data, combined to the lack of activity of manzamine F, suggest that the ability of the C-34 allylic carbon to form a stabilized carbocation may play a
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critical role in the biological activity of the manzamine alkaloids against the malaria parasite. Manzamines have also been reported to be antiinflammatory, antifungal, antibacterial and antitubercolosis agents and to exhibit activity against AIDS opportunistic pathogens (e.g. Cryptosporidium parvum and Toxoplasma gondii) [42, 45-47]. In order to correctly evaluate their antimalarial potential, it should be noted that, apart from the relatively narrow therapeutic index, a major weak points of these compounds is constituted by their extremely complex structures. Although the complete synthesis of manzamines has been recently described [48], obviously, it will not be able to provide the adequate amounts of compounds for complete clinical studies and, hopefully, for introduction in therapy. Thus, the recently initiated efforts aimed at the microbial production of manzamines could represent the better chance for the development of these unique molecules as antimalarial drugs.
,-OH
Fig. (9) Chemical structures of the active lepadin E (35) and the inactive lepadin D (36)
Lepadins constitute a class of recently discovered antimalarial marine alkaloids. These molecules are decahydroquinoline derivatives bearing a linear eight-carbon chain isolated from two marine invertebrates of Australian origin, Clavelina lepadiformis [49] and Didemnum sp. [50]. Lepadin E (35) [Fig. (9)] exhibited antimalarial activity in the high nanogram range (IC50 = 400 ng/mL) while its close analogue lepadin D (36) [Fig. (9)] is almost completely inactive (IC50 = 6100 ng/mL). This
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marked difference of activity highlights the importance of the 2£-octenoic acid ester functionality in place of the secondary alcohol. The mechanism of action of these molecules has not been investigated; however, it could be, in some extent, related to that of the structurally similar aromatic quinoline compounds, as chloroquine. Authors have proposed that the conformationally mobile side-chain could serve to stabilize non-bonding interactions with heme, or with any other "receptor" molecule [50]. However, an exclusive pharmacokinetic effect of the alkyl side chain cannot be excluded. Complete synthesis of lepadins has been accomplished [51, 52]. A class of alkaloids whose chemical structure appears to be related to that of lepadins have been isolated from sponges belonging to the genus Oceanapia [53, 54]. These molecules, called phloedictynes, are 1,2,3,4tetrahydropyrrolo-[l,2-a]-pyrimidinium derivatives bearing at C-6, in addition to an OH group, a variable-length alkyl chain and at N-l a four/five methylene chain ending in a guanidine group, while at C-7 a thioethylguanidine chain may be present or not (37) [Fig. (10)].
Fig. (10) Structural variety of phloedictynes (37) and of the active compound phloedictyn 5,7i (38)
Recently, phloedictynes have been tested against the chloroquineresistant FGB 1 strain of the malaria parasite Plasmodium flaciparum and
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some of them, particularly phloedictyn 5,7i (38) [Fig. (10)], exhibited a good activity (IC50 = 300 ng/mL) with cytotoxicity at concentrations 50fold higher [54]. Although this activity is not exceptional, the simple structure of phloedictyns, that can be obtained through complete synthesis with relative ease [55], encourages further studies. Hopefully, the importance of the different functional groups and the optimal length of the alkyl chains will be thus estimated. For the above classes of marine alkaloids the antimalarial activity is several orders of magnitude higher than the cytotoxic activity and thus, although their mechanism of action has not been determined, it should be likely ascribed to a specific action on Plasmodium. This could not be the case of homofascaplysin A (39) [Fig. (11)], a/?carboline-indole alkaloid isolated from the sponge Hyrtios erecta [56]. Indeed, homofascaplysin A (39) showed activity against chloroquineresistant P. falciparum strains with an IC50 of about 20 ng/mL, but its toxicity toward rat skeletal muscle myoblast cells was estimated to be less than 1 |a.g/mL, and thus the selectivity index of this molecule is very narrow.
39 Fig. (11) Chemical structures of homofascaplysin A (39) and of ascididemnin (40)
A similar reasoning applies to ascididemnin (40) [Fig. (11)] and to 6bromoaplysinopsin (41) [Fig. (12)]. The first is a pyridoacridone alkaloid isolated from several marine sponges, whose antiparasitic activity against P. falciparum falls in the same concentration range required for the cytotoxic activity [57]. The second is a simple indole derivative, first isolated in 1985 [58], recently re-obtained from the sponge Smenospongia aurea [59], whose activity against the D6 clone of P'. falciparum has IC50 = 340 ng/mL with a selectivity index of only 14. Interestingly, compound
188
41 showed also a high affinity for human serotonin 5-HT2C and 5-HT2a receptors. s-s
H3ca 42 Fig. (12) Chemical structures of 6-bromoaplysisnopsin (41) and of lissoclinotoxin A (42)
Analogously, lissoclinotoxin A (42) [Fig. (12)], a sulfur-containing alkaloid isolated from the tunicate Lissoclinum perforatum [60], showed high activity against P. falciparum but was later found to be a DNAdamaging agent [61] at very low concentrations and, thus, its use as an antimalarial agent cannot be proposed. Heptyl prodigiosin (43) [Fig. (13)] is another antimalarial alkaloid isolated from a tunicate. Precisely, this pigment was purified from a culture of a-proteobacteria isolated from a marine tunicate and showed an antimalarial activity similar to that of quinine against the chloroquine sensitive strain P. falciparum 3D7 with an in vitro activity that was about 20 times the in vitro cytotoxic activity against mouse lymphocytes. When this molecule was tested in vivo, a single administration of 5 mg/Kg significantly extended the survival of P. berghei ANKA strain-infected mice but, unfortunately, the same dose caused sclerotic lesions at the site of injection [62].
OCH-,
43
Fig. (13) Chemical structures of the antimalarial pigment heptylprodigiosin (43)
189
CYCLOPEROXIDES The artemisinin inspiration The sweet wormwood Artemisia annua (Compositae), also named qinghao, has been used in Chinese folk medicine for 2000 years, originally as a treatment for haemorrhoids, but starting from the III century also to treat fevers. In 1972, after activity-guided fractionation, the sesquiterpene derivative artemisinin (in China named qinghaosu: "the active principle of qinghao") (44) [Fig. (14)] was isolated; later its structure was elucidated and it was shown to possess a potent antimalarial activity [5, 63]. This molecule soon appeared to constitute a major breakthrough in the antimalarial therapy because of: i) its nanomolar activity on chloroquine-resistant P. falciparum strains (higher than the activity on chloroquine-sensitive ones) even on cerebral malaria; ii) its fast action; iii) the absence of detectable toxicity at therapeutic doses.
45 Fig. (14) Chemical structures of artemisinin (44) and of its semisynthetic derivatives artemether (45) and artesunate (46)
Artemisinin (44) is a structurally complex cadinane sesquiterpene lactone bearing an endoperoxide group embedded in a 1,2,4-trioxane ring. With its unique juxtaposition of peracetal, acetal and lactone functionalities, it has very much to interest organic chemists. Totally synthetic routes to artemisinin have been developed [64], but their complexity suggests that they will very unlikely supplant the natural extract as drug source.
190
An intense scientific activity has been carried out entailed to the chemical derivatization of artemisinin. The aim was to obtain compounds with better solubility, higher stability, and thus with increased formulation characteristics, and, possibly devoid of the neurotoxic side effects detected for the natural molecule [65]. These efforts soon resulted in the recognition that the endoperoxide linkage is an essential feature for antimalarial activity, given that the acyclic diol and the ether (1,3-dioxolane) analogues of artemisinin were completely devoid of activity [66]. Consequently, the lactone group became the main site for chemical variations that bore the preparation of the oil-soluble artemether (45) [Fig. (14)] and the water-soluble artesunate (46) [Fig. (14)]. Although these molecules are now used for treatment of severe malaria with the support of the World Health Organization, unfortunately, they still possess neurotoxic activity. As a result of the continuing synthetic studies, several artemisinin derivatives, some of which surpass the parent compound in antimalarial potency, have been prepared [67] but many of them show toxicity or have unfavourable pharmacokinetic features. An essential requirement to design optimized artemisinin derivatives would be a perfect knowledge of the mechanism of its antimalarial activity. Unfortunately, still today our knowledge appears incomplete. While the crucial importance of the peroxide pharmacophore is no longer questioned, basically two different mechanisms of action, not completely antithetic, are now on the ground.
H ?
Fe"PPIX
alkylation of protozoan biomolecules
Fig. (15) A schematic view of the postulated mechanism of action of artemisinin (44)
191
According to the first hypothesis, artemisinin (or its analogues) would interact, within the parasite food vacuole, with the iron center of the heme unit released during the digestion of hemoglobin. The interaction of artemisinin with the heme ferrous iron would cause the cleavage of the peroxide bridge and the formation of alkoxy radicals that, after several rearrangements, would result in the formation of free C-centered radicals. These should be toxic to the parasite because they should alkylate not better defined "sensitive" macromolecular targets [Fig. (15)]. This hypothesis was based on the evidence that, in several experimental models, artemisinin reacts with iron ions and in particular it interacts strongly with hemin (ferriprotoporphyrin IX) and its ferrous form (ferroprotoporphyrin IX) to give covalent adducts [68]. However, two different recent evidences have weakened this postulated mode of action: i) it has been recently demonstrated that, once in the parasite cell, artemisinin only scarcely accumulates within the food vacuole and, thus, a key role of its interaction with heme is unlikely [69]; ii) some artemisinin derivatives that are extremely active as antimalarials show very low tendency in vitro to form carbon radicals [70]. The second mechanism hypothesized for artemisinin is based on the interaction with a specific target. This has been identified as a Ca2+dependent ATPase specific of P'. falciparum (PfATP6), a trans-membrane protein associated with the parasite endoplasmic reticulum [69]. However, it is still not clear whether artemisinin reacts with this target as it is (and, therefore, the peroxide bridge exerts its key role concomitantly with the binding), or it needs a foregoing reaction with an iron-containing molecule that, however, should not be heme [70]. Further experiments would be required to gain more insights into the mechanism of action of the cycloperoxide-containing antimalarial agents. The isolation of different antimalarial cycloperoxides from natural sources can evidently help in this task. Indeed, it could provide additional information about the structural features required to the carbon backbone of a cycloperoxide-containing antimalarial agent. With luck, this research could afford new natural compounds whose antimalarial activity is higher than that of artemisinin. In this context, with the inspiration of artemisinin, several research groups are currently engaged in the isolation
192
of cycloperoxide-containing compounds from terrestrial plants and active compounds as yingzhaosu A (47) [Fig. (16)] have been obtained and some semisynthetic derivatives, as arteflene (48) [Fig. (16)], have also been prepared.
Fig. (16) Chemical structures of yingzhaosu A (47) and of the semisynthetic derivative arteflene (48)
In the next two sections we will give a survey of the contribution in this field coming from marine sources. Indeed, a number of cyclic peroxides have been isolated from marine organisms and some of them have been tested for antimalarial activity. For clarity, we decided to divide these molecules in two categories according to their postulated (and only in few cases unambiguously demonstrated) biogenetic origin: polyketide derivatives and terpene derivatives. Polyketide derivatives Marine sponges belonging to the family Plakinidae contain a series of simple cycloperoxide derivatives that have been identified as polyketide metabolites possessing six- or five-membered 1,2-dioxygenated rings (1,2-dioxane or 1,2-dioxolane, respectively). A further variation is represented, in some cases, by the presence of a 3-methoxy substitution, building a peroxyketal group. The parent compound of this group of secondary metabolites is plakortin (49) [Fig. (17)] that was isolated more than 25 years ago from Plakortis halichondroides [71]. This interesting secondary metabolite, whose polyketide skeleton suggests the involvement of butyrate units in the biogenesis, has been recently re-isolated in remarkable amounts from
193
the Caribbean sponge Plakortis simplex [72]. In the same study the absolute configuration of the four stereogenic carbons of plakortin has been determined by means of chemical derivatization and reaction with chiral auxiliaries; in addition, a closely related analogue, named dihydroplakortin (50) [Fig. (17)] has been obtained [72].
o
-v-
y
49
O^O/^COOCH3
50
Fig. (17) Chemical structures of plakortin (49) and dihydroplakortin (50) At the time of its first isolation, plakortin was found to be a weak antibacterial agent, while a recent study has finally disclosed the antimalarial potential of this molecule [73]. Using the pLDH assay, plakortin (49) and dihydroplakortin, (50) were assayed against D10, chloroquine-sensitive strain, and W2, chloroquine-resistant strain of P. falciparum. The two compounds showed a good activity, that was more potent on the W2 strain (IC50 = ab. 250 ng/mL on D10; ab. 180 ng/mL on W2). In addition, the two compounds proved to be not cytotoxic in vitro [72]. Interestingly, in the same investigation [73] the structurally related, even more sterically hindered, five-membered cycloperoxide plakortide E (51) [Fig. (18)] was found to be inactive.
OOCH 3
51 Fig. (18) Chemical structure of the inactive plakortide E (51)
The chemical structure of these two antimalarial leads is remarkably simple and thus they could constitute a good probe to establish structureactivity relationships, to check the currently postulated mechanisms of
194
action for antimalarial peroxides and to prepare semisynthetic or totally synthetic derivatives. In this regard, a synthetic study toward this class of cyclic peroxides has recently appeared [74]. Some 1,2-dioxane derivatives structurally related to plakortin have been isolated from Plakinidae sponges and tested for their antimalarial activity. Plakortide F (52) [Fig. (19)] has been isolated from a Plakortis sp. [75] and it has been shown to possess an antimalarial activity that is slightly lower (about one half) compared to that of plakortin: IC50 = 480 ng/mL on D10; ab. 390 ng/mL on W2; however, unless plakortin, this molecule was found to be consistently cytotoxic since the IC50 of toxicity against human colon carcinoma and mouse lymphoma cells is only about double (IC50 = 1.25 \iglmL) than the concentration of the antimalarial activity.
Fig. (19) Chemical structures of plakortides F (52), K (53), and L (54).
A moderate antimalarial activity was also recently reported for plakortide K (53) [Fig. (19)], an 1,2-dioxane derivative substituted at position 3 with an a,(3 unsaturated ketone, isolated from a Jamaican sponge Plakortis sp. [76]. This molecule showed activity against W2 P. falciparum strain with IC50 = 570 ng/mL and a selectivity index > 8.4. Interestingly, plakortide L (54) [Fig. (19)], a closely related analogue lacking the carbonyl function, was completely inactive. Two additional plakortides, named plakortide O (55) [Fig. (20)] and plakortide P (56) [Fig. (20)], have been isolated from Plakortis halichondrioides and tested for antimalarial activity against P. falciparum [77]. These compounds showed a very low activity with an IC50 = 8 (j,g/mL for plakortide O and
195
an IC50 > 50 ug/mL for plakortide P. In addition, these molecules showed toxicity in vitro toward several cell lines at lower concentrations.
55
56
Fig. (20) Chemical structures of plakortides O (55), and P (56).
It should be noted that all these plakortides have a close structural similarity with plakortin and, therefore, their lower level of antimalarial activity can be utilized to gain useful information about the structureactivity relationships within this class of simple cycloperoxide derivatives. The main differences among these compounds are ascribable to the stereochemistry. Indeed, while in the structure of plakortin the most hindered chains attached to the 1,2-dioxane ring are in cis orientation, in the other analogues a trans orientation is present. Most likely, these latter molecules experience a more problematic approach of the cycloperoxide group to its target. However, the chemical structure of the side chains must be also important, as indicated by the marked difference of activity between plakortides K (53) and L (54) and between plakortides O (55) and P (56).
H H3C00C
v°"V 0 C H 3
CH 3
57
58
Fig. (21) Chemical structures of peroxyplakoric acids A3 (57) and B3 (58) methyl esters
Further information on the structure-activity relationships come from data on synthetic and natural 3-alkoxy-l,2-dioxene and 3-alkoxy- 1,2dioxane (both peroxyketals) derivatives that were shown to possess a very good antimalarial activity. In this class of molecules, the alkoxy
196
substituent at position 3 could partly mime the non-peroxidic oxygen atom of the 1,2,4-trioxane ring of artemisinin. The methyl esters of peroxyplakoric acids A3 (57) [Fig. (21)] and B3 (58) [Fig. (21)], isolated from Plakortis sp., showed a very good antimalarial activity against P. falciparum with IC50 = 50 ng/mL and a good selective toxicity index (about 200) [78]. Through the syntheses of some analogues of these active compounds, some conclusions about the structural requirements within these classes of antimalarials were drawn. For example, compound 59 [Fig. (22)] proved to be almost completely inactive, whereas compound 60 [Fig. (22)] retained the in vitro activity of peroxyplakoric acid B3 methyl ester, indicating the importance of the side chain for the antimalarial activity [79]. PCH3 H3COOC
59
60
Fig. (22) Two synthetic analogues of peroxyplakoric acids methyl esters.
When compound 60 was examined through an in vivo system against P. berghei infection, it showed little antimalarial potency because of lability in mouse serum. This undesired finding was demonstrated to be due to the hydrolysis of the ester function to the inactive carboxylic acid. Indeed, the monoethyl amide analogue of 60, that is stable to hydrolysis in the serum, showed a good in vivo activity [80]. Finally, the low antimalarial activity observed for two additional marine cycloperoxides strictly related to peroxyplakoric acid B3 methyl ester, namely chondrillin, (61) [Fig. (23)] [81], and muqubilone, (62) [Fig. (23)] [82], provides other interesting suggestions. The insertion of a double bond within the 1,2-dioxane ring is evidently detrimental for the activity, while the presence of the methoxy group at C-3 exerts a pivotal role in the determination of the antimalarial activity for this group of molecules. Most likely, simple 1,2-dioxane molecules, that, like plakortin, are consistently active, possess other features that are able to compensate the lack of the methoxy group.
197
H3COOC
61 0-0
O
CH-,
62 Fig. (23) Chemical structures of chondrillin (61) and muqubilone (62).
Terpene derivatives Terpene derivatives containing a peroxide group are frequently isolated from natural organisms and marine sources make no exception. Unfortunately, only very few of these molecules have been tested for their antimalarial activity. Sigmosceptrellin A, (63) [Fig. (24)], is a norsesterterpene derivative that showed activity against P. falciparum with IC50 = 470 ng/mL on D6 clone and 420 ng/mL on W2 clone [18] and low toxicity. Interestingly, the C-3 epimer of 63, named sigmosceptrellin B, (64) [Fig. (24)], proved to possess an activity four times lower in the same test with an IC50 = ab. 2000 ng/mL [82]. This is a good demonstration of the importance of relative stereochemistry to determine the antimalarial activity in the series of 1,2-dioxane derivatives.
o
63 64 Fig. (24) Chemical structures of sigmosceptrellin A (63) and and of its C-3 epimer sigmosceptrellin B (64).
198
Methyl-3-epinuapapuanoate, (65) [Fig. (25)], a norditerpene derivative isolated from the New Caledonian sponge Diacarnus levii [83], showed in vitro activity against chloroquine-resistant strains of P. falciparum with IC50 = 1.2 ug/mL [84]. When the molecule was tested against P. berghei in vivo, at the concentration of 25 mg/Kg, a 56% growth inhibition was observed.
65 Fig. (25) Chemical structure of methyl-3-epinuapapuanoate (65)
MISCELLANEOUS COMPOUNDS In this section we have grouped all the marine secondary metabolites that possess a certain antimalarial activity and do not fall in one of the preceding groups, namely they do not contain an isonitrile or a cycloperoxide group and they are not alkaloids. The antimalarial activity of these molecules is generally very low, falling in the jag/mL range; however, since almost all are not cytotoxic, their activity against Plasmodium should be intended as specific and could be used, at least in principle, to elaborate optimized derivatives. Halorsellinic acid (66) [Fig. (26)] is an ophiobolane sesterterpene isolated from the marine fungus Halorsellinia oceanica that showed in vitro antimalarial activity with IC50 =13 ug/mL [85]. (5)-Cucurphenol (67) [Fig. (27)] is a sesquiterpene phenol isolated from different marine sponges belonging to the genus Didiscus [86]. This molecule exhibited a series of biological activities including potent antifungal activity against Candida albicans, inhibition of the protonpotassium ATPase with a possible application to treat peptic ulcers, and in vitro antimalarial activity with MIC of 3.6 ug/mL against the D6 clone of P. falciparum and of 1.8 ug/mL against the W2 clone.
199 HOOC
66
Fig. (26) Chemical structure of halorsellinic acid (66)
Another phenol-containing antimalarial marine metabolite is 15oxopuupehenol (68) [Fig. (27)]. This molecule, isolated from sponges of the genus Hyrtios, is a representative of a distinctive family of sponge metabolites comprising also the quinol-quinone pair of avarol and avarone and biogenetically originating from the junction of a sesquiterpene with a C6-shikimate moiety. Compound 68 exhibited in vitro activity against P. falciparum with MIC of 2.0 ug/mL against the D6 clone of P'. falciparum and of 1.3 |xg/mL against the W2 clone [87].
o.
67
x
x
68
Fig. (27) Chemical structures of cucurphenol (67) and 15-oxopuupehenol (68)
Gorgonians are well known as the sources of unique diterpenes, some of them belonging to unprecedented chemical classes. Some of these molecules have shown a moderate antimalarial activity. Briarellins are a class of eunicellin diterpenes isolated from the gorgonians Pachyclavularia violacea and Briareum polyanthes [88].
200
Among them, briarellin L (69) [Fig. (28)] exhibited activity against P. falciparum with IC50 = 8.0 ug/mL, while, interestingly, the closely related analogue briarellin J, (70) [Fig. (28)], differing only for the lack of an acetoxy group, is practically inactive.
Fig. (28) Chemical structures of the moderately active briarellin L (69) and of the inactive briarellin J (70)
Bielschowskysin, (71) [Fig. (29)], is a diterpene very recently isolated from the Caribbean gorgonian Pseudopterogorgia hallos possessing a highly oxygenated tetracyclic structure based on a previously undescribed ring system [89]. This molecule was shown to exhibit antimalarial activity against P. falciparum with IC50 = 10.0 ug/mL, however, compound 71 exhibited toxicity toward human cancer cell lines at lower concentrations. PHH s-Q
Fig. (29) Chemical structure of bielschowskysin (71)
CONCLUSIONS The marine environment contains compounds that could serve as useful lead structures for the development of new classes of antimalarial drugs.
201
As seen from the preceding discussion, the number of marine lead compounds is lower than that of leads coming from terrestrial plants. This is not the result of an intrinsic "poverty" of the marine sources, while, more likely, it reflects the relatively small number of research group working on marine chemistry. Nevertheless, the antimalarial marine molecules can efficiently integrate the panel of lead compounds isolated from terrestrial sources with new chemical backbones and, sometimes, with "typically marine" functional groups (as isonitriles). In Table 1 we have summarized the antiplasmodial and the cytotoxic activities possessed by the most important antimalarial marine leads encountered in this review. Among them, some isonitrile containing diterpenes, as well as some manzamines and polyketide cycloperoxides emerge as the most promising candidates to future developments. In this regard, the possibility of producing some of these molecules through bacterial cultivation combined to genetic engineering could increase the possibilities of their full pharmacological evaluation and their possible introduction in therapy. As far as the marine antimalarial cycloperoxides, it should be outlined that, in addition to the few molecules tested, many other cycloperoxide-containing molecules have been described from marine sources but they have never been tested for their antimalarial activity. At least nine sponge genera have been recognized to be cycloperoxide producer (or, alternatively, to host cycloperoxideproducing symbiont microorganisms). Terpene cycloperoxides have been described from Prianos, Sigmosceptrella, Latrunculia, Mycale, and Diacarnus sponges; polyketide cycloperoxides have been described from Chondrilla, Xestospongia, Plakinastrella, and Plakortis sponges. These organisms constitute a casket that could keep a treasure: a cycloperoxide compound with antimalarial activity comparable to that of artemisinin and, possibly with simpler structure and better solubility. In the last two or three years we have assisted to a flowering of researches in the field of malaria, and in particular in the field of marine antimalarials. For example, the proposal of a new mechanism of action for artemisinin dates back only to 2003 and the discovery of the antimalarial potential of marine molecules as manzamines and plakortin is a result of the last two or three years. Thus, in conclusion, we are
202
confident that in the near future more marine antimalarials will be disclosed and, hopefully, some of them could also start the long way to become a drug. Table 1. Summary of the antiplasmodial (against D6 and W2 clones) and cytotoxic activities of the main antimalarial marine leads. W2 D6 Cytoa Compound Source IC50(in IC50(in toxicity ng/mL) ng/mL) IC50 (in ng/mL) 142 Axisonitrile-3 Axinella 16.7 >20,000 cannabina (11) 3.2 Isocycloamphilectane Cymbastela 2.5 4300 hooperi (17) Acanthella sp. 0.4 700 Kalihinol A (25) 8.0 Manzamine A Haliclona sp. 4.5 1200 (33) Didemnum sp. 400 Lepadin E > 20,000 (35) Oceanapia 300 Phloedictyn 5,7i 15,000 fistulosa (38) Dihydroplakortin Plakortis 250 180 >20,000 simplex (50) Peroxyplakoric Acid Plakortis sp. 50 10,000 B3 methyl ester (58) Sigmosceptrellin A Sigmosceptrella 470 420 >20,000 sp. (63) Use as reference: 50.5 3.8 17,400 Chloroquine (ref. [331) 4.1 Artemisinin 0.71 >20,000 (ref. [32]) 1 All sponges except Didemnum sp. (a tunicate).
203
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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BIOACTIVE SAPONINS WITH CANCER RELATED AND IMMUNOMODULATORY ACTIVITY: RECENT DEVELOPMENTS MARIE-ALETH LACAILLE-DUBOIS Laboratoire de Pharmacognosie, Unite de Molecules d'lnteret Biologique, UMIB EA 3660, Faculte de Pharmacie, Universite de Bourgogne, BP 87900, 21079 Dijon Cedex, France ABSTRACT: Saponins are natural glycosides of steroid or triterpene which exhibited many different biological and pharmacological actions: e.g. immunomodulatory, antitumor, antiinflammatory, molluscicidal, antiviral, antifungal, hypoglycemic, hypocholesterolemic, to mention just a few. The aim of this review is to summarize recent advances on the bioactivity of saponins related to cancer and immune system, which has attracted a great attention during the last five years. INTRODUCTION Saponins are an heterogenous group of natural products both with respect to structure and properties offering a great molecular and biological diversity. They are structurally composed of a triterpenoid or steroid aglycone moiety and quite complex oligosaccharidic substituents. There has been an increase in the interest of biological effects of saponins which were evaluated by many in vitro and in vivo test systems [1-3]. They are often related to their membrane interacting properties, resulting in potential toxic or specific biological effects which have been reviewed (antiviral, analgesic, antifungal, antibacterial, hypocholesterolemic, hypoglycemic, antitumor, immunoadjuvant etc [1-3]. Hovewer the application of these secondary metabolites as successful therapeutic agents is still very much limited. They are used as wound healing (asiaticoside), veinotonic (aescin, ruscogenin glycosides), antiinflammatory (glycyrrhizin, aescin), expectorant (senegosides). Since some compounds display antitumor activities in association with modification of the immune system, there is no clear distinction between these activities. Hovewer we will report the last research developments on saponins having cancer related and immunomodulatory activity. Some of these compounds have interesting structural features, that may be used as lead structures for the development of further semi synthetic derivatives. The discussion will also focus on the significant achievements
210
in the understanding of their mechanism of action and structure-activity relationships. I. CANCER RELATED ACTIVITY Advances in the treatment and prevention of cancer will require the continued development of novel cancer preventive and therapeutic agents. Cancer chemoprevention is related to the administration of agents to prevent the initiational (mutational) or promotional events that occurs during the processus of neoplastic development (carcinogenesis). The initiation involves the direct action of the carcinogen on target cells (or after metabolic activation) whereas promotion and / or progression means that the initiated cells are stimulated to proliferate. Inhibition of mutagenesis and inhibition of the tumor promotion/ progression have been used as screening methods for the discovery of potent chemopreventive agents. Chemotherapeutic agents in the contrary are administered in order to kill the formed tumor. In vitro cytotoxic or growth inhibitory activity on tumor cells as well as in vivo antitumor activity on transplanted tumors in animals are useful test systems for the discovery of potential antitumor agents. We will report here recent advances in the discovery of saponins as potent chemopreventive, cytotoxic, and antitumor agents. I.I. Chemopreventive activity Antimutagenic activity The bioassays are based on in vitro application of saponins to bacterial or more recently to mammalian cells treated with known mutagens. A fraction, PCC100 (50-250 p-g/ml) consisting of a mixture of group B soyasaponins (Fig. l)and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). These results showed for the first time the antimutagenic activity of soyabean saponins in mammalian cells [4]. Another study on soyasaponin I (1) could bring a potential explanation of the mechanism of action. It was found to be a potent and specific sialyltransferase inhibitor in the concentration range 5-100 |iM. Many studies demonstrated that hyperialylation, which is observed in certain pathological processes, such as oncogenic transformation is associated with enhanced sialyltransferase (ST) activity [5]. Kaikosaponin III (2) from Pueraria thunbergiana (Leguminoseae) showed a potent
211
R1O
2
2
R2
R3 H H
(1)
Soyasaponinl
rha - gal - glcA-
OH
(1Z7) (77)
Soyasaponin II
rha - 2 ara- 2 glcA-
OH
(128) (129)
Soyasaponin IV
2
gal- glcAara- 2glcA2 glc - gal- 2glcA-
Soyasaponinl II Soyasaponin V
rha - 2gal - 2glcA2 2 gal -*glcArha-*gal
(75) (76) Soyasapogenol A
(130)
Soyasaponin A1
(132)
H 2
dehydrosoyasaponi n I
(134)
2
gal - glcA-
OH
H H H
OH
OH
OH
H
gb - ara0-
OH
. 3araO-
OH
2
2
H
3
gb - gal - glcA-
Soyasaponin A2
(133)
H
OH
rha - 2 ara0g rha - 2glcO-
H
Soyasapogenol B
(131)
OH
gto
2
tha - gal - glcA-
=0
Fig. 1 Soyasaponins
COOR 2
CH 2 OH
CH 3 2
2
R =rha- gal- glcA(2) kaikasaponin III
R1
4 (5) (6)
(73) (74)
kalopanaxsaponin kalopanaxsaponin kalopanaxsaponin kalopanaxsaponin saphdoside C hederagenin
A I B H
R2
H rha - 2 araH xyl - 3 rha- 2 ararha- 2 ararha- 2 gb-' xyl - rha- ararha - 2 gb-' aleglc - 4 xyl- 3 rha- 2 araH H
H
antimutagenicity by using the Ames test. At 1 mg/plate, it decreased the number of revertants of Salmonella typhymurium TA100 by 99% against Aflatoxin Bl (AFB1), but by 75% against N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) [6]. In this assay, hederagenin (74) and its glycosides (3-6) exhibited potent antimutagenic activities against aflatoxin Bl (1.5 |a,g/plate), kalopanaxsaponin A (= a-hederin) (3) being the most
212
active (59% inhibition at 0.5 fig/ml, p<0.05) [7]. Since they were ineffective against the direct mutagen MNNG, they were suggested to prevent the metabolic activation of AFB1 or scavenge the electrophilic intermediate capable of inducing mutation [7]. Antitumor promoting activity Few data are available in the literature with regard to chemopreventive effects of saponins on experimental carcinogenesis. The most studied species are several kinds of Panax plants. Among them, Panax vietnamiensis is a source of majonoside-R2 (7) (ocotillol-type saponin), which exhibited a potent anti-tumor-promoting activity on two stage carcinogenesis test of mouse hepatic tumor using N-nitrosodiethylamine as an initiator and phenobarbital as a promoter. It exhibited also a remarkable inhibitory effect on two-stage carcinogenesis test of mouse skin induced by NO donor/12-O-tetradecanoylphorbol-13-acetate (TPA) [8]. In a structure/activity relationship study, it was shown that among tubeimosides I, n, in, (8-10), cyclic bisdesmosides from Bobolstemma paniculatum (Cucurbitaceae), having an original structure with a 3-OH-3-methyl glutarate bridge, tubemioside II (9) may be the most promising agent for cancer chemoprevention [9]. Namely, a single topical application of 7,12dimethylbenz[a]anthracene (DMBA) (lOO^g) to the mice as
xyl_OOH
(8) Tubeimostie I 9) Tubetmosde II 10) Tubeimosdelll
R1 ara 4
?5 "
R2 H H OH
initiator, followed by promotion with TPA (1 |ig per painting) applied twice a week from 1st week after the initiation for 18 weeks resulted in the formation of 10.3 tumors per mouse and 80% percentage of tumor bearing mice. Topical application of tubeimoside I (8), (1 mg/painting), II (9) (0.5 mg/painting) and III (10) (0.5 mg/painting) together with TPA completely
213
inhibited the formation of tumors up to the 18th week. These results suggested some structure/activity relationships: C-16 hydroxyl group of tubeimoside II plays an important role in enhancing its biological activity and decreasing its toxicity. The difference of chemical structure in B and/or in C-position between tubeimosides III and II plays an important role in enhancing biological activity and toxicity of tubeimoside III [9]. If administered orally, tubeimoside III was non active as an inhibitor of tumor promotion. Avicins (11,12), a family of triterpene saponins from Acacia victoriae (Leguminosae) having acacic acid as aglycone and monoterpenic acid linked to sugar units were shown to inhibit chemically induced mouse skin carcinogenesis. Two protocols were used. In the
7"
OH 10" R R1 configuration
HOH2C
(ll)AvicinD (12)AvicinG (69) Konmoonoside A (70) KonmoonosideB
OH H OH OH
SI SI S2 S2
6'S 6'S 6'R 6'S
carcinogenesis model, mice were treated with repeated doses of 100 nmol DMBA (twice a week for 4 weeks). F035, a fraction of triterpenoid saponins containing avicins D (11) and G (12), was applied at doses of 0.5 and 1 mg, 15 min before DMBA twice a week for 4 weeks. In the tumor initiation/promotion model, tumors were initiated in mice by a single application of 10 nmol of DMBA. Avicins were applied 15 min before repetitive doses of 2 jig of TPA (promoter) twice a week for 8 weeks. In both protocols, the fraction F035 at 12 weeks produced a significant decrease of the number of mice with papillomas (>70%) and a reduction in the number of papillomas per mouse (>90%) [10]. Avicins produced a reduction of 62 and 74% in both protocols, respectively in H-ras mutations at codon 61, a significant inhibition of the modified DNA base formation,
214
and a marked suppression of aneuploidy with treatment at 16 weeks in the second model. These parameters are early biomarkers used to determine the efficacy of chemopreventive treatment. This findings, when combined with the proapototic properties of these compounds [57], their ability to inhibit H2O2 generation, nuclear factor-KB ( N F - K B ) activation [11] and inducible nitric oxide synthase (iNOS), suggested that avicins could reduce oxidau've and nitrosative stress, and could emerge as important preventative agents in many clinical cases characterized by chronic inflammation, oxidative stress and high risk of neoplastic development. Panax ginseng showed significant anticarcinogenic effect in a 9 weeks medium-term anticarcinogenicity test in mice lung tumor using benzo [a] pyrene and ginsenosides Rg3, Rg5 and Rh2 (Fig. 2) were shown to be the active constituents [12]. The antitumor promoting activity of Rg3 was mediated possibly by down-regulation of NF-KB and activator protein-1 (AP-1) transcription factors [13]. 1.2. Cytotoxic/antitumor activity In vitro cytotoxic saponins Steroid or triterpene saponins were shown to present growth inhibitory activity or cytotoxic activity in many cancer cells line as listed in the Table 1 (13-77; Fig. 2) [14-45]. In vivo antitumor saponins Among the most promising compounds giving a potential activity in vivo we can found methyl protoneodioscin (49) which is in the list for the
215
20 - S - Protopanaxadiol R 2 gb-
R,
glc
G-Rb2
glc
G-Rc G-Rh2
gic gb-
G-Rg3
g l c — glo
H
G-RS3
6A=glc — 2 g k>
H
2 glc2 glo
6 ara — g b 0
araf—gbH
M1 =IH901
H
R
2 rtia — glc2 glc — gb-
G-Re G-Rf
glcH H
glo
G-Rg,
gic-
glo
H
G-Rh,
glo
gfcglC
OR
gb — 6 gb-
3-Rb,
G-F2 G-Rd
20 - S - Protopanaxatri d
gic-
gic-
rtia —2gic-
H
G-Rg2
glcHO
COOR1 tedc—
gb-O R glc — glcA-
G-Ro
oleanoSc acid H
R1 gb H
OH
u/-.'
-v 4r
glc —
- OH
gb-O
nctoglnsenaside G
glc —
— '''
notoginsenoside H
'
O-glc^-xyl 6 6 x y l — gb — g b - O HO
2 2 xyl — glc — g i c - 0
glc-0 notoginsenoside K
Fig. (2) Stmctureof Panax saponins
notoginsenoside D
216
ma—gb — O
(1 4) galtonioade A OMe
OAc
x/-OR2 OR1
(21) gb-
Rcr 3 R1
R
K
(15)
glc
(E)cinnamoyl
H
(16)
gfc
3,4-d imethoxyclnnamoyi
H
(1^
9b
H
H
(1^
H
3,4-dimethDxybenzoyl
gb
(19)
H
3,4,5-trimethoxybenzoyl
gb
(20)
H
p-methoxybenzcyl
H
217
'-.. 20
-- fl
Ti3
22 /
T16
4 4 S1 = xyl—gb —gal-
- 14
I
|3
fl
1R
xyl 3 4 S2 = xyl—gb—gai-
R*0' H =1 R R1
(22) (23) (24) (25) (26) (27) (28)
OH
iucreastatin chrcmalosideA
R3
R4
corfig.
H,H
S1
25R
H H
H H
=O
S1
25R
-
=0
H H
H H
25R 25R 25R
H
H
=o =o =o
S1 S2 S3 S2
25S
H
H,H
S4
25R
OH O
(29) (30)
R2
9b 4 3 S3 = rha —gb—gb I2 b
4 4 3 S4 = rha — gb—gb — g b gb
2
R1 =H R1=glc-
^ ^
RO*^ R
R
corfig.
H (34)
dosan
(35) gradllin (36)
rha—gc-
H
25R
rha—glo
H
25R
rha—gbj rha
H
25R
glc—rh rha-%
(39)
rh
25S
H
25S
4
H
25R
H
25R
OH
25R
rha H
25R
(40)
g«i—gjc-
m a
4
H
rtia
rha
4
rha -irha— gb2rha
config.
rha
(38)
4
3
R1 3
(37)
2
(32) progenn II (33) .
R
6 1
H
25R
(41)
4
gal rha
218
R
0
-"
6-Fuc
(42)
H
(43) (44) (45)
3-O-Acxyl4-O-Acxylxyl-
,021
OAo AcO-^gal
,5
AcO^-gal
Acc/V
4
(48)
.
R H
protoneodoscm
R
rtia—glcJ^
25S
H
rtia—gjijrtia
25R
protodosan
4
config. 25S
4
rha^—gjc,rtia
CH3 metryiprotoneodbscin
3
rtia — glc—gfc- glo-
R —0*^3
• 14
H (51) methyl protogradllin
CH3
glc—gK j^j
(53)
25R
pseudo protodbsdn
(54a)
dioscoresideA
(54 b)
dicscoresideB
R1
R2
glc—glcrtia rtia—gl^ rtia
H OH
219
COOcJc
COOR,
COOR1 •"R
3
RO'
(62)
R2
R1
R 3
2
glc- rha- ara-
3
4
6
glc- rtia- glc- gfc-
H
H
rtia - g l c - Po-
H
H
rtia - 4 glc- 6glc-
H
H
OH
H
4
(63) (64)
6
4
6
(65)
qu- ara-
(66)
2
glc-%a- ara-
rtia
H
OH
(67)
glc-3rha- 2ara-
rha- 4 qui- 6 glc-
OH
H
(68)
rha- ara-
rtia-4gfc-6gV;-
H
H
H
H
OH
(70)
2
6
xyl- ara- glc-
ttia- gic- glc6
- glc-
lilHAc H
(71) HAc
OH
220
Table 1. Cytotoxic steroid and triterpene saponins (structure) Cell lines Source STEROID SAPONINS cholestane Galtonia HL-60 (candicanoside A, candicans 13) •• " 5(3 cholestane (galtionoside 14)
A,
Activity (IC,n)
Ref
0.032 pM [14] (etoposide 0.025 \iM, methotrexate 0.012 MM) 0.057 \iM [15] etoposide 0.025 \iM, methotrexate 0.012 yM)
"
12xlO"5- 0.014 (iM etoposide 0.025 j i M
[16]
Ornithoga-lum cholestane saundersiae (18-20)
HL-60
[17]
Ajuga salicifolia
Jurkat T
2.5 10-4-1.6.10-2jiM etoposide 2.5.10~2fJ.M adriamycin7.2.10~3fjM methotrexate 0.012 ^M 3^M
HL-60
2.6-6.1 |Xg/ml
[19]
HL60
4.3 Hg/ml etoposide 0.22 |0.g/ml
[20]
cholestane
(15-17)
Polianthes tuberosa
coprostigmastane (ajugasalicioside C) (21) spirostane
[18]
(22-24)
spirostane (25) Fucraeafoetida spirostane (fucreastatin, 26)
Agave americana
Chlorophy turn malayense Camassia leichtlinii Allium porrum
chromaloside (27)
Allium jesdianum
spirostane (29)
HSC-3 (human EC50 carcinoma 2.6-4.0 Jig/ml cells); Ca9-22 (oral squamous) BT549 (breast) A Lu-1, KB, ED 50 1.4-4.8 Mg/ml LNCaP, BC1, Col2
[21]
[22]
spirostane (28)
HSC-2
CC50 0.7-2.2 MM
[23]
spirostane
J774 (monocyte, macrophage), WEHI-164
1.9-5.8 ng/ml
[24]
HL-60
1.5 (Xg/ml; etoposide 0.3 ug/ml
[25]
(29-31)
221 Table 1 contin. Dracaena drago spirostane (32, 42) spirostane Dracaena angustifolia (43- 45) Dioscorea spirostane, furostane panthaica (32,33,34, 35,
1.3; 2.5 ug/ml
[26] [27]
A 375-S2, L- 1.8-8.6 jag/ml 929, HeLa
[28]
HSC-2 (oral LD50 2.0-2.8 ug/ml squamous carcinoma), HGF (human gingival fibroblast) 1.8; 2.1uM, etoposide HL-60 0.37 uM MDA-MB 435, 0.8-7.5 uM HeLa, H-14, HL-60
[29]
53, 54a,54b) Cestrum noctumum
spirostane (36)
Tacca chantrieri Polygo natum zanlanscianens e Triteleia lactea
spirostane
(37,38) spirostane (34)
[30] [31]
Lung, colon, CNS, renal prostate, cancer cells) HL60
GI 50 0.21-0.81uM
HL-60
3.1-3.7 ug/ml
[33]
Most cell lines GI50 0.5- 9uM from leukemia and solic tumors , NCI anticancer drug screen methyl proto- NCI screen GI50 <2uM neodioscin (49) protodioscin (50) NCI screen GI5n <2uM methyl proto- NCI screen KM GI50<2 uM gracillin (51) 12, U251 MDA-MB-231
[34]
spirostane (32)
Triteleia lactea spirostane
[32]
1.8-3.3 ug/ml
(32,35,39) Ruscus aculeatus Dioscorea colletii var. hypoglauca
"
spirostane (46, 47) furostane (protoneo-dioscin) (48)
[35] [36] [37]
222 Table 1. Con tin.
TRITERPENE SAPONINS Aralia dasyphylla
oleanane (55)
KB, HeLa-S3
Pulsatilla chinensis Trevesia palmata
oleanane
HL-60
Acacia concinna
oleanane
1.2, 0.02 ng/ml [38] 5 FU 0.93 ^g/ml (KB), 0.44 ng/ml (HeLa); cytosine arabinoside 0.81 Hg/ml (KB), 0.23 ng/ml (HeLa) 2.3-4.4 (Xg/ml [39]
(56-61) [40]
(69-70)
J774(macrophag 0.06-0.52 uM es) HEK-293 6 MP 0.003-0.017 nM (kidney), WEHI-164 (fibro sarcoma) HT-1080 ED50 0.7-2.82[iM 5 FU ED,n 8|iM
Acacia tenuifolia
oleanane
M 109 (lung)
[42]
Kalopanax pictus
oleanane
Kalopanax pictus
oleanane
Soybean concentrate
oleanane
oleanane
(62-68)
1 ^M
[41]
(71-72) (3,4,73) (3,4,74)
(75-77)
Colon 26, 3LL
[43] EC50 1.1-12.5 ^M (cisplatin 11.3-12 uM) 1.7-1.4 jiM U937, [7] 56.6-110.6 L 1210, HL 60, (cisplatin P 388, HEPG- |iM) 2, SNU-C5 Stomach, breast, prostate
ED50 2.11-4.84 \ig/m\
[44]
Lung GLC4
ll-155^iM
[45]
(Fig. 1) Ginseng and synthetic
dammarane (Fig. 2)
cisplatin IJJM
COLO 320
21-320fiM, cisplatin 3p,M
NCI's in vivo Helllow Fiber Assay in nude mice after evaluation of nontoxicity (maximum tolerated dose, MTD > 600 mg/kg) [35]. In the in vivo experiments, kalopanaxsaponin A (referenced as a-hederin) (3) from
223
Kalopanax pictus (7.5,15 mg/kg, i.p.) apparently increased the life span of mice bearing colon 26 (115, 169 T/C %) and 3LL Lewis lung carcinoma (175, 205 T/C %), as well as cisplatin (3 mg/kg, i.p.) [43]. It was recently reported that a fraction CC-5 (50 mg/kg-400 mg/kg) from the seeds of Nigella sativa caused dose-dependent inhibition of tumor induction and inhibited tumor growth time- and dose-dependently when given before tumor transplantation (i.p. implanted P388 leukemia, s.c. implanted LL/2 (Lewis lung carcinoma) cells). CC-5 at doses of 200 and 400 mg/kg prolonged the life span of P-388 tumor bearing mice by 153% in comparison with 5 FU (184%) [46]. a-hederin (3), isolated from this bioactive fraction was found to inhibit tumor growth more significantly than the alkilating agent, cyclophosphamide (CP) at 20 mg/kg. These results were confirmed in vivo. Namely, after administration to mice with formed tumors, a-hederin ( i.p. for 7 days at doses of 5 and 10 mg/kg) produced significant dose-dependent TIR values (tumor inhibition rate) of 48 % (p<0.05) and 65 % (p<0.01) respectively on days 8 and 50 % (p<0.01) and 71 % (p<0.001), respectively on day 15, compared to 81 % (p<0.01) on day 8 and 42% (p<0.01) on day 15 in the CP-treated group (20 mg/kg) [46]. A study has demonstrated that this molecule stimulates NO release and is able to upregulate iNOS expression through N F - K B transactivation, which may be a mechanism, whereby a-hederin elicits its biological effects [47]. It was observed in the author's group that the combined use of some acylsaponins from Silene fortunei (78,79; 80,81, Fig. 3) and cisplatin increased the intracellular platinum concentration and potentiated the in vitro cytotoxic effect of cisplatin in HT-29 human colon cancer cells [48]. The absence of potentiating activity of deacylated saponins suggested the importance of the p-methoxycinnamoyl group for the activity. Such effect was also observed in vivo with a combination of digitonin and cisplatin during isolated lung perfusion [49].
224
R = S1 ,S2 CO OR2
OCH 3
R1-0
O
CHO
H
R3
(78,79)
OR 2 2 [4 glc — rha —-fuc,OR
gal— glcA-
jenisseenssoside A,B (80, 81) jenisseenssoside C,D
I2
x y l — xyl — rha _3 fucglc
gal (104)
2 * 2! ma — ara — glc-
gb-
2 gaUglcA-
(105)
AcO-6glc- OAc 4 ^ J A ara— ara— xyl— rha -2- fuc2
2
2
2
AcO (106, 107) jenisseenssoside
OH
rha xyl — gbA-
(103)
2 4 rha —^fuc-
gal— glcA-
OH
, , A
gal— glcA-
4
OH
OR ^ 4
ana— ara— xyl — rha — fuc-
OH
Fig. 3 Caryophyllaceae saponins
Multidrug resistance has been a major problem in cancer chemotherapy. A study has shown that ginsenoside Rg3 (Fig. 2) inhibited vinblastine efflux and reversed MDR to doxorubicin, colchicine, vincristine and etoposide in KBV20C cells. Furthermore, Rg3 increased life span in mice implanted with DOX-resistant murine leukemia P388 cells [50]. Apoptosis inducing activity of saponins The cytotoxic activities of saponins may be due to their non-specific detergent effects with changing in membrane architecture, as shown by the aggregation of tumor cells at early stages after saponin treatment. Hovewer, mechanisms other than membrane damage are also involved in the cell death, such as apoptotic process.
225
Apoptosis is a highly organized cell death process characterized by ultrastructural modification (cytoskeletal disruption, cell shrinkage and membrane blebbing), nuclear alteration (internucleosomal DNA cleavage and chromatin condensation), and biochemical changes including activation of proteases. At the molecular level, drug-induced apoptosis involves the mitochondrial release of cytochrome c that combines in the cytosol with the protein APAF-1 in the presence of (adenosine triphosphate) ATP to mediate caspase activation. The release of cytochrome c is regulated by the interaction of proapoptotic proteins (including Bax, Bid) and antiapoptotic proteins including Bcl-2 and Bcl-XL [51]. Since cancer chemotherapeutics were reported to exert part of their pharmacological effect by triggering apoptotic cell death, the search for apoptosis-inducing compounds in tumor cells have become useful for the development of anticancer drugs. There were few reports on the apoptotic activity of saponins, except those on ginsenosides. We will present here recent results obtained with other saponins from Liliaceae, Mimosaceae, Araliaceae, and Caryophyllaceae. Earlier results showed that dioscin (34) isolated from Polygonatum zanlanscianense induced differentiation and apoptosis in HL-60 cells [35]. It was shown later to dose-dependently inhibit the proliferation of HeLa cells with an IC50 of 4 jiM [52]. Quantification of apoptosis by flow cytometry analysis of phosphatidylserine exposure annexin V/propidium iodide (PI) staining, showed that approximatively 70 % of the cells were apoptotic when treated with 2 jiM dioscin. Among them, two thirds were in an early stage of apoptosis and were labelled by annexin V only. This is in contrast to the highest concentration of 8 ^M at which about 90% of the apoptotic cells were in the late stage, so they were stained by both annexin V and PI. The FACS analysis of the cells showed that 36.4% of them were detected in the sub-Gl region after exposure to 5 jxM dioscin for 12 h. The low activity of caspase-8 in Hela cells and the high activity of caspase-9 after treatment with dioscin suggested that it was a potent apoptosis inducer in HeLa cells through activation of the mitochondrial pathway [52]. All these results indicated that HeLa cells underwent apoptosis in a dose- and time- dependent manner. A natural analog, the protodioscin (50) from Trigonella foenumgraecum L. was shown to have a growth inhibitory effect of human leukemia HL-60 cells which results from the induction of apoptosis [53]. The fragmentation of DNA was observed to be both concentration- and time- dependent. Two spirostanol saponins isolated from Tritelia lactea (40, 41) were shown to cause a concentration- and timedependent apoptosis of L1210 cells in the concentration range 0.3 to 10
226
jiM (EC50 = ~ 5 ^M) [54]. Morphological characteristics of apoptosis, including chromatin condensation, cell shrinkage and DNA fragmentation were observed [54]. The apoptotic effects of both compounds were almost equal, suggesting that the OH group at C-17 is not important for the activity. Since another compound having a different oligosaccharidic moiety did not induce apoptosis, the triglycoside part of both spirostanol glycosides plays an important role in this activity [54]. It was shown that the apoptotic effect of saikosaponin d (82) (3xl0"6-10~5M) from Bupleurum falcatum in human CEM lymphocytes may not be mediated by caspase activity but partly by increases in c-myc and p53 mRNA levels and a decrease in bcl-2 mRNA level. Saikosaponin-d (82) at higher concentration (>10"5M) also induced necrosis as demonstrated by annexin V staining [55],
CH 2 OH
R1
OH
(82) saikosaponin d
(102) songarosaporin c
R2
glc— fucglc
Gypenosides, triterpenoid saponins from Gymnostemma pentaphyllum were reported to induce apoptosis in human hepatoma cells [56] through the up-regulation of the apoptotic - inducing proteins (Bax and Bak) and down regulation of Bcl-2 leading to release of mitochondrial cytochrome c and activation of caspase cascade [56]. Avicins D (11) and G (12), acacic acid glycosides having a monoterpenic acid substituent at C-21 were shown to inhibit the growth of several cancer cells, avicin G being more active (IC50 0.12-1.49 (ig/ml, p<0.05) [57]. They were able to induce apoptosis of Jurkat cells (human T-cell leukemia) and MDA-MB-435 breast cancer cell line in a dose-dependent manner on the basis of the Annexin V binding assay [57]. 15-17 % of cells treated with avicin D or G (lug/ml) bound to
227
Annexin V-Fluorescein isothiocyanate (FITC), suggesting early stages of apoptosis. 16 to 37% of these cells demonstrated binding to both Annexin V and propidium iodide, suggesting cell death. Avicin G was 50% more active than avicin D in mediating cell death. Additional work of the authors revealed that avicins induced apoptosis in Jurkat cells by directly perturbing the mitochondria while reducing generation of reactive oxygen species. The inhibition of the phosphatidyl-inositol 3-kinase (PI3K)/Akt pathway was suggested as a part of the mechanism of action [57]. Gleditsioside E (83) from Gleditsia sinensis (Leguminosae), showed significant cytotoxicity against Bel-7402, BCG-823, HeLa, HL-60 and MCF-7 cell lines, using paclitaxel as positive control (IC50 3.0- 8.0 |iM).
xyl— 2 aia— 6 gl<
(83) gleditsioside E
OH
The results of cytometric analysis showed an increase of early apoptotic cells from 3.27 to 59.7 % instead 2.25 to 12.83 % for the middle apoptotic cells and 0.14 to 2.82 % for the late apoptotic cells. Gleditsioside E treatment resulted in a prominent increase of G2/M population in HL-60 cells (48h, Gleditsioside E 15 |aM), and a accumulation of the sub-Gl peak was observed [58]. Securiosides A and B (85, 86, Fig. 4), two triterpene saponins from Securidaca inappendiculata (Polygalaceae) were shown to inhibit macrophage growth through induction of apoptosis only when they were stimulated by macrophage colony stimulating factor (M-CSF) [59]. The 3,4-dimethoxyl group at the Fucose-4 on the 28-glycosyl ester moiety in both molecules was found to be essential for the death-inducing activity. These compounds might offer new perspectives for the treatment of pathological states in which macrophage proliferation occurs such as tumors, inflammation and atherosclerosis [59]. The importance of such cinnamoyl group was also observed in the author's group with jenissenssosides C,D (80,81, Fig. 3), which induced apoptosis in Jurkat cells, since the loss of this group abrogated this activity [60]. Another
228
H3
S1=
S2=
OCH3
OCH3
R1
(85) Securioside A
S3
(86) Secu reside B
S3
Ac-gbH
H
xyl-
H
gal — xyl2
I
ana (110,111)
H
gal — xyl|3
gb (112,113)
S 3 ,S 4
H
4
H
gal — xyl-
api-
I glc gal — xyl-
3
(123) onjisaponinA
S'.S 2
rha-
(124) onjisaponinE
S 5 ,S 6
H
H
gal — xyl-
(125) onjisaponin F
SS,S
H
api-
a r a — xyl-
(126) onjisaponin G S 5 ,S 6
H
api-
xyl-
Fig. 4 Pdygalaceae saponins
structure-activity relationship study showed that glycosylation at the C-3 OH group and esterification at C-28 COOH group of ursolic acid (84) led
229
COOMe glc-O
to methyl ursolate (3-glucoside with decreasing apoptosis and caspase-3 inducing activity in A 431 human epidermoid carcinoma cells [61]. A metabolite of ginseng, protopanaxadiol saponin Ml with antimetastatic property was shown to inhibit the proliferation of B16-BL6 mouse melanoma cells in a time- and dose-dependent manner and to induce apoptotic cell death within 24h at 40jiM. [62]. The induction of apoptosis by Ml (Fig. 2) involved the up-regulation of the CDK-inhibitor p27 as well as the down-regulation of c-Myc and cyclin Dl [62]. Ginsenoside Rh-2 (Fig. 2) was shown to induce apoptotic manifestations in rat C6 gliomal cells, evidenced by changes in morphology, DNA fragmentation, production of ROS and activation of caspase pathway in a Bcl-XL-independent manner [63]. Ginsenoside Rg3 was shown to inhibit the growth of human prostate carcinoma LNCaP cell line through a caspase-3 mediated apoptosis mechanism by the activation of the expression of cyclin-kinase inhibitors, p21 and p27 and arrest of LNCaP cells at the Gl phase [64]. Ginsenosides Rs4 and Rs3 (Fig. 2) were shown to induce apoptosis in SK-HEP-1 cells. This effect was related to significantly elevated protein levels of p53 and p21 and downregulated activity of both cyclins E- and A-dependent kinase resulting in cell cycle arrest at the Gl/S phase [65, 66]. A novel intestinal metabolite IH-901 (20-0-|3-D-glucopyranosyl-20S protopanaxadiol) (Fig. 2) formed after oral administration of Rbl, Rb2, Re (Fig. 2) in rats, dramatically suppresses HL-60 cell growth by inducing programmed cell death through activation of caspase-3, which occurs via mitochondrial cytochrome c release independenly of Bcl-2 modulation [67]. A structure/activity relationship study has shown that the presence of sugars at C-3 or C-6 position of protopanaxadiol and protopanaxatriol such as in ginsenosides Rh2 and Rhl (Fig. 2) reduces the potency to induce apoptosis in cultured human leukemia cell line (THP-1) [68]. II. IMMUNOMODULATORY ACTIVITY Several in vitro and in vivo studies have evaluated the immunomodulatory properties of saponins. Immunomodulators may be divided into two
230
categories, the specific immunomodulators which include the adjuvants in vaccine where they enhance the antibody and cell mediated responses to an antigen while the non specific immunostimulants are given on their own in order to elicit a generalized state of resistance to pathogens or tumors [69]. Suitable systems for in vitro screening of immunostimulants are cultures of human granulocytes, macrophages and lymphocytes which are obtained from donor blood. II. I Saponins as immunostimulants Macrophage activation Macrophages play a significant role in host defense mechanisms. When activated, they inhibited the growth of a wide variety of tumor cells and microorganisms. Nitrite oxide (NO) has been identified as the major effector molecule involved in the destruction of microorganisms and tumor cells by activated macrophages during the non-specific host defense of the immune system. In macrophages, nuclear factor KB ( N F - K B ) in cooperation with other transcription factors has been found to coordinate th expression of genes encoding iNOS. Moreover, NF-KB plays a critical role in the activation of immune cells by up-regulating the expression of many cytokines essential for the immune response. It was shown that glycyrrhizin (87) stimulates macrophage-derived NO production, and is able to upregulate iNOS expression through N F - K B transactivation in murine macrophages. These actions may provide a mechanistic basis for the antitumor properties of glycyrrhizin (87) [70]. Similar results were obtained with ct-hederin (3) [47].
O
2
glcA-glcA-0
X,
„
. (87) glyccyrhizin
Among cycloartane glycosides from Astragalus Turkish species, only astragaloside I (88) was able to stimulate N F - K B expression in macrophages. This implies that they are critical structural features responsible for macrophage activation by saponins [71].
231
Action on the lymphocyte proliferation The immunostimulant activity of saponins was assessed by examining their effect on mitogen-induced lymphocyte proliferation. Lymphocyte proliferation or transformation is a process thereby de novo DNA synthesis takes place in response to a mitogene or any other stimulator (concanavallin A). Two cycloartane - type triterpene saponins (89,90, Fig. 5) from Astragalus peregrinus were shown to stimulate the proliferation of mouse splenocytes at a dose of 0.1 jo,g/ml in presence or absence of Con A [72]. Supporting this conclusion a structure/activity relationship study of the modulation of lymphocyte proliferation has been undertaken with Astragalus saponins (89-94, Fig. 5). Active in the concentration range of 0.01-10 jxM, the cycloastragenol 3-0-glycosides preferentially carrying a substituent in position 2 of the 3-0-linked sugar are the most active compounds in the presence of a growth stimulator [73]. About cycloastragenol- 6-0-xylosides, they are active both in the presence or in the absence of Con A whereas the 6-O-glucosides lack activity in presence of Con A. In the contrary, aquilegiosides C-F (95-98) from Aquilegia vulgaris have been found to suppress the proliferation of lymphocytes in mouse allogeneic mixed lymphocyte reaction with IC50 ranging from
R-0
R
R1
(88) astagabside I
AcO-xyl-
(89) peregrinoside I
OAo rhaJgfc. H
(90) peregrinoside II
rhalgfc.
(91) astraversianine VI
AcO-2xyl- gk>
(92) trigonoside II
H
ara-xyl- xyl3
(93) trigonoside III (94) astraversiarine XV
0 K>gB— o
R<
R2
(95) aquilegbsideC
H
alo-
(96) aguSegtoside D
H
QIC"
*=-
AcO-xyl-
xyl-
rha-xyl-
xyl-
(97) aquilegbside E OH (98) aquilegioside F OH
H
R H R-O-xyl-0
gb-glc6
2
dimetioxycinnamoyl-glc-glcRg.5 Cydoartane-type saponins
232
3.7 x 10"5 to 2.2 x 10~4 M [74]. These results corroborated those already obtained with Cimicifuga cycloartane glycosides (99-101, Fig. 5) [75]. In a structure/activity relationship study, songarosaponin C (102), a saikogenin derivative from Verbascum songaricum was shown to possess the highest immunosuppressive activity (SI = 0.1, c = 10 |ig/ml) and is more active than cyclosporin A in vitro, used as a reference compound (SI = 0.4, c=12 Hg/ml) [76]. The immunosuppressive effects of ginsenosides Rbl, Rb2, Re and Rg (Fig. 2) from Panax ginseng were measured on CD4+ and CD8+ lymphocyte proliferation. In term of inhibitory potency, Rb2 seems to be a main principle that shows potent imunosuppressive effect (IC50 21.8-24 ^iM). This effect may be in part due to the interruption of the IL-2 production induced by mitogenic treatment [77]. The latest research developments from the author's laboratory concern triterpene saponins from Caryophyllaceae, Mimosaseae, and Polygalaceae. They have been tested in an in vitro lymphocyte proliferation test system [78]. In this test the percentage of lymphocyte proliferation was quantified by determination of the formazan from exogenous MTS in lymphocytes. The octaglycoside of gypsogenin (103, Fig. 3) from Acanthophyllum squarrosum showed a moderate concentration-dependent immunomodulatory effect. It displays immunostimulant activity in the concentration range 1 (Xg/ml-100 pg/ml, whereas at higher concentration a marked cytotoxicity was noted (58% at 100 |ig/ml). In the concentration range (10.1 jig/ml), the immunostimulant activity of the pentaglycoside (104, Fig. 3) was low compared with the octaglycoside (103) and it was not cytotoxic at 100 jig/ml [78]. New acylated quillaic acid saponins from Silene fortunei were tested in an in vitro lymphocyte proliferation assay (105-107). The proliferation was measured by 3H-thymidine incorporation in Jurkat T cells [60]. Jenisseenssosides E and F (106, 107, Fig. 3) showed an immunomodulatory effect dependent of the concentration. In the concentration range lO'MO"1 |iM, jenisseenssosides E, F stimulate weakly Jurkat cells proliferation with SI = 1.36 and from 1 jxM an inhibition of the proliferation was observed [60]. Jenisseenssosides C,D (80,81, Fig. 3) showed a significant inhibition of Jurkat cells proliferation from 5 \iM and a proliferation activity in the concentration range lO^-lO'pM with SI = 1.44. The p-methoxycinnamoyl group linked to the fucosyl residue was crucial for this immunomodulatory activity as the deacylated derivative showed a lower proliferative activity and was found to be not cytotoxic from 5 pM to Jurkat cells [60]. In our in vitro lymphocyte proliferation assay, glycosides of acacic acid acylated by an o-hydroxybenzoyl unit isolated
233
29
. 30
HO Me
HO HO
(108) NHAc (109) glc-
from Albizia adianthifolia (108,109) were found to exhibit a dosedependent immunomodulatory effect in the concentration range 10~2-10 jxM,whereas the prosapogenin of 108 showed a lymphoproliferative activity in the same concentration range and no cytotoxic effect at all the tested concentrations [79]. This underlies the importance of the esterification at C21 and C-28. In the same assay on Jurkat cells, a fraction containing two pairs of presenegenin glycosides acylated with cis/trans- pmethoxycinnamic and cis/trans- dimethoxycinnamic acids showed a concentration-dependent immunomodulatory effect (110/lll;112/113, Fig. 4). This effect was abrogated with the prosapogenin (tenuifolin = presenegenin-3-O-P-D-glucopyranoside underlying the importance of the acyl-oligosaccharide moiety in the activity [80]. II.2. Saponins as immunoadjuvant An immunological adjuvant may be defined as any substance that, when incorporated to a vaccine formulation, acts generally to accelarate, prolong or enhance the quality of specific immune responses to vaccine antigens [81]. An adjuvant suitable for use in human and/or veterinary vaccine should lack toxicity, stimulate strong and lasting humoral and T-cell immunity, induce a good memory response, lack immunogenicity and have a good stability. Adjuvant can elicit either a Thl or a Th2 type immune response [82]. A Thl immune response, mediated by Thl cells is characterized by production of cytokines such as interleukine-2 (IL-2), tumor necrosis factor-^ (TNF-(3) and interferon-y (IFN-y) and an enhanced production of IgG2a, IgG2b and IgG3 in mice. A Thl immune response is necessary for the production of cytotoxic lymphocytes (CTL). A Th-2 response mediated by Th-2 helper cells is characterized by the production
234
of the cytokines IL-4, IL-5 and IL-10 and an enhanced production of IgGl and IgA. A Th-1 response is required for protective immunity against intracellular infectious agents such as certain viruses, bacteria and protozoa and presumably malignant cells. The only licensed adjuvants for use in human vaccines are aluminium based-salts which elicit only a Th-2 response which is ineffective against intracellular pathogens and malignant cells. Hovewer hundered of natural and synthetic compounds were characterized with adjuvant activity, some of them being in preclinical evaluation. A saponin fraction consisting of several acylated bisdesmosides quillaic acid glycosides obtained from the bark of Quillaja saponaria Molina tree (Quillajasaponins) was shown to present adjuvant activity, stimulating a Th-1 response and producing antigen-specific CTL [69]. The compounds present in the mixture differ mainly in their oligosaccharide composition. The presence of an aldehyde group at C-4 of the aglycone of the saponins plays a key role in stimulating Th-1 type immunity since modified saponins at C-4 lack adjuvanticity. It might form an imine group (Schiff base) with aminogroups on certain T-cell surface receptors to provide a co-stimulatory signal that is transducted by the receptor [83]. Furthermore the oligosaccharide chains apparently mediate the targeting to APCs via the selectins localized on the surface of these cells. Although quillajasaponins induced adjuvanticity, they presented serious toxicity and instability. When these compounds are deacylated after mild alkaline hydrolysis (liberating the lipophilic monoterpene ester groups from the fucosyl residue) there is still a stimulation of antibody response, a significant decrease of toxicity and a concommitant loss in the ability to stimulate a lymphoproliferative response and a CTL production [84]. QS-21 (114, Fig. 6) isolated from this saponin mixture was identified as an acylated saponin at the 4-OH position of fucose with two linked 3,5dihydroxy-6-methyloctanoic acids. It showed a potent adjuvant activity against a wide variety of antigens with low toxicity in preclinical studies in mice, guinea pigs, monkeys and baboons [85, 86]. This molecule stimulates antibody and cytotoxic T lymphocyte responses when added to systemic vaccine formulations given either by the subcutaneous or intramuscular routes. It was also reported that QS-21 (114, Fig. 6) can induce both systemic and mucosal immunity to a nasally administered DNA vaccine, suggesting that it can exert adjuvant activity vhen administered by non parenteral routes [87]. Namely, it was shown that oral administration of QS21 (114, Fig. 6) enhances immunity to co-adminitered antigens such as tetanus toxoid and that different doses of QS-21 (114, Fig. 6) lead to distinct patterns of cytokine and serum antibody responses. This dose-
235
dependent mucosal adjuvanticity requires early IL-4 help [88]. The observation that orally administered QS-21 enhanced immune responses clearly suggested that QS-21 resists degradation in intestinal environment and can be incorporated into oral vaccine formulations [88]. Extensively studied in animal model, QS-21 has been evaluated in a large number of parenterally administered vaccines in Phase I and Phase II human clinical trials including cancer immunotherapeutics, HIV recombinant envelope, and malarial antigens [85]. To date it has been tested in more than 2600 individuals in 60 clinical trials at doses ranging from 50 to 100 \ig [85,86]. Gangliosides GM2, GD2, and GD3 are expressed on the cell surface of human malignant melanomas, GD3 being the most abundant [89]. These antigens are considered to be potential targets for treatment with vaccines in the field of immunotherapy. It was
o
R3 (114)
QS21
H
r
OH O ara(f)
(115)
QS7
rha-
Ac
(116)
DS1
H
H
OH
(117)
RDS1
H
H
NH - CH2-(CH2)10 -C H,
(118)
DCC adduct
H
Fig. 6 Natural and semi synthetic qii llajasaponhs
OH
236
shown that antibodies against GM2 obtained by vaccination with GM2 covalently conjugated to keyhole limpet hemocyanin conjugate (GM2KLH) and the immunological adjuvant QS21 has been associated with an unexpectably favorable disease free and survival [89]. This vaccine is now in phase III clinical trials. The superior immunogenicity of the KLH conjugate vaccine plus QS-21 has been also demonstrated in melanoma patients immunized with GD3-KLH [89]. Another fuc-GMl-KLH conjugate plus 10 \ig QS-21 vaccine was found to be optimal to induce production of antibodies against human small - cell lung cancer cells in mice, suggesting the initiation of a clinical trial in patients with small-cell lung cancer [90]. Other Phase I trial studies have shown the superiority of QS21 as immunological adjuvant for vaccination with tyrosinase 370 D peptide or gp 100 peptide in HLA A0201 melanoma patients [91,92]. A study undertaken in HIV-1 uninfected persons has shown that QS-21 may provide a means to reduce the dose of the recombinant soluble gpl20 HIV1 protein immunogen. Moderate to severe pains were observed in majority of volunteers [93]. Three double blind, randomized Phase I clinical trials in healthy adults with various QS-21 containing formulations have shown that it was possible to improve the acceptability of QS-21 through reformulations with certain excipients [94]. Other saponins from Quillaja saponaria are known to have adjuvant activity for the stimulation of antibody responses. A saponin QS-7 (115, Fig. 6) is of particular interest [95]. This saponin is more hydrophilic than QS-21 (114) having a shorter acyl chain substitution than QS-21 and was shown to induce at a dose of 40 (ig a cell-mediated immune response to HIV-lgpl20 and OVA in mice (similar to that induced by a 5-10 jig dose of QS-21). In a continuing search for adjuvants with enhanced adjuvanticity and low toxicity, some authors have prepared new chemical semi-synthetic saponins and had compared their adjuvant properties with original saponins. The deacylsaponins which are more hydrophilic and less adjuvant active than the native saponins offer a practical source of starting materials for the production of semi-synthetic adjuvants. The incorporation of a C-12 alkyl chain through a stable amide bond at the carboxyl group of the glucuronic acid residue of the deacylated saponins yielded a family of saponin analogs referred to as GPI-0100 [82,96]. As example, according to the procedure of Marciani et al., 2000 [82], coupling of the aliphatic amine, dodecylamine with the deacylated quillajasaponin's glucuronic acid, in presence of dicyclohexylcarbodiimide/N-hydroxysuccinimide (DCC/NHS) as catalyzator produces the stable GPI-0100 mixture that stimulates Thl
237
immunity as well as CTL production. This analog's stability is a consequence of the resistance of the dodecylamide moiety to the hydrolysis. Furthermore, GPI-OlOO was shown to be at least 20 times less lethal than quillajasaponins in mice. It appears to be devoid of toxicity in mice at a dose up to 1 mg. As mechanism of action, it was postulated that an antigen/saponin analog complex may interact with the membrane lipid bilayer to form a transient opening for delivery of the antigen inside the cell. In contrast to GPI-OlOO, saponin amide derivatives containing longer and more lipophilic alkyl side chains than dodecyl stimulate an IgG profile similar to that of the deacylated saponins [96]. It was reported in a study about the development of novel synthetic multivalent vaccines containing different tumor-associated carbohydrate antigens that the immune enhancer GPI-OlOO stimulated higher antibody titers than the adjuvant QS-21 [97]. GPI-OlOO less toxic than QS-21 is currently in a Phase I trial for the treatment of prostate cancer. GPI-OlOO has been developed and licensed to several companies [99]. Liu et ah, 2002 [98] have prepared semi-synthetic derivatives of saponins allowing a structure/activity relationship study of this series of adjuvants. The adjuvant activity of DS-1 (116, deacylated QS-21, Fig. 6), RDS-1 (117, Fig. 6) (dodecylamide at the position C-6 of glucuronic acid of DS-1) and GPI-OlOO were evaluated in mice over a wider doses range [98]. This comparative study showed that amidation of glucuronic acid (RDS-1 or GPI-OlOO) does not substantially improve the diminished adjuvant activities (antibody and CTL responses) of the deacylated compounds (DS-1 or crude deacylated quillajasaponins) and that QS-21 is a better adjuvant than RDS-1 and GPI-OlOO, especially for stimulation of IgG2a response [98]. Significant differences between both chemical procedures used to prepare GPI-OlOO [82, 98] might explain the highly divergent immune stimulatory properties of this mixture [99]. Some DCC intermediates (118, Fig. 6) could be present in the preparation and could explain the poor T-cell immune response stimulated by the product [99]. Such affirmation was also the subject of controversy since GPI-OlOO mixture was shown to have a very similar HPLC profile in both cases [100]. Furthermore, pure HPLCRDS1 synthetized from the pure DS1 obtained from the pure QS 21 probably doesn't contain any byproducts as ascertained by NMR and MS spectra [100]. To try to elucidate this controversy, the reverse phase lowpressure liquid chromatography (RP-LPLC) fractionation of the GPI-OlOO derivatives has been made yielding two fractions RP18-1 and RP18-2 which were characterized by LC/MS [106]. RP18-1 which contains DS saponin adducts of N-dicyclohexylurea while RP18-2 fraction contained only the
238
dodecylamide derivatives of DS saponins. Since all the Th-1 adjuvanticity (IgG2a, IFN-Y, 11-2, CTL) resides in this fraction, the term GPI-0100 has been proposed to be applied to this fraction [106]. If the saponins from Quillaja saponaria (Rosaceae) and their anologs have been extensively studied as immunoadjuvants, few saponins from other sources have been reported to possess this property. In the following we will summarize results on studies of adjuvant saponins from Rhamnaceae, Araliaceae, Polygalaceae, and Fabaceae. The dammarane-type triterpene glycosides, jujubosides A (119), B (120), C (121) and protojujuboside A (122) from Zizyphus jujuba var spinosa were found to show potent immunological adjuvant activity on the base of the serum anti-OVA antibody levels in OVA - immunized mice [101]. Jujubosides A (119), Bl (123) and protojujuboside A (122) were most equivalent to QS-21 used as a positive control and jujuboside C (121) was found to show an exceptionally potent effect. 21
'''-. IS' 18 T 13H
T 27
-16
15 \ %
RO
3
/
•
b '*
6
(119) jujuboside A
(120) jujuboside B
R gte- 6 gbI2 glc
glc - 3 ata|2
glc (121) jujuboside C (123) jujuboside B1
ttia
6
glc- gb
(122) protojujuboside A
glc-glcara|2 I2 xyl rtia
I2 rha 3
I2 xyl rha glc - 3 ara|2 ale
I2 p-D-fuc
These results showed the importance of the nature of the 3-glycosidic moiety in the molecules. Other dammarane-type triterpene-glycosides from Panax notoginseng notoginsenosides -D, -G, -H, and -K (Fig. 2) were found to increase the serum IgG levels in chiken ovalbumin (OVA)immunized mice [102]. The two saponin fractions PS-1 and PS-2 of Polygala senega were shown to increase specific antibody level to the antigens, both in mice immunized with OVA and in hens immunized with rotavirus [103]. In mice, there was a preferential increase of the IgG2a subclass. The in vitro production of IL-2 and IFN-y by lymphocytes in
239
response to OVA antigen was also enhanced. Since the saponins were found less toxic at the same dose than their conterpart Quil A, these saponin fractions might represent a promising source of adjuvants and needed further structural requirements. A study showed that onjisaponins A, E, F, and G (124-126, Fig. 4) (10 \xg each) from Polygala tenuifolia (Polygalaceae) have mucosal adjuvant activities by intranasal inoculation in mice with influenza HA vaccine and diphteria-pertussis-tetanus (DPT) vaccine by inducing antigen-specific IgA antibodies in nasal washes [104]. All onjisaponins showed no hemolytic activity up to 100 jig/ml suggesting that they may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines. The presence of a carboxyl group at C-23 instead of an aldehyde group can be just as effective for inducing adjuvant activity. A correlation between adjuvant activity and amphipathic structure of saponin was established by comparison the antibody response against chicken ovalbumin (OVA) in mice and hydrophile-lipophile balance (HLB) of structurally analogs of soyasaponins [105]. Soyasaponins (1, 127, 77, 128, 129, Fig. 1) bearing sugar chains showed adjuvanticity stimulating anti-OVA total IgG and IgGl antibody responses, while their corresponding aglycones soyasapogenols A and B (130, 131, Fig. 1), did not. These results suggested that the sugar side chain is essential for the adjuvant activity of soyasaponins. To quantify the structure/function relationship in amphipathic soyasaponins, the ratio of hydrophilic sugar side chain to lipophilic aglycon was converted to an HLB value. Among bisdesmosidic soyasaponins, soyasaponin Al (132) (HLB: 26.9) with a long sugar side chain induced stronger total-IgG and IgGl antibody responses than soyasaponin A2 (133) (HLB: 21.4). In the case of monodesmosidic soyasaponins the following order in term of total IgG and IgGl antibody response was dehydrosoyasaponin I (134) > soyasaponin I (1) > soyasaponin II (127) > soyasaponin III (77). The adjuvant activity increased with the HLB value. This finding supports a relationship beetween adjuvant activity and HLB value not only for soyasaponins but also for many other saponins, for instance, Gypsophila saponins, saponaroside A from Saponaria, lablaboside F and onjisaponins from Polygala tenuifolia [105]. CONCLUSION Cancer-related and immunomodulating activities are among the most studied pharmacological properties of saponins in the last years. Several purified saponins extracted from plants were shown to be cytotoxic against a large panel of cancer cells and the efficiency of the antitumor compounds
240
seems to be related to the propensity of tumor cells to respond to these compounds by apoptosis. Spirostanol glycosides from Liliaceae, saikosaponins from Bupleurum falcatum, acacic acid glycosides from Acacia victoriae, quillaic acid glycosides from Silene species and several ginsenosides from Panax ginseng were shown to induce many apoptotic manifestations in cancer cells. Only a few compounds, such as oc-hederin, have shown antitumor properties in mice transplanted with colon 26 and 3LL lung carcinoma cells. The immunomodulatory potential of saponins was evaluated by using in vitro lymphocyte proliferation assay and macrophage phagocytosis assays. The cycloartane glycosides from Astragalus species, the quillaic acid glycosides from Acanthophyllum squarrosum, the saponins from Albizia adianthifolia and those of Polygala arenaria were found to exhibit dose-dependent immunomodulatory effect in Jurkat cells in the concentration range of 102-10 ^iM. Some structureactivity relationship study showed that this effect was abrogated after deacylation of these compounds, underlying the importance of acylation in the activity. The immunoadjuvant activity of QS 21, a quillajasaponin received a great attention since it was shown to be active in clinical trials such as in malaria, HIV, or melanoma vaccines. It has the unique capacity to stimulate a Thl immune response as well as the production of cytotoxic T lymphocytes against exogenous antigens. To allow a structure/activity relationship study, some semi-synthetic analogs were prepared and the GPI0100 (a deacylated mixture of quillajasaponins which has been coupled with a C-12 alkyl chain through an amide function at the C-6 of glucuronic acid) was shown to be more stable, less toxic than QS-21 and to present an equal or better adjuvant activity than QS 21, depending of the mode of preparation. To understand the mechanism of action of saponins, some hypotheses have been formulated. They concerns membrane changes which can in vitro and in vivo modify the cellular functions of the immune system. The work on Quillajasaponins and their analogs showed that those saponins with carbonyl groups have the capacity to form imines (Schiff bases) leading to T-cell activation and are capable of acting as adjuvants, but the precise mechanism involved in the adjuvanticity still remain undefined. REFERENCES [ 1] [ 2]
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Marciani, D.J.; Press, J.B.; Reynolds, R.C.; Pathak, A.K.; Pathak, V.; Gundy, L.E.; Farmer, J.T.; Koratich, M.S.; May, R.D. Vaccine 2000,18,3141-3151. Press, J.B.; Reynolds, R.C., May, R.D.; Marciani, D.J. in Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed., Elsevier Science: Amsterdam, 2000, 24,131-174. Marciani, D.J.; Pathak, A.K.; Reynolds, R.C.; Seitz, L.; May, R.D. Int. Immunopharmacol. 2001, 1, 813-818. Kensil C.R. Methods Mol. Med. 2000, 42: 259-271 Kensil, C.R.; Kammer, R. Expert Opin. Invest. Drugs 1998, 7: 1475-1482. Sasaki, S.; Sumino, K; Hamajima, K.; Fukushima, J.; Ishii, N.; Kawamato, S.; Mohri, H.; Kensil, C.R.; Okuda, K. J. Virol. 1998, 72:4931-4939. Bokaya, P.N.; Marinaro, M.; Jackson, R.J.; van Ginkel, F.W.; Cormet-Bokaya, E.; Kirk, K.L.; Kensil, C.R.; McGhee, J.R. /. Immunol 2001, 166, 2283-2290. Ragupathi, G.; Meyers, M.; Adluri, S.; Howard, L.; Musselli, C ; Livingston, P.O. Int. J. Cancer 2000, 85, 659-666. Capello, S.; Liu, N.X.; Musselli, C ; Brezicka, F.T.; Livingston, P.O.; Ragupathi, G. Cancer Immunol. Immunother. 1999, 48, 483-492. Schaed, S.G.; Klimek, V.M.; Panageas, K.S.; Musselli, L.B.; Hwu, W.J.; Livingston, P.O.; Williams, L.; Lewis, J.J.; Houghton, A.N.; Chapman, P.B. Clin. Cancer Res. 2002, 8, 967-972. Slingluff, C.I.; Yamshcikov, G.; Neese, P.; Galavotti, H.; Eastham, S.; Engelhard, V.H.; Kittlesen, D.; Deacon, D.; Hibitts, S. Grosh, W.W. etal. Clin. Cancer Res. 2001, 7, 3012-3024. Evans, TG; McElrath, M.J.; Matthews, T.; Montefiori, D.; Weinhold, K.; Wolff, M.; Keefer, M.C.; Kallas, E.G. et al. Vaccine 2001, 19,2080-2091. Waite, D.C.; Jacobson, E.W.; Ennis, F.A.; Edelman, R.; White, B.; Kammer, R.; Anderson, C ; Kensil, C.R. Vaccine 2001, 19, 39573967. Kensil, C.R.; Wu, J.Y.; Anderson, C.A.; Wheeler, D.A.; Amsden, J. Dev. Biol. Stand. 1998, 92, 41-47. Marciani, D.J. US Patent 5,977,081,1999. Ragupathi, G.; Coltart, D.M.; Williams, L.J.; Koide, F.; Kagan, E.; Allen, J.; Harris, C ; Glunz, P.W.; Livingston, P.O.; Danishefsky, S.J. PNAS 2002, 99, 13999-13704.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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CHEMICAL AND BIOLOGICAL ASPECTS OF IRIDOID BEARING PLANTS OF TEMPERATE REGION NEERAJ KUMAR, BIKRAM SINGH, V. K. KAUL, P. S. AHUJA Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India INTRODUCTION Naturally occurring iridoids and their glycosides are widely distributed in plants and serve as important taxonomic markers. The iridoids were named after ants of the genus Iridomirmex, from which iridodial, iridomyrmecin, and related compounds were isolated and found to be involved in the defense mechanisms of these insects. Similar structures also exist in plants for example; nepetalactone from Nepeta cataria L. (Lamiaceae), or teucriumlactone C from Teuchum marum L. have marked properties (the effects of the former on cats earned it some evocative names; catnip, Katzenmelisse, herbe-aux-chats). Iridoids are monoterpene compounds characterized by a cyclopenta [c] pyranoid skeleton, also known as the iridane skeleton (cis-2oxabicyclo-[4, 3, 0]-nonane). These are also found to occur in a variety of animal species. In a broad sense, it is acceptable to include in this group the secoiridoids, which arise from the latter by cleavage of the 7, 8 bond of the cyclopentane ring. This group (of about 500 known structures) chiefly comprises iridoid glycosides (>300), secoiridoid glycosides (>100), and non glycosidic compounds (>100). The group is biosynthetically homogeneous, and is represented by the number of orders and plant families within the dicotyledons. They are elaborated preferentially by gamopetalous plants e.g. Dispsacales, Gentianales, Lamiales, Scrophulariales, which makes them interesting chemotaxonomic markers [1,2].
248
STRUCTURE OF IRIDOIDS Generally iridoids are divided in two categories, iridoids and secoiridoids and these mostly exist as glycosides. Iridoids are also found in aldehydes, alcohols (iridodial (1) iridodiol (2)), alcoholic esters (valepotriates), lactones (irridomyrmecin (3), nepetalactone (4)) and alkaloids (actinidine (5), skytanthine (6)) forms. Secoiridoids on the other hand are formed by cleavage of cyclopentane ring of iridoid.
Secoiridoid carbon skeleton
Iridoid carbon skeleton
.CH,
COOH COOH COOH
COOCH,
9 R=H 10R = OH 11
Secoiridoids are also known to exist as alkaloid glycosides formed by the addition of tryptamine or phenyl ethylamine moieties. They are further divided into following groups. - bearing vinyl group at C9 e.g. sweroside (7), gentiopicroside (8)
249
- bearing ethylidene or hydroxyl ethylidene group at C9 e.g. oleoside (9), 10-hydroxy oleoside (10) - alkaloidal glycosides formed by the condensation of secologanin with tryptamine, tryptophan or phenylethylamine to form e.g. Ipecoside (11), strictosidin (12) - and hydrangenosides formed by condensation of secologanin with p-hydroxy-cinnamic acid and acetic acid e.g. hydrangenoside A (13), hydrangenoside E (14). Iridoids generally have ten carbon atoms. If C-ll is present, it is considered part of a carbomethoxyl group (loganin (15), geniposide (16)) or part of a carboxylic acid group (monotropein (17); in rarer cases, this group is replaced by a hydroxymethyl group (Valerianaceae, Caprifoliaceae), or by an aldehyde or methyl group (lamioside (18)).
HO.
y ^
r
II
H ..OH
COOCH3
In certain cases, the C-ll is absent (aucubin (19), catalpol (20), harpagoside (21)). The pyran ring is only exceptionally open (for example, in the case of iridodialogentiobioside (22) and of nepetariaside (23), the precursor of nepetalactone). The majority of iridoid glycosides in a broad sense, of the term are glucosides, with the glycosidic linkage established between the hydroxyl group on the anomeric carbon of D-glucose and the hydroxyl in the 1 position of the aglycone. A small number of structures are now known in which the sugar portion of the molecule is an oligosaccharide (e.g. rehmaniosides). There are multiple structural variations. The methyl group which is normally at C-8 can be more or less oxidized; examples include a hydroxymethyl group (aucubin (19), monotropein (17)) and epoxides (valtrate (24), deutzioside (25)). There may be an unsaturation at C-7(8) (geniposide (16), aucubin (19)), which may become a center of
250
oxidation (catalpol (20)) or hydration (lamioside (18)). Note the possible oxidation of C-6 (aucubin (19), verbenalin (26), harpagoside (21)) and the potential unsaturation at C-6(7) (monotropein (17)) [1, 2]. COOCH,
COOCH 3
-Glc HO
16
HO.
HO O-p-gentiobiosyl
COOCH3
OGlc
COOH OGlc OGlc
26
BIOSYNTHETIC ORIGIN The incorporation of labelled mevalonic acid (MVA), as well as labelled geraniol derivatives, into iridoid-type structures and indole alkaloids, demonstrates the terpenoid character of these metabolites. Several mechanisms have been proposed, such as the one demonstrated-involving the cyclization of the dialdehyde resulting from the oxidation of 8hydroxygeraniol to iridodial (or to 8-epiiridodial (27)). The oxidation and glucosylation of iridodial leads to the formation of loganin (28), the immediate precursor of most iridoids. The same process applies to 8epiiridodial to lead via 8-epiloganin, antirrhinoside (29), as well as to aucubin (19) and gardenoside (30).
251
Loganin is the intermediate that undergoes the ring opening reaction which leads to the formation of secoiridoids. This step occurs by a mechanism that remains to be elucidated and affords secologanin (31) as the precursor of all of the secoiridoids and consequently, of the indole alkaloids that incorporate this pattern [1, 2, 3]. General biosynthesis of iridoids is given in fig. 1. OOCH,
COOCH,
OGlc
8-Hydroxygeraniol OGIc
Monoteipene-indole alkaloids
Fig. (1). General biosynthesis of iridoids
EXTRACTION AND CHARACTERISATION Extraction of these glycosides is particularly delicate due to their great instability. This instability also explains the darkening that takes place soon after plant collection in any species containing iridoids. In addition, it explains the name of pseudoindican or chromogenic glycoside that used to be applied to some of these compounds. Extraction is achieved with polar solvents (alcohols of various concentrations) and frequently, an initial separation is obtained by redissolving the extraction residue in water, then re-extracting this with immiscible solvents of increasing polarity. The fractionation is generally done by chromatography on alumina, on charcoal (with a risk of irreversible adsorption), on porous polymers (e.g., XAD-2) with polar
252
eluents and more by reverse phase HPLC. The purification is achieved using classical procedures (TLC, HPLC). BIOLOGICAL AND PHARMACOLOGICAL PROPERTIES Numerous iridoids are involved in plant-animal interactions e.g. the defensive function of this type of compounds in ants. From the pharmacological standpoint, the applications of this class of compounds are rather limited. Some iridoids have anti-inflammatory activity, which is weak by the oral route and stronger by topical application e.g. 1 mg of aucubin, verbenalin, or loganin have an activity almost similar to that of 0.5 mg of indomethacin on the TPA-induced mouse ear edema. Some are ingredients in various forms of allopathic medications (valerian), others are typically, phytotherapeutic products (devil's claw, olive tree). Others receive attention for their non-pharmaceutical applications (yellow gentian). The hepatoprotective effects of picrosides I and II from kutkin, the crude active fraction in Picrorhiza kurroa is well known and documented. MAJOR IRIDOIDS CONTAINING DRUGS Picrorhiza kurroa Royle Ex Benth (Scrophulariaceae) Picrorhiza Royle is a genus with two species, P. kurroa and P. scrophulariflora endemic to the Himalayan region of Pakistan, Nepal, India and China. In India, P. kurroa is distributed from Kashmir in west and Arunachal Pradesh in east within an altitudes range of 2,700-5,000 m. Vernacular and common name for trade is Kutki. The unregulated overharvesting has threatened the status to near extinction [4]. Root extracts of P. kurroa have been used to treat liver disorders, caused by the ever increasing environmental pollution, exposure to industrial toxicants, food adulteration, malnutrition, injudicious use of drugs, excessive consumption of alcohol and certain infections. Such disorders pose a major challenge in the health care programmes of any country. Presently, however, no effective therapy is available for the treatment of various liver ailments [5, 6]. PicrolivR (also referred as kutkin), is a standardised bioactive fraction of P. kurroa with proven hepatoprotective, choleretic, anticholestatic,
253
anti-hepatitis B virus and immunostimulant activities [7-12]. The hepatoprotective activity of picroliv has been evaluated against hepatic damage induced by various agents such as galactosamine, paracetamol, thioacetamide, carbon tetrachloride, lanthanum chloride, monocrotaline, ethyl alcohol, aflatoxin, Amanita phalloides toxin and cycloheximide in rats. The LD50 of picroliv by i.p. route in mice was found to be 2030 mg/kg. By the oral route it was 2500 mg/kg in both mice and rats [6, 7, 10,13,14]. In sub-acute toxicity study of over 90 days, picroliv was found to be safe in rats and monkeys [6, 15-18]. Of the minor components, apocynin has been shown to inhibit neurophil oxidative burst in addition to being a powerful anti-inflammatory agent, and the curcubitacins to be highly cytotoxic. P. kurroa contains iridoid glycosides such as picroside-I (32), picroside-II (33), picroside-III (34), picroside-IV (35), kutkoside (36), minecoside (37), veronicoside (38), 6-O-t-ferulloyl catalpol (39), 6-O-cferulloyl catalpol (40), pikuroside (41), cucurbitacin glycosides and phenolic compounds [18,19-30] and very recently a new iridoid glycoside picroside-V (42) has been reported [31]. Picroliv is a mixture of 60% of two iridoid glycosides, picroside-I (6-O-trans-cinnamoylcatalpol) and kutkoside (10-O-vanilloylcatalpol) present in a ratio of 1:1.5, along with several other uncharacterised glycosides as minor constituents [6]. Picroside-I and II were also determined as the major constituents in the commercial samples [25] and in the capsules containing 50% of P. kurroa root powder [8].
H3C0,
254
R,0
32 33 34 35 36
H vanilloyl
H H
Cinnamoyl H
H
H
t-Fenilloyl
H
H
p-OH-Cinnamoyl
H
Vanilloyl
H
t-Isofemlloyl
H
H
38
Benzoyl
H
H
39
t-Ferulloyl
H
H
40
c-Ferulloyl
H
H
37
OH
CH,0
Vanilloyl
CH3O
t-lsofenilloyl
255
Valeriana (Valerianaceae) The genus Valeriana contains about 250 species in the world. Majority of representatives of this genus are distributed over the temperate regions. The three most important species that play a role in herbal medicine are V. offwinalis L., V. jatamansi Jones (synonym V. wallichii DC.) and V. edulis Nutt. ex Torr. and Gray ssp. procera H.B.K. The subterranean parts of three valerian species, namely Valeriana offwinalis L., V. jatamansi Jones and V. edulis Nutt. ex Torr., are used as mild sedatives, but show large difference with regard to their constituents. Consequently, phytomedicines prepared from these species are characterized by different chemical compositions. It is still not clear as to which constituents are responsible for the sedative action, but valepotriates, valerenic acid and their derivatives are generally considered to contribute to it. Valerenic acid has spasmolytic and muscle relaxant effects and inhibits the breakdown of gamma amino butyric acid (GABA) in the central nervous system (CNS). In addition, the essential oil may also play a role in the biological activity of valerian. Quality assurance and quality control of the crude drug and its preparations should therefore be based on these major groups of secondary metabolites [32-36]. Valeriana jatamansi commonly known as Mushkbala grows in temperate zone of western Himalaya at altitudes from 1,300-3,300 m and in Afghanistan and Pakistan. It is a perennial herb used in Ayurvedic and Unani systems of medicine. Roots and rhizomes of V. jatamansi are used for the preparation of phytomedicines with mild sedative action [37-39] used in perfumery and tobacco flavouring industry for its musky, woody and balsamic notes. Essential oil and the resinoid are used for flavour and fragrance purposes. Its essential oil is reported to have antibacterial and antifungal activities [40]. Having wide application in perfumery & phytomedicines, it has been the subject of numerous investigations. The presence of chatinine and valerine (alkaloids), linarin (flavonoid), aliphatic acids, steroids, phenols, tannins, saponins, sugars, valepotriates and a naphthoic acid [41, 42] have been reported from the plant. In view of the increasing demand of this plant by pharmaceutical industries, it is mostly collected from wild sources due to which this plant has attained a threatened status and is listed in the endangered category. Cultivation of valerian was attempted in India and Germany [39, 43]. Valerian species growing in temperate climate contain the iridoids (valepotriates) and their
256
degraded products (baldrinals) along with the volatile compounds mainly valeric, isovaleric and hydroxyvaleric acids [43]. In the late 1960s, Thies [33] and co-workers isolated a novel group of natural products from subterranean parts of V. jatamansi, and called these compounds valepotriates. Valepotriates are triesters of polyalcohols with an iridoid structure possessing an epoxy group (valerianaepoxy-triesters). Differences are found in the number of hydroxyl groups, the type of ester groups, and the degree of saturation. As a result of dehydration or esterification of various alcohol functions, species-dependent mixtures of valepotriates are yielded. CH2OR :H2OR.
CH,OR,
OHC 24 R, = R2 = COCH2CH(CH3)2 R3=COCH3 43 R,=R3=COCH2CH(CH3)2 R2= COCHj 44 R,=COCH2C(CH3)2OCOCH3 R2=COCH2CH(CH3)2 R3= COCH3 52 R,=R3=COCH2CH(CH3)2 R,=H
45 R,=R3=COCH2CH(CH3)2 R2=COCH3
47 R=COCH3 48 R=COCH2CH(CH3)2
46R,=COCH(OCOCH2CH(CH3)2)CH(CH3)2 R2=COCH2CH(CH3)2 R3=COCH3 R1=OH
Based on their chemical structure valepotriates are divided into two main groups, namely the diene type (including valtrate (24), isovaltrate (43) and acevaltrate (44)) and the monoene type (including didrovaltrate (45) and isovaleroxyhydroxydidrovaltrate (46)). Valepotriates are unstable compounds; they are thermolabile and decompose under acidic or alkaline conditions, as well as in alcoholic solutions. After hydrolysis valeric and isovaleric acids are found among other compounds. The main decomposition products of the valepotriates are the yellow-coloured baldrinals (baldrinal (47), homobaldrinal (48)). The baldrinals are chemically reactive and may subsequently form polymers. Although valepotriates were once thought to be the active ingredients, these compounds are chemically unstable and are not found in teas and tinctures. Instead their degraded products, baldrinals, are
257
found in such preparations, and may account for much of valerian's sedative effect [44-51]. V. officinalis contains valerenic acid (49) and its derivatives hydroxyvalerenic acid (50) and acetoxyvalerenic acid (51) as well as valtrate (24), isovaltrate (43) acevaltrate (44), and deacetylisovaltrate (52). Small amounts of didrovaltrate (45) valerisodatum (53), valechlorine (54), kanokoside A (55), kanokoside C (56), kanokoside D (57) and isovaleroxyhydroxydidrovaltrate (IVHD) (46) may also be present [34, 52-59]. V. jatamansi and V. edulis lack valerenic acid and its derivaties, but contain valepotriates in higher amounts than V. officinalis. V. jatamansi is reported to contain valtrate (24), isovaltrate (43), acevaltrate (44), isovaleroxyhydroxydidrovaltrate (IVHD) (46) and didrovaltrate (45) [33, 50, 51, 60]. In V. edulis, valtrate (24), isovaltrate (43), acevaltrate (44), didrovaltrate (45) and isovaleroxyhydroxydidrovaltrate (46) are present [34,61]. :H,OR,
HO" OR COOH 49R = H
S3 R= Glc, Rl= COCH2CH(CH3)2 R2=H 57 R= COCH2CH(CH3)2, R,= Gentiobiose R2=OH
50 R = OH 51R = OCOCH3
CHjOCOCH,
H3C
CH 2 OH OR 55 R= COCH2CH(CH3)2 Rl= Gentiobiose 56 R=COCH2CH(CH3)2 R,=Glc
258
V. officinalis and V. jatamansi are also rich in essential oils. V. edulis carries only a trace amount of the volatile compounds. Table I, shows the content of valepotriates and essential oils in the three valerian species. All the three species are used for the production of solid oral dosage forms, while from V. officinalis also tinctures and teas are also made. Another relevant group is formed by the baldrinals which are yellowcoloured decomposition products of the valepotriates. Baldrinal (47) originates from valtrate and acevaltrate and homobaldrinal (48) from isovaltrate. As cytotoxic and mutagenic effects have been described for baldrinals, their absence in the crude drug and in preparations has to be proved in order to avoid possible hazardous effects. Table 1. Contents - based on dry weight - of the major groups of compounds in three valerian species used in herbal medicine Valeriana officinalis wallichii edulis
Valerenic acid and derivatives (%) 0.05-0.9 absent absent
Valepotriates
0.8-1.70 1.8-3.5 8.0-12.0
Essential oil (%,v/w) 0.5-2.0 0.1-0.9 <0.10
V. officinalis contains 0.8-1.7% of a mixture of valepotriates, consisting mainly of valtrate and isovaltrate in a ratio of 1:1-1:4 [56]. V. jatamansi contains 3-6% of valepotriates [62]. Next to valtrate and isovaltrate, didrovaltrate is also present in these species. Two chemotypes are distinguished for V. jatamansi i.e. the monoene and diene types. V. edulis is rich in valepotriates (8-12%) and reported to have mutagenic properties in vitro [56, 63]. Valtrate, isovaltrate, acevaltrate, didrovaltrate and IVHD are present in this species. Valerosidate, an iridoid glycoside, is found in V. officinalis (up to 1.5 %) and in V. jatamansi (up to 5%). Valepotriates are not only present in valerian but also in species of Centranthus. In addition, they do not occur exclusively in subterranean parts of the plants, but also in the leaves of Valeriana and Centranthus species. In contrast to valepotriates, valerenic acid and its derivatives are chemically stable. In future, they may play an important role in the standardization of valerian preparations of V. officinalis. Using the guidelines of the German Pharmacopoeia (9th edition), Schimmer and Roder (1992) [64] investigated valerian roots and valerian tincture. Valerenic acids were detected by thin-layer chromatography (TLC) in 19
259
out of 23 commerical plant drugs derived from V. officinalis. In two plant products containing extracts from V. jatamansi and V. edulis, these compounds could not be detected. Valerenic acid and its derivatives were also detected in several self prepared aqueous extracts and in commercial tinctures. In the subterranean parts of V. officinalis, a number of alkaloids (0.050.1%) occur; actinidine, 8-methoxyactinidine (valerianine) and naphthyridylmethylketone have been found as well as several other, yet unidentified alkaloids. Furthermore, isoferulic acid, y- aminobutyric acid, free fatty acids and short-chain carboxylic acids have been isolated. In the leaves of V officinalis, the presence of four flavonoids has also been demonstrated. From the subterranean parts of V. jatamansi, isomers of lanarin isovalerate and 4-methoxy-8-pentyl-l- naphtholic acid were isolated and characterized [50, 51]. The difference in secondary metabolites between the three medicinally used Valeriana species imply that the Pharmaceuticals prepared from the respective crude drugs also differ largely with regard to their chemical composition. However, legal demands do not exist on this point. Neverthless, several manufacturers have standardised their products, either on valepotriates or on valerenic acid and its derivatives. Another important factor is the dosage form. When a herbal tea was prepared by extraction of the subterranean parts of V. officinalis with hot water, up to 60% of the valepotriates remained in the root material and only 0.1% could be recovered from the tea. In another study no valepotriates could be detected in the tea, whereas valerenic acid and its derivatives were present. This leads to the conclusion that teas from subterranean parts of V. officinalis will be practically devoid of baldrinals. For other valerian species that are not commonly used in the form of tea no data are available. However, as V. jatamansi and V. edulis contain considerably larger amounts of valepotriates than V. officinalis, it cannot be assumed that a tea prepared from V. jatamansi or V. edulis will also be devoid of baldrinals. Film-coated tablets and capsules of Valerian contained small amounts (
260
that the baldrinals react further to form condensation products with other constituents from the tinctures [65]. Valerian products are marketed and used worldwide and comprise one of the best-selling entities of the health food/natural medicine sector in the industrialised world. More than two hundred commercial preparations containing Valeriana species have been reported [65]. One report of cultivation of V. jatamansi in India exists [48] but no data is available about quality of essential oil from cultivated populations. Wienschieva [52] and R. Bose et al, [66] have reported cultivation of V. officinalis by United Dutch Herb Cooperatives in Netherlands reporting difference in the composition of volatile oils from wild vis-a-vis cultivated populations. To improve yield and quality of volatile oil of V. jatamansi, studies were conducted at IHBT, Palampur, India to assess the effect of shade management, planting geometry, and plant nutrient management on yield and quality. Studies on influence of application of inorganic fertilizers on growth and yield of the crop indicated increase in number of slips per plant, total plant biomass and weight of roots (on dry weight basis) with application of NPK fertilizers @ 150:75:75 kg/ha as compared to lower rates or untreated check. Overall, different growth parameters showed marked seasonal variation. The fresh weight of roots was higher in July, at 9 months after transplanting (MAT), as compared to that in April (18 MAT), which could be attributed to higher moisture content in the roots at the former stage owing to peak monsoon period. On an average, yield of dry roots, root/shoot ratio, and proportion of roots in whole plant biomass showed increment with advancement in the age after transplanting. Overhead shade due to nylon-nets provided better crop growth as compared to upper storey shade vis-a-vis the crop in open field. Performance of V. jatamansi under different tree species indicated allelopathic influence of the upper storey plantation on root production. Analysis of rhizomes of cultivated plants showed significantly higher content of valepotriates and patchouli alcohol than reported in wild plants. The maximum valepotriate content was found at 4.3% (dry matter basis) under nylon net shade. A marked difference in other constituents of ar-curcumene, (3-patchoulene, and y-patchoulene was found in higher percentage than reported from wild plants [62].
261
Swertia (Gentianaceae) The genus Swertia was founded by Linnaeus in 1753. The plant species are erect annual, biennial or perennial herbs, generally distributed in tropical to temperate Himalayas between 1,200-3,000 m. The genus Swertia contains different bitter principles which are generally the iridoids and secoiridoids. There are many species of Swertia that are sources of iridoids and secoiridoids. About 250 species occur throughout the world. Nearly 32 species occur in India of which 15 occur in N-W Himalaya. Almost all the species have medicinal properties. Important among them is S. chirata which is used as a medicinal remedy since long and is known as 'chirata' [67, 68]. S. chirata Buch Ham. Syn. Gentiana Chyrayta Roxb., Ophelia chirata Grisebach, Agathotes chirayata D. Don. is an erect annual herb, 0.6-1.5 m high, branching stem, leaves smooth entire, opposite, very acute, lanceolate, flowers numerous, peduncles yellow. It is found in temperate Himalaya, 1,200-3,000 m, from Kashmir to Bhutan and Assam. It is commonly known as Chirata, Chiretta, Charayata, Kiratatikta [6769]. The important constituents of this plant are bitter principles namely chiratin, ophelic acid, amarogentin (58), amaroswerin (59) and gentiopicrin (gentiopicroside) (8). Amarogentin isolated from S. chirata in 1955, is highly bitter plant product known till date. Structurally amarogentin is sweroside - 2'-3", 5", 3'"- trihydoxydiphenyl -2"carboxylic acid ester. The biphenyl carboxylic acid moiety of amarogentin is biosynthesized from three acetate units and a 3hydroxybenzoic acid. The latter is derived from phenylanine via benzoic acid [67, 68, 70-73]. Swertiamarin (60), a secoiridoid, 5-hydroxy derivative of sweroside (7) is also reported. It is involved in number of chemical studies to determine the biogenetic relationship of secoiridoid glucosides. The labelled DL-mevalonolactone-2-14C is metabolized to sweroside (7), swertiamarin (60) and gentiopicroside (8) by S. japonica which suggests the biogenetic origin of gentiopicroside from sweroside via swertiamarin. The pathway involves hydroxylation of sweroside to yield swertiamarin which on dehydration results into gentiopicroside [7476]. Moreover, it contains polyoxygenated xanthones; 1-hydroxy 3, 5, 8trimethoxy xanthone, 1-hydroxy -3, 7, 8- trimethoxy xanthone (decussatin), 1, 8-dihydroxy-3, 7-dimethoxy xanthone (III), 1, 8-
262
59
Rj
R4
H
H
H
H
feruloyl
H
Ri
R2
60
H
62
H
dihydroxy-3, 5-dimethoxy xanthone, 1, 3, 5, 8-tetrahydroxy xanthone; 1, 3, 7, 8 - tetrahydroxy xanthone and 1,3,6,7-tetrahydroxy xanthone - (2-PD-glucose (mangiferin)- isolated from the roots and aerial parts [77-81]. S. chirata is used as an effective remedy for chronic fever. The bitter principles from the plant used as bitter tonic which promotes the flow of bile, stomachic, febrifuge, anthelmintic antipyretic, laxative and galactagogue. Chirata also exhibits insecticidal activity [68, 82]. Swertiamarin (60) should have anticholinergic properties [83-85], sweroside and gentiopicroside have hepatoprotective activities and both are being used as antihepatitis drug [86, 87]. The drug is used in atropy, emaciation or cachexy, gonorrhoea, phthisis, bronchitis, asthma, dry cough and tonic in chronic diarrhoea and dropsy. Hypoglycemic activity has also been
263
reported. The roots impart yellow colour to cotton and is accordingly used to dye linen. [88]. S. ciliata (G. Don) B.L. Burtt syn. 5*. purpurascens Clarke a species of Swertia, is an annual, erect, solitary or tufted, branched, scaberulous herb, used as a substitute for true "chirata". It is most commonly distributed in temperate N-W Himalaya, 1,700-4,100 m, from Kashmir to Kumoan [67]. The roots contain alkaloids. Xanthones, reported from the plant showed activity against mycobacterium tuberculosis. The extracted xanthone-Oglucosides showed noteworthy CNS depressant, cardiovascular stimulant and anticonvulsant activities. Swertisin and an iridoid swertiamarin (60) have been reported from this species [89]. S. alata (D. Don) Clarke is an annual, erect solitary or tufted, branched herb, distributed in temperate N-W Himalaya at altitude 1,500-3,100 m, from Kashmir to Kumaon. Similarly swertisin, iridoid swertiamarin (60), with oleanolic acid and belidifolia are reported from the aerial parts and roots [90]. Infusion of the plant is largely used as a tonic and febrifuge. [68]. S. nervosa Wall. Ex DC is an annual, erect, branched, glabrous herb, distributed in north temperate Himalaya. Sweroside (7) and swertiamarin (60) and secoiridoid glycoside; vegeloside (61) have been reported from this species [91]. Similarly, swertiamarin from S. angustifolia [92], swertiamarin (60) and sweroside (7), secoiridoid glycosides; augustiamarin (62), augustioside (63) and eustomoside (64) from S. augustifolia [93], swertiamarin, sweroside (7) amarogentin B (65), amaroswerin (59) and
OH OH
HO. HO,
65
264
COOCH,
OH COOH
O 67 Ri~ m-hydroxybenzoyl 68 R[= m-hydroxybenzoyl; R 2 = 2,3-dihydroxybenzoyl 69 R,= B
OH
78
swertiapunimarin (66) from S. punicea [94-96] and three iridoids, swertiaside (67), senburiside-I (68) and senburiside-II (69) have been reported from S. japonica [97-99]. Gentiana (Gentianaceae) Annual, biennial or perennial, glabrous-scabrous, branched-unbranched, grass like herbs, often with their root stock, leaves radical or cauline or both. About 400 species are known in the world. Most of the species occur in temperate areas in Asia, but the genus is also common in Europe and North America. Nearly 62 species are found in India of which 25
265
species have been ascertained in N-W Himalayas [67], it is commonly known as gentian. Plants from this genus have wide uses as medicinal herbs; bitter principles of Gentiana constitute many pharmacologically important compounds which justify the use of most species of this genus in traditional medicine for the preparation of bitter tonic. Gentianae radix, the dried rhizomes and roots of Gentiana lutea, has been used in folk medicine in many countries for long time [100]. Underground parts of G. scabra and eight allied species, commonly known as longdan in Chinese traditional medicine, have been used in the treatment of hepatic and cholesteric diseases [101]. Mostly the iridoid glycosides of gentian species are secoiridoids. Biosynthetically they are derived from iridoidal (1) via deoxy loganic acid (70) and loganin or loganic acid (71). Earlier work established the terpenoid origin of the iridoid glucosides in gentian species. Thus feeding 14 C labelled mevalonate (MVA) to G. triflora gave incorporation in to gentiopicrosides (8) [102]. Gentiopicroside and swertiamarin are two important secoiridoid glucosides in Gentianaceae, the former being quantitatively predominant [101]. G. lutea Linn, a perennial herb with large tap root and at least upto 1 m tall. Leaves are large and deeply veined, in basal rosette until flowering. This species grows wild in the mountain areas of Europe at an altitude of 1,000- 2,500 m. It is commonly used for medicinal purpose and to flavour alcoholic drinks (bitters). Gentianae radix is the pharmacentical name of the the root of Gentiana lutea. It contains bitter principles of secoiridoid types e.g. gentiopicroside (8), amarogentin (58), swerorside (7) and swertiamarin (60) and alkaloid (gentianine (72)) [103,104].
coocH 3
72
266
The yellow colour of the root is due to the presence of xanthons (gentisin, gentisein, isogentisin, gentoside etc). Medicinally, it is a gastric stimulant, specifically indicated in dyspepsia with anorexia, but to digestive atony of any sort. It may be used in the treatment of insufficient gastric secretions intestinal and gastric inflammation, hepatic and gall bladder disease. Its ethnobotanical use in Europe is as digestive, poison antidote, against snake bites, anti-rabies, liver and stomach ailments, intestinal worms, wound washing and improvement of appetite [105]. The seeoiridoid bitter principles, particularly amarogentin, stimulate gustatory receptor in the taste buds, causing a reflex increase in the secretion of saliva, gastric juice and bile, thereby stimulating the appetite. Gentiopicroside isolated from different Gentiana species showed the anti-inflammatory activity in carrageenin-induced foot edema in rats, smooth muscle relaxing and fungicidal activity when treated with Pglucosidase [106-107]. Antitumour properties and activity against staphylococcus aureus for aglycones of gentiopicroside and swertiamarin have been reported [108-109]. Gentiopicroside was found to be capable of suppressing chemically and immunologically induced hepatic injuries [110]. G. kurroa Royel is a perennial, glabrous, branched herbs, roots stock stout thick, completely covered by bases of radical leaves commonly known as Indian Gentian root. It is generally distributed in Jammu & Kashmir and N-W Himalaya at an altitude of 1,200-3,660 m [68-70].
82
HO'
267
Two iridoid glucosides, morroniside (73) and gentiopicroside (8) have been reported from this species [111-113]. The plant has a bitter taste, emmenagogue; useful in syphilis and leucoderma (yunani). The roots are medicinally used as a bitter tonic, and as substitute for the true gentian, acts as an aperient in larger doses. It is used as a bitter tonic for improving appetite and stimulating gastric secretion. The drug is also administered in fever and urinary complaints [68, 69]. G depressa D. Don generally distributed in Nepal at an altitude of 3,000- 3,500 m. From the aerial part of this species mixed iridoidsecoiridoid glucoside, depresteroside (74), iridoid glucoside such as depressoside (75), 3"-glucosyl depresteroside (76) and depressin (77) have been isolated [114-116]. G. tibetica King, a Chinese species is traditionally used in the preparation of Chinese medicine, called "Quinjiao" is used for the treatment of fungal and bacterial infections, hepatitis, constipation, rheumatism, pain and hypertension [117]. Iridoid glycosides reported from this species are loganic acid (71), gentiopicroside (8), sweroside (7), 2'-(2, 3- dihydroxybenzoyl) sweroside (78), trifloroside (79), rindoside (80), macrophylloside A (81) along with two secoiridoid glycosides namely 8-hydroxy-10-hydrosweroside (82) and isomacrophylloside (83) [118].
OH
86
268
G. linearis is found in Canada and is one of several species endemic to North America. The roots of this species is reported to contain two novel iridoids, 7-O-coumaroyl-loganic acid (84), 7-O-(4"-O-glucosyl)
269
coumaroyl-loganic acid (85) and a secoiridoid 6"'-O-glucosyltrifloroside (86) with trifloroside (79) and gentiopicroside (8) [119]. Moreover, gentiopicroside, sweroside and swertiamarin were isolated from different parts of Gentian species. Gentiopicroside and total bitter glycosides content were found to be higher (4.1-6.7 %) in the roots of G. rigescens, G. triflora, G. atuntsiensis, G. manshurica and G. scabra [120]. Amarogentin (58) from the roots of G. atuntsiensis and G. rigescens [121,122], two secoiridoid glycosides trifloroside (79) and scabraside (87) from the roots of G. triflora var. japonica and G. scabra var. Burgeri respectively and 2'-(2, 3-dihydroxybenzoyl) sweroside (80) from G. scabra was also reported [123-126]. Iridoids and secoiridoid glycosides reported from different species of Gentiana are shown in table 2.
OH
Centaurium erythrea Rafn. (Gentianaceae) The Red Centaury (Erythrcea centaurium, Pers.) is an annual herb, indigenous to Europe, Western Asia and North Africa and naturalised in North America, with a yellowish, fibrous, woody root, the stem is stiff, square and erect, 3 to 12 inches in height, often branching. The leaves are pale green, smooth and shiny, their margins undivided. The lowest leaves are broader than the others, oblong or wedge-shaped, narrowed at the base, blunt at the end and form a spreading tuft at the base of the plant, while the stalkless stem-leaves are pointed and lance-shaped, growing in pairs opposite to one another at somewhat distant intervals on the stalk, which is crowned by flat tufts (corymbs) of rose-coloured, star-like flowers, with five-cleft corollas.
270 Table 2. Iridoids and secoiridoid glycosides reported from different species of Gentian. Sr. No.
Gentiana species
1
G. lutea
Gentiopicroside (8), amarogentin swertiamarin (60), gentianin (72)
2
G. kurroa
Gentiopicroside (8), morroniside (73)
Compounds reported
References
(58),
sweroside
(7),
(75),
3"-
103,104 111-113
G. depressa
Depresteroside (74), depressoside glucosyldepresteroside (76), depressin (77)
4
G. tibetica
Loganic acid (71), gentiopicroside (8), sweroside (7), 2'-(2,3dihydroxybenzoyi) sweroside (78), trifloroside (79), rindoside (80), macrophylloside A (81),8-hydroxy-10-hydrosweroside (82), isomacrophylloside (83)
118
5
G. linearis
Gentiopicroside (8), 7-O-coumaroyl-loganic acid (84), 7-O-(4"O-glucosyl) coumaroyl-loganic acid (85), 6'"—Oglucosyltrifloroside (86), trifloroside (79)
119
6
G. rigescens
3
114-116
Gentiopicroside (8), amarogentin (58)
120,122
Gentiopicroside (8), scabraside (87), swertiamarin (60), 2'-(2, 3-dihydroxybenzoyl) sweroside (80)
123-125
7
G. scabra
8
G. thflora
9
G. atuntsiensis
Gentiopicroside (8), amarogentin (58)
G. manshurica
Gentiopicroside (8)
G. campestris
Gentiopicroside (8), 5-desoxyeustomoside (88), swertiamarin (60), eustomoside (64), eustomoruside (89), eustoside (90)
12
G. gelida
Gentiopicroside (8), trifloroside (79), swertiamarin (60), gelidoside (80), eustomoside (64), gentomoside (91), eustomoruside (89)
128
13
G.lactea
Gentiopicroside (8), loganic acid (71), sweroside deacetylcentapicrin (92), swertiamarin (60)
129
14
G. macrophylla
10 11
Gentiopicroside (8), trifloroside (79), swertiamarin (60)
Gentiopicroside (8), sweroside (7), gentiopicroside (8), quinjiaoside A (93)
126 120,121 120
swertiamarin
127
(7), (60), 130,131
15
G. pedicellata
Goganic acid (71), 4'-p-coumaroylloganic acid (94), loganin (15), 2'-p-coumaroylloganin (95), 2'- caffeoylloganin (96), 2'ferulloylloganin (97), 4'-p-coumaroylloganin (98)
16
G.punctata
Gentiopicroside (8), gentioside, sweroside (7), swertiamarin (60), gentiopicroside (8), gentioflavoside (99)
134
17
G. purpurea
Gentiopicroside (8), amaropanin (100), amarogentin (58), amaroswerin (59), gentiolactone (101)
135,136
18
G. pyrenaica
Loganic acid, 6'-[2(R)-methyl-veratroyloxypropanoyl]-loganin (102), moroniside, 4'-p-coumaroylmorroniside (103), 6'-O[2(R)-methyl-3-veratroyl] morroniside (104), kingiside (105), 6'-vanilloylkingiside (106), epikingiside (107), 6'vanilloylepikingiside (108) [134-137]
137-140
19
G. septemfida
Loganic acid (71), sweroside, swertiamarin (60), gelidoside (80), eustomoside (64), eustomoruside (89), eustoside (90), septemfidoside (109)
141
132,133
271 COOR
A= OCH 3
R,O'
R3 15 71 94 95 96 97 98 102
CH3 H H CH3 CH3 CH3 CH3 CH3
H H H H H H H H
R4
H H H H H p-coumaroyl p-coumaroyl H caffeol H feruloyl H H p-coumaroyl H H
R5 H H H H H H H A
7 66 92 110 111 112 113
H H D D H H H
D=
R2 H H H Ac D H H
R4 H Glc H H H H
D
272
101
The name of the genus to which it is at present assigned, Erythraea, is derived from the Greek erythros (red), from the colour of the flowers. The genus was formerly called Chironia, from the Centaur Chiron, who was famous in Greek mythology for his skill in medicinal herbs, and is supposed to have cured himself with it from a wound he had accidentally received from an arrow poisoned with the blood of the hydra. The English name Centaury has the same origin.The presence of bitter secoiridoid glycosides justifies its usage in gastrointestinal tract [142]. It contains secoiridoid glycosides, gentiopicroside (8), centapicrin (110), deacetylcentapicrin (92), decentapicrin A (111), decentapicrin B (112), decentapicrin C (113), swertiamarin (60), 6'-mhydroxybenzoylloganin (114), dihydrocornin (115), secologanin (31), centauroside (116), gentioflavoside (99); alkaloids such as gentianin (72) and xanthone derivatives [142-148]. The plant is aromatic, bitter, stomachic and acts on the liver and kidneys, purifies the blood, and is an excellent tonic used to treat jaundice, wounds and sores.
OH
273
The dried herb is given in infusion or powder, or made into an extract and used extensively in dyspepsia, for languid digestion with heartburn after food. The same infusion may also be taken for muscular rheumatism [149-151]. The chief iridoids and secoiridoid glycosides, sweroside, swertiamarin and gentiopicroside are also reported from other species of Centaurium such as C. maritimaum, C. pulchellum, C. quintense, C. spicatum, and C. tenuiflorum [152]. COOCH
COOCH
ROR=m-hydroxybenzoyl
COOCH
CHO
COOCH,
HO HO OH 116
Lonicera (Caprifoleaceae) A genus of erect climbing and scrambling shrubs distributed chiefly in the sub tropical and temperate regions of the northern hemisphere. It is commonly known as honeysuckle. Some species of Lonicera are highly valued in gardens for their handsome foliage and fragrant flowers. About 200 species are known in N. America, Europe and Africa, 45 species are known in India. Lonicera species are medicinally important, used in indigenous system of medicine as an antipyretic, stomachic, diuretic and in dysentery [68,153]. Different species of Lonicera have been chemically investigated and various iridoids, bis-iridoids, sulfur containing monoterpenoids, alkaloids,
274
glucosides, triterpeoids saponins, coumarin glycosides and flavone glucosides have been isolated [154-167]. Iridoids and secoiridoid glycosides are reported to possess hypotensive, sedative, antipyretic and antitussive activities [158,159]. L. japonica is a perennial trailing or climbing woody vine of the honeysuckle family that spreads by seeds, underground rhizomes, and aboveground runners [168]. It has opposite leaves that are ovate, entire (young leaves often lobed), 4-8 cm long, with a short petiole, and variable pubescence. Young stems are reddish brown to light brown, usually pubescent, and about 3 mm in diameter. Older stems are glabrous, hollow, with brownish bark that peels in long strips. The woody stems are usually 2-3 m long, (less often to 10 m). L. japonica is native to East Asia, including Japan and Korea [169,170]. From this native range it has spread to Hong Kong [171], England [172], Wales [173], Portugal [174], Corsica [175], Hawaii [176], Brazil, [177], Argentina [178], possibly the Ukraine [179] and the continental United States, primarily by way of horticultural introductions. It contains loniceracetaldehyde-A (117), loniceracetaldehyde-B (118), loganin, secologanin, sweroside (7), secologanoside-7-methyl ester (119), kingiside (105), morroniside, 8epiloganin, vogeloside, epivogeloside (120) [180]. Several tannins such as caffeoylquinates (CQs) isolated from L. japonica possibly have inhibitory effects on HIV [181]. It is used effectively for fever and skin ailments and rashes.
275
Lonicera angustifolia Wallich ex DC is a deciduous, erect shrub 1.83.6 m high, with sweet edible berries, found in the Himalaya from Kashmir to Kumoan, Nepal, Arunachal Pradesh at an altitude of 1,800 3,600 m. [67,153]. Oleanolic acid, ursolic acid and methyl 4-hydroxy benzoate with iridoids and secoiridoids; sweroside, loganin and 6"-O-Papiofuranosyl sweroside (121) have been isolated from leaves [182]. Its fruits are used in folk medicine to relieve gastric troubles in cattle, flowers are useful source of be-forage. Lonicera periclymenum Linn. (Woodbine Honey Suckle) is a small climbing shrub cultivated in hill states in India for its sweet-scented flowers. A new biosidic ester iridoid glucoside, periclymenoside (122) has been isolated along with loganin and loganic acid [183] and four secoiridoid glucosides namely secologanin, morroniside, secoxyloganin (123) and secologanoside (124) were characterized from stem [184]. The flowers contain mucilage and possess antispasmodic diuretic and sudorific properties, flowers are also used in the form of syrup in diseases of the respiratory tract and spleen [67]. L. Quinquelocularis Hardw. (Himalayan Honey Suckle) is a large deciduous shrub, rarely a small tree, distributed in the Himalaya from Kashmir to kumaon and Bhutan at an altitude of 1,200-3,600 m [67]. 1- inositol, hexacosanol, n-triacontanol, nonacosane have been isolated from the aerial part of the plant. Recently well known iridoids; loganin, sweroside and new iridoid 6'-O-[3-apio furanosyl sweroside have been reported from the roots [185]. H,CO, GlcO
H,CO 122
COOCH 120
276 COOCH
H
\HO R 7 H OH 121 Apiofuranosyl
COOR, H
COOCH3
COOR2
0 HO Rl 119 CH,
R2 H
123 H
CH 3
124 H
H
OH OH
AcO'
COOCHj
277
OOCH3
RO
136 R-=H 148 R= p-coumaroyl ISO R=O-vanilloyl
129
L. alpigena CB Clarke, syn. L. webbiana wall is a medium sized to large shrub found in the Himalaya from Kashmir to Kumaon at an altitude of 2,100-3,900 m. Xanthones such as rhudoxanthin, loniceraxanthine and webbiaxanthi and two secoridioids, alpigenoside (125) and kingiside have been reported from L. alpigena [186]. L. caerulea, a Japanese species reported to have 7ketologanin (126) epivogeloside, caeruloside A (127), caeruloside B (128), caeruloside C (129), loganin, secologanin and sweroside [187-189]. Plantago (Plantaginaceae) The genus has about 270 species mostly cosmopolitan in distribution, about 10 species are known in India [153]. All herbs are found in moist or marshy places. The name Plantago is derived from the Latin "Planta", which means foot sole, comparing the leaves of/5, major with the print of foot. It is commonly known as Plantain. Iridoids and caffeoyl phenylethanoid glycosides can be used as valuable taxonomic markers in the Plantaginaceae. There are many species known to contain iridoids.
278
P. major Linn, is a perennial herb with erect, stout root stock found in the temperate and alpine Himalaya from Kashmir to Bhutan at an altitude of 600-3,500 m. Leaves are radical, ovate or ovate oblong, entire or toothed, 8-20 cm long and 4-7 cm broad, flowers are small, green, crowded or scattered in long slender. P. major has almost cosmopolitan distribution. Chemical studies on plant samples of P. major from various origins revealed the presence of iridoids. Its aerial part is found to contain aucubin (0.2-1.3), asperuloside (130), gardoside (131), geniposidic acid (132), majoroside (133), melittoside (134), and 10 -acetoxy majoroside (135) [190-194]. The presence of catalpol has been confirmed [192] as well as denied [193]. 3, 4-dihydroxyaucubin (136) and 6'-O-P-glucosyl aucubin (137) have also been isolated from this species [195]. COOH
131
138 R,=R2= H 151R,= H, R 2 -CH 3
20 R=H 139 R=O-coumaroyl
HO
279
The plant is used in the treatment of rheumatism and in griping pain of bowels.The seeds are useful in dysentery [68]. The plant is considered haemostatic and wound healing in burns and inflammation of tissues. The leaves are considered cooling, alternative, febrifuge, diuretic, astringent and vulnerary and used in diarrhea and piles. In Switzerland it is a local remedy for toothache. In Tuscany (Italy) decoction of leaves is reported to be used in eye wash. An ointment of powdered leaves in vaseline or peach seed oil is used in the treatment of suppurative skin diseases as an anti-pruritic, in ecthyma and as anti-inflammatory. The roots are considered astringent and febrifuge, and their decoction is used for coughs [68, 69]. Aqueous fractions from P. major leaves were found to be biologically active. They activate the complement system and induced the production of tumor necrosis factor alpha (TNF-a) by human moncytes in vitro [196]. Aucubin (19) reported to have liver protective effect and it was shown to be antidote for fatal mushroom poisoning caused by Amanita phalloides. It exhibited low toxicity [197]. In India, the leaves and roots of P. major are used for their astringent properties and to bring down fever. The seeds are taken as a cure for dysentery and are considered to have stimulant effects.
COOH
COOCH
,OH
280
Plantago lanceolata Linn. (Rebwort plantain, Isabgol (H)) is a perennial glabrous herb. Leaves are all radical in a rosette, ovatelanceolate, 6.5-16 x 2.5-3 cm entire, base decurrent in to short petioles. It is distributed in the Western Himalaya, Kashmir to Nepal, Srilanka, Europe and N. Asia at an altitude of 1,500-2,400 m. The iridoids reported from the leaves are agnuside (138), aucubin (19), catalpol (20), asperuloside, 10-cinnamoylcatalpol (139) and methyl desacetylasperulosidic acid (140). Agnuside is characterized as p-hydroxy benzoate of aucubin. [190,198,199]. Caffeoyl phenylethanoid glycosides reported are verbascoside, isoverbascoside, plantamajoside, and lavandufolioside [200,201]. The leaves and roots of the plant are considered astringent, vulnerary and alternative and used in the treatment of cough, pulmonary diseases and asthma. The leaves are applied to the wound, inflamed surfaces and sores. The aqueous extract of leaves promote epithelial growth, diminishes hypererimia and accelerates promotion of scab. Their alcoholic extract exhibits anti bacterial action against Streptococcus betahaemolyticus, Micrococcus pyogenes var. aureus and Bacillus subtilis, thus confirming their wound healing properties. [69]. P. depressa Willd. syn. P. tibetica Hook is a central Asian species and occurs in Ural Mountains through Siberia, Mongolia, Manchuria, Sakhalin and western Himalaya to Garhwal at altitude of 2,500 m. The iridoid, aucubin (19) with six phenylethanoids namely acteoside, P-hydroxy-acteoside, P-oxoacteoside, campenoside I, cestanoside I and orobanchoside have been isolated. The major constituents were found to be acteoside and aucubin. Medicinally anti-bacterial activity was attributed to acteoside [202, 203]. Aucubin (19) is also reported from other Plantago species such as P. renformis, P. cornuti, P. asiatica, P. media, raoulii, P. australis, P. alpina, P. maritima, P. uniflora, P. arborescence, P. atrata, P. altissima, P. argentia, P. ovata, P. lundborgii and P. patagonica. Similarly catalpol (20) is reported from P. uniflora, P. atrata, P. nivalis, P. altissima, P. argentia, P. ovata, P. lundborgi, and P. patagonica [194,198, 200, 204-207]. Vitex (Verbenaceae) A genus of small trees or shrubs, leaves is 3-7 foliage, long petioled. Flowers are paninculate cymes, centrifugal. The species of this genus are
281
widely distributed in the tropics and temperate regions of both the hemisphere. About 250 species are known all over the world and about 14 species are known in India. [70]. A number of species of vitex are used in medicine. V. negundo is one of the common plants, fairly widespread throughout the greater part of India, and its leaves and roots are sold as commercial drugs. Vitex has been used since ancient times as a female remedy. Vitex is believed to suppress libido or sexual desire. It also inspires chastity, which explains one of its common names, chaste tree, thus the plant became then a symbol of chastity. Other than this, Vitex stem acts on the pituitary gland to produce a hormone called luteinizing hormone (LH). It also keeps prolactin secretions in check which may benefit some infertile women as well as some women with breast tenderness associated with premenstrual syndrome (PMS). Vitex stabilizes the cycle after withdrawal from progesterone birth control pills. Likewise, many studies note that it controls acne. It is helpful in regulating ovulatory cycle. It is also used in the treatment of menopause, menorrhagia, menstrual difficulties, premenstrual syndrome, amenorrhea, dysmenorrheal, endometriosis and fibrocystic breast disease among women [208-211]. Vitex negundo Linn, a large, aromatic deciduous shrub with quadrangular, densely whitish tomentose, branchlets, upto 4.5 m in height or sometimes a small, slender tree distributed in Eastern Africa, Madagascar to Iran, Afghanistan, Pakistan, throughout the greater part of India in the outer Himalayas, Sri Lanka, Burma (Myanmar), Indo-China, Thailand, throughout Malaysian region, east to the Palau Islands, the Caroline Island and the Mariana Islands. It is widely cultivated in Europe, North America and in West Indies and also found in other Asian countries such as China, Japan and Taiwan, ascending to altitude of 1,500 m. Different flavonoids and essential oil compounds have been reported from bark and leaves. From the leaves a number of compounds have been reported viz. glucononitol, p-hydroxybenzoic acid, 5-hydroxyisophthalic acid and 3, 4-dihydroxybenzoic acid along with two glucosides [212]. The reports indicate the presence of terpenes, a-pinene, camphene, citral and beta-caryophyllene in the essential oil from the leaves and flavonoids such as casticin, orientin, isoorientin, luteolin, luteolin-7-Oglucoside, corymbosin, gardenins A and B, 3-O-desmethylartemetin, 5-Odesmethylnobiletin, 3',4',5,5',6,7,8-heptamethoxyflavone, 3',5-dihydroxy4',7,8-trimethoxyfiavanone and 3',5-dihydroxy-4',6,7-trimethoxy
282
flavanone from the leaves and twigs [213-218]. Anti-inflammatory activity is attributed due the presence of a diterpenoid, 8, 11, 13abietatrien-6-ol and three triterpenoids, 2, 3-dihydroxy-5, 12-oleanadien28-oic acid, 2, 3-diacetoxy 5, 12-oleanadien-28-oic acid and 2, 3diacetoxy-18-hydroxy-5, 12-oleanadien-28-oic acid in the seeds [219]. Five Iridoid glycosides namely aucubin (19), agnuside [215] 2'-phydroxybenzoyl mussaenosidic acid (negundoside) (141), 6'-phydroxybenzoyl mussaenosidic acid (142) and nishindaside (143) isolated from leaves and other parts of V. negundo [220-222]. The leaves also contain two alkaloids, nishindine and hydrocotylene in addition to phydroxy benzoic acid, 5-hydroxyisophthalic acid 3, 4-dihydroxy benzoic acid, an amorphous glucoside, tannic acid. From the roots acetyloleanolic acid was characterized [223]. COOH
HO'
R2O 141 Rj=p-hydroxybenzoyl, R 2 = H 142 Ri=H, R2= p-hydroxybenzoyl
149 R,=H, R2= OCH3
It is widely used in the indigenous system of medicine in India. Various medicinal properties are ascribed to leaves and roots of this plant, for example, the leaves are aromatic and vermifuge and the roots are used as expectorant, febrifuge and tonic [68, 224]. The shrub is one of the common plants used in Indian medicine and commomly known as Shambhalu in Hindi and Nirgundi or Sephali in Sanskrit. Almost all parts are employed, but the leaves and the roots are more important and are sold as drugs. A decoction of the leaves, with the addition of Piper longum, is given in catarrhal fever with heaviness of the head and dullness of hearing; the leaves are also smoked for the relief of headache and catarrh. A decoction of the leaves was found to prevent the development of swellings of joints in experimental arthritis in adult albino rats, caused by formaldehyde injection [68]. The drug is also reported to possess tranquillizing effect. It is a constituent of the Ayurvedic preparations called Visha garbha thaila [69].
283
Recently, observations on rats revealed that fresh leaves of V. negundo have anti-inflammatory, pain suppressing, antihistamine and antioxidant activities [225]. The leaves are applied to rheumatic swellings of the joints and in sprains. The juice of the leaves is used for the treatment of fetid discharges. They show anti-inflammatory, antibacterial, antifungal and analgesic activities. It is useful in the treatment of superficial bruises, injuries, sores and skin infections [68, 69, 226]. An extract of the leaves showed anticancer activity against Ehrlich ascites tumour-cells. A clinical trial of a powder from the rhizome of Alectra parasitcia which grows on the root of V. negundo is found to be effective in the treatment of leprosy with no toxic effect [69, 227]. An iridoid glycoside, 10-O-p-hydroxybenzoylaucubin (agnuside) isolated from Vitex negundo reported to have hepatoprotective activity. The roots possess tonic, febrifugal, expectorant and diuretic properties. They are used in dyspepsia and rheumatism, and also for boils. The powdered root is prescribed as an anthelmintic and as a demulcent in dysentery. It is also given for piles [68]. Different Vitex species, V. agnus-castus V. lucens, V. trifoliata etc, are found to contain iridoids; aucubin (19), agnuside. Vitex agnus-castus I . is a small tree or shrub, which is widely distributed along the Anatolian coastal lane [228]. COOH
R,O'
144 R=foliamenthoyl 145 R=6,7-dihydroxyfoliamenthoyl 147 R=H
146 Rj= p-coumaroyl, R2= caffeoyl
From the flowering stems of V. agnus-castus various iridoids have been reported such as 6'-O-foliamenthoylmussaenosidic acid (agnucastoside A) (144), 6'-O-(6,7-dihydrofoliamenthoyl) mussaenosidic acid (agnucastoside B) (145),7-O-trans-p-coumaroyl-6-O-trans-caffeoyl-8-epiloganic acid (agnucastoside C) (146), aucubin (19), agnuside, mussaenosidic acid (147) and 6'-O-p-hydroxybenzoylmussaenosidic acid. However, the investigations of some other Vitex species have resulted in
284
the isolation of iridoid glycosides named agnuside, eurostoside (148), negundoside (2'-p-hydroxybenzoylmussaenosidic acid), 6'-phydroxybenzoylmussaenosidic acid, nishindaside and isonishindaside (149) from leaves; agnuside and 10-O-vanilloylaucubin (150) from fruits; agnuside, limoniside (151) and pedunculariside (152) from stem bark [220-222, 229-236]. This plant has important medicinal properties and is especially used for treatment of premenstrual problems and hyperprolactinemia because of its hormone-like effect [237-240]. In Anatolian folk medicine, V. agnuscastus is used as diuretic, digestive, antifungal and also against anxiety, earlybirth and stomachache [241, 242]. Verbena (Verbenaceae) A large genus of herbs or sub-shrubs distributed in the tropical and temperate regions. One species, V. qfficinalis is indigenous to India. The herbs, commonly known as Verbenas, are grown as annuals for their clusters of showy and often fragrant flowers. Verbena officinalis Linn. (Vervain) is an erect or decumbent perennial herb with a woody root stock, 30-150 cm tall, found in all temperate region of the earth in the Himalaya from Kashmir to Bhutan and the Khasi, Aka and Lushai hills, at an altituded of 300-1,800 m. Stems quandrangular; leaves opposite or ternate, oblong or ovate, coarsely toothed, pinnatifide, lilac, in dense, slender, elongated spikes; pyrenes oblong, 3-ribbed, dorsally smooth. The iridoid glycosides, verbenalin, hastatoside (153) and the phenyl propanoids verbascoside and eukovoside have been isolated from this plant [243-245]. The plant is valued for different actions e. g. relaxant, sedative, nerve tonic, galactogogue, diaphoretic, antispasmodic and hepatic action. Verbena strengthens the nervous system whilst relaxing tension and stress. It is used in the treatment of depression, melancholia paralysis, amenorrhea, dysmenorrhea and in healing of wounds [68, 245, 246]. It is used as relaxant and antispasmodic remedy in asthma, migraine, insomnia and nervous coughing. The fresh leaves are used as febrifuge, tonic and as rubefavient in rheumatism [68]. The glycosides also have a reputed galactagogue and emmenagogue action. Chinese use Verbena to treat migraine associated with female sex hormones fluctuations. The
285
galactagogue properties are attributed due to aucubin (19). Verbena has been documented to possess weak parasympathetic properties causing slight contraction of the uterus, and verbenalin exhibits uterine stimulant activity. Verbena is used as the contrinent for liver conditions, jaundice and gallstones, [245, 246] and as a gentle but effective laxative. Verbenalin, one of the constituents has a direct action on smooth muscle and also has a potential hypotensive effect. Petroleum ether, chloroform and methanol extracts of aerial parts were found to exhibit antiinflammatory activity with the chloroform extract being the most active. Methanol extract yielded two iridoid glucosides, verbenalin and hastatoside, a phenylpropanoid glycoside, verbascoside and P-sitosterolD-glucoside [247]. The production of free radicals in brain and skeletal muscle in the animal model after exercise to exhaustion could be reduced by pre-treatment of verbascoside [248]. It is a traditional remedy for infected gums, tooth decay and halitosis. In many parts of Europe it is used in the treatment of early stages of fevers, colds and in nervous disorders. In Tuscany, it is used as poultice for liver complaints, taken inernally for same disease and for dropsy [69]. COOCH3
H3C0,
154
HO,
OH
286
Verbascum (Scrophuleriaceae) Verbascum is a large genus of herbs or shrubs, commonly known as Mullein, distributed in Asia, Africa and Europe. The iridoid glycosides are widely distributed in genus Verbascum and it is well known for its variety of iridoids, which are of value for the taxonomic evaluation of this genus. Four species are found wild in India. V. thapsus Linn, is an erect, tomentose biennial herb, 90-180 cm tall, native to Europe and to western and central Asia and Britain, but now occurring in most temperate areas of the world [249]. In Himalayas it is distributed in the temperate region from Kashmir to Bhutan at an altitude of 1,500-3,600 m. Leaves entire or crenate, thickly covered with soft, whitish, stellate hairs, radical leaves large, stalked, obovate- lanceolate, cauline sessile, obovate- lanceolate acute or acuminate. Flowers yellow, crowded, cone shaped, finely pitted. Constituents of V. thapsus includes rotenone and coumarin from leaves, polysaccharides; oligosaccharides such as heptaose, octaose, nonaose and verbascose from the roots, iridoid glycosides including harpagoside, harpagide (154), aucubin (19), 6-O-P-xyloxyl aucubin (155) and catalpol (20); fiavonoids, including 3'-methylquercitin, hesperidin and verbascoside; saponins and volatile oils [250-257]. Verbascum is a valuable remedy for most conditions affecting the respiratory system.The leaves are heart stimulant, anti-inflammatory and are used in pectoral complaints, dysphomia, piles and sun burn. The leaves make an excellent poultice for boils and sores. Flowers are antispasmodic, antitussive, emollient, antineuralgic and also effective for throat inflammations and roots are demulcent, astringent and febrifuge. Both leaves and flowers are used in the treatment of pulmonary diseases such as bronchitis, dry coughs, whopping cough, tuberculosis, asthma, hoarseness, bleeding of the lungs and bowels. Seeds are aphrodisiac, narcotic. The roots are employed as febrifuge. [226, 258-261]. The plant is mildly diuretic and has a soothing and ant-inflammatory effect on the urinary tract, and acts as a mild sedative. Oil made from the flowers is used to help sooth earache, and applied externally for eczema and other types of inflamed skin conditions [258, 260-263]. It is a domestic remedy for pneumonia, fever, congestion, allergies, migraine, tumor formation, throat ailments, tonsilitis, skin ailments, catarrhs and colic [258, 259, 262]. Antiviral activity against infleunza in chicken embroys has been
287
reported [264]. Its leaf extracts have been shown to be active against bovine herpes virus type 1, and showed slight antibacterial and antifungal activity [265, 266]. Its methanol extract has been shown to be effective against mosquito larvae [267]. In the trans-Indus region the herb is much employed for the treatment of asthma and other pulmonary complaints. In Europe and USA, the thick wholly leaves are much valued as demulcents and emollients. It is a relaxing expectorant for dry, chronic hard coughs such as in whooping cough, tuberculosis, asthma and bronchitis. The leaves of Verbascum were once made into herbal 'tobacco' and smoked for asthma and tuberculosis [69]. Veronica (Scrophulariaceae) A large genus of herbs or sub shrubs, commonly known as speedwells, distributed mainly in the North Temperate Zone, many of them being alpine. About 250 species are known and 40 species are found in India [69,70,153]. Veronica anagallis-aquatica Linn, is a stoloniferous, succulent herb, found near the watery places throughout the greater part of India, chiefly in the Himalayan region. Stem 15-45 cm tall, leaves oblong lanceolate or linear oblong, flowers pink, purple or white. The plant contains the glucoside rhinathin (aucubin 0.08%) [69]. Other constituents are Benzoic acids; p-hydroxybenzoic, Protocatechuic, caffeic, vanillic, ferullic, isoferulic and p-coumaric acid; aucuboside (aucubin) (19), catalpol (20) and its acyl derivatives [268]. The herb is credited with anti-scorbutic properties. The herb is also used for bladder troubles, as it is supposed to pulverize the stones in the bladder [69]. The leaves are used in Japan as a salad. The roots are used in Indo-China countries for the preparation of gargles. V. beccubunga Linn. (Brook lime) or another species found to contain iridoids aucubin (19) has similar medicinal use as that of V. anagallum. V. arvensis Linn, is distributed in western Himalayas and the Nilgiri hills at an altitude of 2,000-3,000 m. It is credited with diaphoretic, diuretic and expectorant properties, its medicinal uses are almost similar to those of V. beccabunga. V. arvensis contain the glucoside aucubin.
288
Lamium (Labiatae) A genus of annual or perennial herbs distributed in Europe, North Africa and temperate Asia. Three species occur in India. Lamium album Linn. (White deadnettle) is an ascending or decumbent glabrous or hispidly hairy perennial herb, 25-50 cm. high, with a creeping rootstock, found in waste places and along road sides in western Himalaya from Kashmir to Kumaon at an altitude of 1,500-3,000 m. Leaves are ovate-cordate, crenate or serrate; flowers are white or pale pink, in axillary whorls; nutlets (4 mm long), smooth, black or dark brown. The plant contains, flavonoids, iridoid glucosides; lamalbid (156), two secoiridoid glucosides; alboside A (157) and alboside B (158) and 6deoxy-lamalbid (caryoptoside) (159), condensed tannins and mucilage; phenolic and fatty acid, polysaccharides, triterpene saponins [269-280]. Moreover cysteine, aspartic acid, glutamic acid, serine, asparagine, glycine, threonine alanine, Y~amm°t>utyric acid, methionine, valine, leucine, proline, ornithine and lysine have been isolated from flowers leaves and roots. Feeding experiments with deuterium labelled precursors showed that caryoptoside, lamalbid, and the secoiridoid; alboside B are derived from 8-epi-deoxyloganic acid in lamium album and that caryoptoside is an intermediate in the biosynthesis of lamalbid and alboside B. The plant possesses astringent, antispasmodic and antiinflammatory properties [281, 282] and is reported to be used in decoction against hemorrhages of the uterus, nose etc. The flowers are sweetish in taste and are used as mild astringent, haemostatic, hypnotic and depurative in bleeding piles; they are useful against fluor albus, chlorosis and debilities [69]. The tannins in Lamium are responsible for its tranquillizing, mildly astringent and haemostatic actions, while the saponins are responsible for a mild expectorant action. It is a useful remedy in menorrhagia and intermenstrual bleeding, and for the regulation of intestinal activity and bowel movement. It is also used in the treatment of abnormal vaginal discharge. L. amplaxicaule Linn. (Henbit) is a decumbent, much branched annual herb, 10-30 cm high, with arbicular leaves and purple red flowers, found in temperate Himalaya from Kashmir to Kumaon upto 3,000 m, Arunachal Pradesh and Aka hills in Assam. The plant is considered
289
stimulant laxative diaphoretic, antirheumatic and cephalic. It contains iridoid glucoside, 5-deo-xylamioside (160), 6-deaxylamioside (161), lamiide (162), lamiol (163), lamioside (18) and ipolamiide (164) [283285]. Nepeta (Labiateae) A genus of perennial or annual herbs found in Europe, N. Africa and Asia. About 30 species occur in India. Nepeta cataria Linn. (Catnip) an erect, hoary, pubescent, perennial herb 60-100 cm, high, found in western temperate Himalayas from Dalhousie to Kashmir, upto altitude of 1,500 m. Leaves ovate, coarsely, crenate; flowers white, dotted with purple; nutlets broadly oblong, smooth, brownish black. It is grown for its scented leaves and flowering tops are used for flavouring purpose and in medicine. Leaves and shoots are used for flavouring sauces and cooked foods; leaves and flowering tops are considered carminative tonic, diaphoretic, refrigerant and soporific. It is a traditional remedy for colds and flu. It is also a safe remedy for the infectious diseases of childhood, such as measles. A catnip tea is useful for many children's ailments, including measles, chicken pox, colic fevers, indigestion hives, nervousness, headache insomnia and hyperactivity [286]. COOCH 3
COOCH
HO
COOCH3
O. OH
HO'
OH 158
290
The sesquiterpene lactonesdihydronepetalactone (165), isodihydronepetalactone (166), nepetalactone, epinepetalactone (167). Nepetolglucosyl ester (168) from leaves, 1,5,9-epideoxy loganic acid (169), nepetalic acid (170), nepetariaside, nepetaside (171) have been reported [287-292]. Galium (Rubiaceae) A large genus of straggling herbs chiefly distributed in the temperate regions of the world, commonly known as Bed straw. About 25 species are reported from India. Galium aparine Linn, is a delicate trailing or climbing herb distributed in temperate Himalaya upto an altitude of 3,650 m. Leaves arranged in whorls of 6 or 8, midribs and margins minutely prickly; flowers white tinged with green on axiliary stalks; fruits small with hooked bristles. COOCH 3 3 '
HO
AcO
HO
160 R= H, R'=OH 161 R=OH, R'=OH
18 163
COOCH3
164
291 COO-GIc 4,
H
COOH
OGIc 171
The plant contains iridoid glycosides; asperuloside (120) monotropein and aucubin (19), phenolic acids; caffeine, gallic acid, anthraquinone derivatives, flavonoids, coumarins, citric acid and red dye. It has been employed in the form of an infusion, as aperient, diuretic, refrigerant, alternative and antiscorbutic. Extract of leaves used as astringent, plant paste applied on skin disease [69,153]. Galium verum Linn. (Cheese Rennet) A slender perennial herb, 1-3 ft high, with erect angular stems, found in Kashmir, Lahul and other western Himalayan regions at an altitude of 1,500-3,000 m. It contains palustroside, rutin, asperuloside, chlorogenic acid, quercetins, 3- glucosyl quercetin, 3-rutinosyl quercetin, 3, 7, diglucosyl quercetin and 7-glucosyl luteolin. Two iridoids; VI (172) and V3 (173) along with asperuloside and daphylloside (174) have been isolated from aerial parts of the plant. [293-298].
rn: 172
COOH,C HO, OH OH HO 173
292 COOCH
HO.
HjCOOC 174
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[12]. [13]. [14]. [15].
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
303
IRIDOIDS AND SECOIRIDOIDS FROM OLEACEAE JOSE A. PEREZ, JOSE M. HERNANDEZ, JUAN M. TRUJILLO, HERMELO LOPEZ Instituto de Bio-Orgdnica "Antonio Gonzalez". Avda. Astrofisico Francisco Sanchez, 2, 38205. Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez, 3, 38205, La Laguna, Tenerife, Spain ABSTRACTS: The iridoid and secoiridoid derivatives of Oleaceae have been grouped regarding structural similarities. The biosynthetic routes leading to these compounds and their most relevant biological activities, described up to now, have also been reviewed.
INTRODUCTION The family Oleaceae consists of about 600 species grouped in 25 genus. This family is almost cosmopolitan but it is best developed in Asia and Malaysia. The members of this family are mainly trees and bushes, which are very much appreciated, not only by their timber and oils, but also by their ornamental uses. The most recent classification, based on the molecular phylogeny [1] (sequence rps 16 and trnL-F) changes the level of a subfamily into that of a family and establishes that some genus of uncertain classification, such as Nyctanthes, Dimetra, and Myxopyrum are considered Oleaceae. Among the most frequent compounds isolated from species of this family are iridoids and secoiridoids. The term iridoid is used to design a wide group of monoterpenes, in most cases, as glycoside derivatives, whose structure may be considered as deriving from iridane (cis-2oxabicycle-[4.3.0]-nonane (1). The secoiridoid-type of compounds derive from iridoids by elimination of the link 7-8, to give rise to the basic structure (2).
304
(1)
There are plenty of publications regarding the isolation and structure determination [2], chemistry [3,4], biosynthesis [5] and biological activities [6] of these compounds. The main feature accounting for their classification are based in their chemical structure and biosynthetic postulates. Thus, El-Naggar and Beal [7] compiled 258 compounds, that they divided into ten groups, according to either of the different number of carbons that are contained in the iridane skeleton, the increase in the degree of oxidation and the different standards of substitution. Both, iridoids and secoiridoids were included in this classification, irrespective if they are or not glucoside derivatives. The iridoids containing nitrogen were excluded. Hegnauer [8] classifies them in nine structural groups, including pseudoalcaloids and complex compounds of the type of indol and isochinolone alkaloids. Boros [9] compiled a complete list of the spectroscopic data of the iridoids known up to 1989. Even though, the classification based on chemical structures is important, the biosynthetic classification seem to be more convenient. Thus, a lot of these compounds, related from a chemical point of view, in many reviews are included in the same group, though they come from very different taxonomic sources. Those products must have been originated through quite different biosynthetic routes. The biosynthetic approach was used by Inouye [5], who divided these compounds in non-glycoside iridoids (including pseudoalkaloids of the skytantin type), glucoside iridoids and glucoside secoiridoids. The two •first groups have not been subdivided but the third group has been subdivided into four subgroups regarding the biosynthetic routes and structural similarities. Non-glycoside iridoids and gentianin-type pseudoalkaloids were excluded. Jensen uses also the biosynthetic approach to classify iridoids, whose systematic relevance had already been pointed out by Jensen and col [10,11]. One recent revision by Jensen and col [12] correlates the distribution of the iridoids of the different
305
biosynthetic routes in Oleaceae with the phylogenetic classification cited above[l]. In this work, we want to keep the structures of the iridoids and secoiridoids which are present in Oleaceae, their biosynthesis and the biological activities, so far described, up to date. STRUCTURES The tables 1 - 7 contain the structures of the iridoids and secoiridoids from Oleaceae. The reference and the natural origin correspond to the first cite in which the compound has been named. In any case, secoiridoids such as oleuropein (81), ligustroside (82) and many others can be obtained from several species. Table 1.- Iridoids COOMe
CH 2 OR 4
HO
3 4 5 6 7 8 9 10 11 12 13 14
R, OH A B C D H OH OH OH OH OH D
R2 OH OH OH OH D D D OH OH F D D
CH,OH CH3 CH,OH CH, CH, CH, CH, E CH, CH, CH, CH2OH
R4 H H H H H H H H F H H H
15
F
OH
CH,
H
R-i
OH
Name Nyctanthoside Arbortristoside A Arbortristoside B Arbortristoside C Arborside A Arborside B Arborside C Arbortristoside D Arbortristoside E
6,7-di-fl-benzoyl nyctanthoside 6-O-transcinnamoyl-6-P-
Origin N. arbortristis N. arbortristis N .arbortristis N. arbortristis N. arbortristis N. arbortristis N. arbortristis N. arbortristis N. arbortristis N. arbortristis N. arbortristis N. arbortristis
Rf 12 14 14 15 16 16 16 17 17 18 18 19
N .arbortristis
19
306
16 17 18 19 20 21 22 23 24
OH OH OAc F H 11 H H H
OH OH A/1 OAc .1 K L M N
G COOCH, COOCH, CH, CH3 CH-, CH, CH3 CH3
hidroxyloganin Arborside D Myxopyroside
H 11 H H H 11 H H H
N. arbortristis M. smilacijolium M. smilacifolium N. arbortristis P. excelsa P. excelsa P. excelsa P. excelsa P. excelsa
Picconioside I Picconioside 11 Picconioside III Picconioside IV Picconioside V
A OCH,
B
H,C
c
K
D
L
OGIc
H,C
H»C
COO-
H3C
COO-
H,C
COO-
M -CH,
OH
20 21 21 22 23 23 23 23 23
307
F
N
H3C
COO-
-CH
COOMe
O=<
COOMe
H O ••"•
OGlc
H,C
OGlc
(25)
(26)
7-ketologanin
6-p-hydroxy-7-epiloganin P. excelsa [23]
P.excelsa [23] COOH
HOOC
OGlc
(27) Forsythide F. viridissima [24]
COOGIc
HOOC
OGlc
(28) 11-glucosyl forsythide F. europea [25]
COOMe
HOOC OGlc OGlc
308
(29) 11-methyl forsythide F.europea [25]
(30) Syringopicroside S. vulgaris [26] COOMe
H3C
(31) R=aOH, la-7-dehydrologanetin[31] (32) R=(3 OH, ip-7-dehydrologanetin[31]
33
34
R P-D- glcpglcp p-D-glcp-(3 glcp
35
p-r glc/7
36 ( 6 - -l)-a-Dgalp
Name 6'-0-a-Dglucopyranosylsyringopi croside 3'-0-a-Dglucopyranosylsyringopi croside 4'-O-a-Dglucopyranosylsyringopi croside 6'-<9-a-Dgalactopyranosylsyringo picroside
Origin reticulata S.
Rf 28
S. reticulata
28
s. reticulata
28
s . reticulata
29
309
H2Cx
37 38 39
Name R H(6"S:6 "R=53:47) Jashemsloside A Glc (6' 'S) Jashemsloside C Glc (6' 'R) Jashemsloside D
Origin J. hemsleyi J. hemsleyi J. hemsleyi
Ref 27 27 27
COOMe
H3C
OH OR
R H3C
40
HO
CH
Name Jashemsloside B
Origin J. hemsleyi
Ref 27
6"-O-transJ. hemsleyi coumaroylloganin
27
0
41 0
42
,f r ^Y' 0 H
6"-O-cis-
J. hemsleyi
coumaroylloganin
H
(43) Jashemsloside E ./. hemsleyi [27]
3C
OGIc
27
310
Table 2. Monomeric secoiridoid glucosides COOR2 COOR.,
OGIc
Ri
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
R2
R3
A CH3 B CH3 C CH3 D CH3 E CH3 F CH3 I CH3 A CH3 G CH3 G CH3 H CH3 CH3 CH3 CH2OAc J CH3 CH3 CH3 K CH3 L CH3 CH3 CH2OH CH3 M CH2OH G G CH2OH CH3 H H S CH3 CH3 0 CH3
CH3 CH3 CH3 H H H H H H G CH3
64 CH3
N
CH3
65 CH3 S 66 67 G 68 H
P G S S
CH3 CH3 CH3 CH2OH
Name
Jaslanceoside C Jaslanceoside D Jaslanceoside A Jaslanceoside B Jaslanceoside E Jasmultiside Jaspolyside Jaspofoliamoside A Jaspofoliamoside B Jaspolinaloside Multifloroside Multiroside Demethylligustroside (2 R)-2 Hidroxyoleuropein (2 S)-2 Hidroxyoleuropein Frameroside Hidroxyframoside A Hidroxyframoside B Udhenoside
Origin Ref 30 (i) 30 (i) 31 (i) 31 (i) 32 (ii) 32 (ii) JJ (ii) 33 (ii) 32 (ii) 34 (iii) (iv) 35 36 (i) (iv) 37 (iv) 37 (iv) 37 (iii) 38 (iii) 38 38 (iii) 39 (v) 39 (v) (v)
39
(v) (vi) (vi)
39 40 40 41
(Vil)
311
69 CH3 70 CH3
T U
71
S
u
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
S
OH
s
s
96 97
CH3 M CH3 V S T CH3 T H CH3 CH3 Glc CH3 G CH3 G S CH3 S Glc G T G U S w CH3 X CH3 Y CH3 s CH3 G CH3 Z CH3 Q CH3 s CH3 G CH3 M G H
M CH3
CH3 CH3
Formoside
1 -o-p-
glucosylformoside CH3 1 -<9-pglucosylfraxiformoside Isoligustrosidic acid CHa Framoside CH3 Angustifolioside A CH3 Angustifolioside B CH3 Fraxiformoside CH3 Excelsioside CH3 Oleoside CH3 Methylglucooleoside CH3 CH2OH 10-hydroxyoleuropein Oleuropein CH3 Ligustroside CHa Safghanoside C CH3 Safghanoside D CH3 Safghanoside E CH3 Safghanoside F CH3 Safghanoside A CH3 Safghanoside B CH3 CH2OAc 10-acetoxyligustroside CH2OAc 10-acetoxyoleuropein Lucidumoside C CH3 Lucidumoside D CHa Lucidumoside A CH3 Lucidumoside B CH3 CH3 6"-6>-(3glucopyranosyloleuropein Angustifolioside C CH3 C Jasminoside
(viii) (viii)
42 42
(viii)
42
(viii) (viii) (ix) (ix) (viii) (x) (xi) (xii)
(xvi) (xvi) (xvi) (xvi) (iv)
42 42 43 43 44 45 46 47 48 49 50 51 51 51 51 51 51 52 52 53 53 54 54 55
(ix) (xvii)
56 57
(Xli)
(xi) (xiii) (xiv) (xiv) (xiv) (xiv) (xiv) (xiv) (XV) (XV)
(i) J. odoratissimum, (ii) ./. lanceolarium, (iii) ./. multiflorum, (iv) J. polyanthum, (v) F. americana, (vi) F. ornus, (vii) F. udhei, (viii) F. formosana, (ix) F. angustifolia, (x) F. excelsior, (xi) O. europea,(x\\) L.
312
japonicum, (xiii) L. obtusifolium, (xiv) S. afghanica, (xv) O. fragans, (xvi) L. lucidum, (xvii),/. humile
A
N
-CH,
OH
O
B
HOOC
C
-CH;
HOOC
D
OCH,
Q
OCH,
-CH
OH
HO H 3 CO
T
HO
G
U OGIc
313
V
-CH
-H,C OGIc
OCH,
w
H,C OGIc
n u
HO
K
X
H,C OGIc
-CH.
OH
o / HO
HO'
L
o
OH
-CH.
Y -CH
M
-CH
OGIc -CH: OH
COOR,
COOMe
OR 2
HO
2
Name (i)
Origin (*)
Ref 58
2
(ii)
(*)
58
Ri
R2
98
H
99
H
CH OH XX^ XX^ :JX^ CH OH
R4
314
100
Ac
XT'
101
H
Ac
CH2OH
(iii)
CH3
(*)
58
(•*)
31
/***")
59
CH 3
102
H
H
103
H
H
HO
.
(*) F. oxycarba (**) J. odoratissimum l***)J. officinale
COOMe
(104) (8Z)-nuzhenide S. reticulata [28]
(IV)
CH3
(v)
~ ^ ^ H
CH3
CH3
OGIc
. OMe
(i) (ii) (iii) (iv) (v)
59
Fraxicarboside A Fraxicarboside B Fraxicarboside C (2' 'R)-2"-methoxyoleuropein (2' 'S)-2 "-methoxyoleuropein
•O.
,0
CH,
(105) 8Z-ligustroside S. reticulata [28]
OGIc
315
COOMe
I-UC
(106) Ri=H, R 2 =Glc (108) Fraxamoside Insularoside-3 "(9-f3-D-glucoside F.americana [39] Xb : R,= R2= Glc (107) Insularoside-3 ,6 -di-(9-[3-diglucoside F. insularis [40]
OH
OH
OH
"OH
,0
O,
OGIc
(109) Insuloside F. insularis [40]
316
(110) Desrhamnosyloleacteoside F. insularis [40]
(111) Isooleoactoside J.nudiflorum [60]
317
OGlc
Ri
112 113 114 115 116 117 118
OH OGlc OH OH OH OGlc OH
R2 H H H H H
R3
CH3 CH3 CH3 CH3 CH2OH H CH3 OH CH3
Name Insularoside Fraxuhdoside Ornoside Uhdoside A Uhdoside B Oleayunnanoside Hydroxyornoside
Table 3.-Dimeric and trimeric secoiridoid glucosides
Origin Ref F. insularis 61 F.udhei 62 F. ornus 63 F. udhei 64 F. udhei 64 F. udhei 65 F. ornus 66
318
COOMe
R,0
Ri
119 H V" 120
COOMe
R2 H H
Name Origin Jasamplexoside A J. amplexicaule Jasamplexoside B J. amplexicaule
Ref 67 67
OGIC
OH Jasamplexoside C J. amplexicaule
121 H
R,0
Ri
R2
122 A 123 H 124 A
H A H
R3 A H H
Name Oleonuezhenide Isonuezhenide Nuezhenide \y
COOMe
A= OGIc
Origin L. japonicum L. japonicum L .japonicum
Ref 68 68 69
67
319
H3C. OGIc
R 125
Name Safghanoside F
Origin S. afganica
Ref 51
126
Safghanoside G
S. afganica
51
(127) Neopolyanoside J.polyanthum [3 7]
(128) Jaspolyoside J.polyanthum [70] COOMe COOMe
H3C OGIc
320
(129) Jaspolyanthoside J.polyanthum[70]
H3C
130 131 132
R, A H H
R2
H A
H
R3 H H A
Name Isojaspolyoside A Isojaspolyoside B Isojaspolyoside C
Origin ./. polyanthum J. polyanthum J. polyanthum
A=
COOMe
COOMe
OG!c
Ref 55 55 55
321
(133) Jaspopolyanoside ,/. polyanthum [55] COOMe COOMe
H3C
OGIc
(134)
Oleopolyanthoside A J.polyanthum [71]
(135) Oleopolyanthoside B J.polyanthum [71]
(136) Jaspolyoleoside A
322
J. Polyanthwn [71]
H,C
Ri
137
R2 Y
COOMe
H
Name Jaspolyoside B
Origen ./. polyanthwn
Ref 71
Jaspolyoside C
J. polyanthwn
71
OGIc
138
H
^ j ^
COOMe
H3C
(139) Polyanoside J. polyanthun [55]
323
(140) Fraximalacoside F.inalacophylla [72]
(141) Austrosmoside O. ausl7ocaledonica[73] Table 4.- Ciclopentane derivatives
H,C
R-i
(142)
(143)
324 Ri
142a A 142b A 142c OH 142d A 143 A
R2 A A A OH A
R3 OH A A A OH
Name Jasnudifloside A Jasnudifloside B Jasnudifloside D Jasnudifloside E Nudifloside A
Origin J. nudifolrum J. nudifolrum J. nudifolrum J. nudifolrum J. nudifolrum
Ref 74 74 75 75 75
COOMe
(144)
144a 144b 144c 144d 144e 145
R2 Ri A OH OH A A A OH A A LA OH A
(145) R3 OH OH OH OGlc OGlc OH
Name Jasnudifloside F Jasnudifloside G Jasnudifloside H Jasnudifloside K Jasnudifloside L Nudifloside D COOMe
A= OGlc
Origin J. nudiflorum J. nudiflorum J. nudiflorum J. nudiflorum J. nudiflorum J. nudiflorum
Ref 60 60 60 60 60 60
325
H,C OGIc Ri
146 147 148
CH3 CH3 H
Ri
149 150 151 152 153 154
A B OH A A A
R2 H H H
R2 A A A OH OH A
R3 H OH OH
Name Jasmesoside 9 "hidroxyjasmesoside 9 "hidroxyjasmesosidic acid
R.I
R4
OH A A A OH A
OH OH OH OH A OH
Name Molihuaside A Molihuaside B Molihuaside C Molihuaside D Molihuaside E Sambacoside A
Origin J. mesnyi J. mesnyi J. mesnyi
Origin J. sambac J. sambac J. sambac J. sambac J. sambac J. sambac
Ref 76 77 77
Ref. 78 78 78 78 78 79
326
155 156 157 158 159
A A C C OH
OH A C C
c
A OH C OH C
A A OH OH OH
Sambacoside E Sambacoside F Sambacein I Sambacein II Sambacein III
COOMe
J. sambac J. sambac J. sambac J. sambac J. sambac
79 80 80 80 80
COOMe
B =
A=
OGluc
C =
CHO
CHO
H3C
H,C
OGIc
Ri
R2
Name
Origin
Ref.
327
160 161 162
H H OH H 0 H ^V^ [
COOH
Jasminin 2 -hidroxyjasminin .Tasmosidic acid
./. mesnyi J. mesnyi J. mesnyi
81 82 82
Jasmoside
J. mesnyi
74
Jasminin-10 -[3-Dglucoside
J. mesnyi
77
H
3
OGIc
163
H
0 **Y
COOMe
1 H 1^ D
OGIc
164
H
Glc
H,C
H,C
165 166
R, H Glc
R2 CH3 CH2OH
Name 9'' -deoxy-j asminigenin 8,9-dihydroj asminin
Origen J. azoricum J. sambac
Ref. 83 84
328 H,C,
H,C
OGIc
Ri
167 168
H OH
Name Isojasminin 4-hidroxy-isoj asminin
Origin J. mesnyi J. mesnyi
Ref. 82 82
H,C H,C,
OGIc
(169) Jasnudifloside I J. nudiflorum [60]
OGIc
(170) Jasnudifloside K J. nudiflorum [60]
329
H,C
...OR
H,C
R =
(171) Jasnudifloside C ,/. nudiflorum [74]
X
n
(172) Nudifloside B R=H (173) Nudifloside C ./. nudiflorum [86]
Table 5.- Epi-kingiside derivatives COOR
R= (174) Lilacoside S. vulgaris [87]
330
R= (175) Fliederoside S. vulgaris [87]
R= (176) 6'-O-trans-p-8-coumaroyl-8-epi-kingiside ./. odoratissimum [30]
R= (177) 6'-O-cis-;>8-coumaroyl-8-epi-kingiside, J. odoratissimum[30]
(178) Multiflorin J. multiflorum [88]
331
Table 6.- Secologanoside derivatives and lignan and coumarin secoiridoids COOR,
CH 2
CH3
180
CH3
181
CH3
Name Oleuroside
Origin O. europea
Ref. 89
Frachinoside
F. chinensis
90
Secologanoside 7-methyl ester
S. reticulata
29
R2
Ri
179
OGIc
TX OH
CH3
(182) Sambacolignoside J .sambac [91]
(183)
332
Escuside Fraxinus ornus [92] Table 7.-Non glucosidic secoiridoids and jasmolactones
COOMe
OHC H,C
(184) O. europea[46]
(185) O. europea[46]
COOMe
CHO H,C
CHO
(186) Ligstral O. europea [43]
(187) Oleacein O. europea [93] COOR,
Ri
188 CH3 189 CH3
R2 A B
Name Jasmolactone A Jasmolactone B
Origin J. multiflorum J. multiflorum
Ref 94 94
333
190 A 191 B
A B
Jasmolactone C Jasmolactone D
J. multiflorum J. multiflorum
94 94
A=
Iridoids and secoiridoids, as it happens in other groups of natural products, may show an ample variety of structures. It is well known that the biochemistry, the physiology, and the morphology of a plant is subdued to evolutional pressure. During millions of years of evolution, countless number of products have been synthesized by different species, and we can wonder what may be the sense for the plant to contain such as enormous amounts of variants in a sort of "metabolic orgy" that seems to be senseless. We must not forget that the variability is the bases for natural selection. The physiologic or morphologic evolution is based on the expression of new structural proteins or new enzymes which are translated in cumulative advantages or disadvantages. But the biochemistry evolution is more complex and we, not only should have in mind the appearance of new enzymes or new activities, but the evolution of "metabolic environment". The accumulation of products from the main metabolic routes provides the ideal concentrations in order that the hardly viable reactions which are catalyzed by low specific enzymes, may be carried out, giving rise to new substances, originally in small amounts. These compounds are of an irrelevant interest to the energetic needs of the living organisms in which they are contained. However, a change in the environment produces a selective pressure on the organism that favours the production of these compounds, which become important and even indispensable to their survival. At the same time, mechanisms such as genetic duplication allow that the copies follow a diverting evolution without any risk of the survival of the living beings and their descendants. This evolution has as an aim, to provide a greater specificity and efficacy to the catalytic process of the enzymes which are expressed. The evolutional stages of such proteins would explain their great "promiscuity" and the variety of reactions that one enzyme could catalyzed.
334
The role of these enzymes seems to be important in the rate of accumulation of the different types of iridoids in the plant. In the ripening process of the fruit of Olea europea [95] (olive tree), a variation in the rate of three of its iridoids is detected. During the growing process oleuropein is accumulated. In the following stage, the ripening in green, a reduction in the levels of chlorophyll and oleuropein are found. Elenolic acid and dimethyl oleuropein ( if this last is present in the plant) begin to accumulate at this point, due to an increase in the esterase activity. In the ripening in black, antocianins begin to appear and the levels of oleuropein keep on going down. The accumulation of elenolic acid and dimethyl oleuropein reaches its highest point due to greater esterase activity, leading to an increase in the content of flavonoids, verbascoside and 3,4dihydroxyphenyl ethanol. On the other hand, the fruit accumulates only glycoside iridoids, that are provably less toxic than their aglycones, which are present in the leaves, being this the reason for it to be stable[96]. The variability of the iridoids present in Oleaceae is mainly determined by two types of reactions: (a) Esterifications with alcohols in their metabolic surroundings, being the most frequent esterifications with tirosil (2-(4-dihydroxyphenyl)ethanol) and dopalol (2-( 3,4-dihydroxyphenyl)ethanol) and iridane. But, in the same way, they can act as alcohols with acteoside, lignans and even with other iridoids. (b). Acylation of the hydroxyl groups, where the most common moieties are acetyl, cinnamoyl, coumaroyl, feruloyl and caffeoyl. Other iridoids can act also as acyl groups. Acylation can occur with the hydroxyl groups of the base skeleton or with the hydroxyl groups of the carbohydrate moiety, being the hydroxyl group at C-6' the most common site in Oleaceae. These modifications act as blocking agents for fundamental compounds in the biosynthetic routes, preventing them from being consumed, leading to their accumulation up to concentrations high enough to be detected. These modifications provide us "photograms" of the biosynthetic succession from which we can deduce standards for its development. Relevant products in the biosynthesis of iridoids such as loganin or epiloganin are frequently detected by the presence of acyl derivatives, even though they certainly are in all species that contain iridoids. From the taxonomic point of view, both the nature of the acyl moiety and the position of the acylation can be used as indicators. Though the enzymatic "machinery" and the metabolic environments provide collections of natural products which characterize the genus, families and other taxons
335
of a superior rank, as iridoids are for the Oleaceae , not everyone of the species of the taxon express them. For instance, iridoids are not detected in Ligustrum pedunculare and L. robustum. BIOSYNTHESIS The starting compound for the biosynthesis of iridoids is the mevalonic acid, and the first step seems to be common to all the structures, but they divert from the eyclization step onwards, where the iridane skeleton is formed. Dahlgreen [11] proposed three routes based on biosynthetic data and by an analysis of taxons of the reported iridoids (see scheme 1).
336 H,C
OH
AMV
10-OH geraniol/ nerol OH H,C
10-OH-geranial/neral
10-oxo-geranial/neral
337 Route I CH,
CH,
H,C
H3C
Iridoidai
O
O
OH
CHO
OH
H,C
COOH
COOH
O
o
OH
Scheme 1 (Cont.)
Deoxiloganic acid
H3c
OGIu(OH)4
338 Route II CH, CH,
epi-iridoidal
H,C H,C
OH
CHO
.0 H,C
OH
CHO
H
I
COOH
./
lib
.0 H,C
OGIu(OH)4
OH
COOH
.0 H,C
Scheme 1 (Cont)
OGIu(OH)4
epi-deoxyloganic acid
339
The route II includes the 8-epi series and can be divided into two subroutes. The subroute a includes 10-hydroxygeraniol and goes via epiiridoidal and epi-iridotrial, up to the 8-epi-deoxiloganic acid. Decarboxylation at C-ll frequently occurs after the elaboration of the acid. This route is fundamentally represented in the superorder Lamianae and in some families of the superorders Cornanae and Ericanae. The subroute b coincides with a up to the point of formation of epi-iridotrial, where the glucosilation takes place; that is, some steps before and subsequent oxidation to C-ll carboxylic acid. There is nothing reported about the decarboxylation. The iridoids produced by this route are not extensively represented, they are found mainly in the family Rubiaceae and perhaps in Apocinaceae, as is the case of the iridoids already found in Gardenia jasminoides . There is another route described that includes the 10-hydroxycitronelal e iridoidal. Pagoni and col. found that (S)-ra(-)-citronellol and (S)-(-)-10-hydroxycitronellol, but not their corresponding 3,4-unsaturated analogous, have a role as intermediates to dolichodial, dolicholactona and teucrin in Teucrum marum and nepetolactone and dihydronepetolactone en Nepeta cataria [97-101]. The iridoids in Oleaceae must be formed after the route 1, with 10hydroxygeraniol as the starting compound, via iridoidal and iridotrial up to the deoxyloganic acid. From this point onwards, Jensen et al. [12] have put forward five routes to explain the origin of all the iridoids found in this family. The first of these proposals (Scheme 2) is based on feeding experiments [25] in Forsythia viridissima and Forsythia europea. The C10 site of deoxyloganic acid is oxidised to alcohol to give adoxosidic acid, and further oxidation at the same position result in forsythide which, in turn, must be the common origin to a series of derivatives found in Forsythia. This route may be exclusive for this tribe. COOH
pnnu OUUH
COOH
HOOC
340
Deoxyloganic acid
Adoxosidic acid
Forsythide
Scheme 2.- Biosynthetic pathway to Forsythia iridoids The second route is based on the structural similarity of the iridoids contained in the tribe Myxopyrae (Scheme 3). The clue intermediate is loganin, which would have been formed by oxidation and esterification of the deoxyloganic acid. Successive oxidations of loganin would lead to nyctanthoside, and a further esterification at C-ll would originate myxopyroside. Even though it is only a hypothesis, this sequence is a logic reason for the great variety of iridoids of this type found in Myxopyreae. COOH
COOMe
COOMe
OGIc
OGIc
OGIc
Deoxyloganic acid
6-OH-loganin
^oganin COOMe
HO
COOMe
1
HO«
Me00C
OGIc
Nyctanthoside
Myxopyroside
Scheme 3.- Hypothetical biosynthetic pathway to iridoids in Myxopyreae The secoiridoids from Fontanesia differ from those found in other genus of Oleaceae. The typical secoiridoids derived from oleoside 78 are absent; but, in their place, others derivatives closer to secologanic acid, very similar to those of Gentiales , are present. Damtoft and col. [102]
341
have put forward a third route (Scheme 4), in which loganic acid is transformed into secologanic acid and successively into de secoiridoids present in this genus; more exactly, in F. phillyreoid.es and F. fortunei [103], the only two species so far studied. COOH
COOH
"*•
HO
OGIc
OGIc Loganic acid
Deoxyloganic acid
CHO
H,C
COOMe
COOH
OGIc
Secologanic acid
H,C H,C
OGIc
OGIc
5-Hidroxyloganol
Scheme 4.- Biosynthetic pathway to Fontanesia secoiridoids As it has already seen, the two last routes are based on the common intermediates loganin and loganic acid, respectively. However, it has been found that the couple 7-epiloganin/7-epiloganic acid are the intermediates for the biosynthesis of the majority of the compounds oleoside-type found in Syringa and Fraxinus [104-106]. This two intermediates may have been formed, the first by oxidation at the 7-epi-site and esterification at C11 and, the second, by oxidation at C-7-epi position. Each of these two intermediates give rise to a corresponding route. One of these routes (Scheme 5) involves oxidation of 7-epi-loganic acid to ketologanic acid
342
and further transformation into epikingisidic acid and its derivatives and even in addition, secologanoside and its derivatives. This route is supported by experiments in Syringa josikaea. COOH
*-
OGIc
COOH
COOH
OGIc
OGIc
HO-i-
Ketolosanic acid
7-epi-loganic acid COOR COOH
COOR
OGIc OGIc
R=H, Epikingisidic acid R= H, Secologanoside R=Me, Epikingiside R= Me, Secoxyloganin
Scheme 5.- Biosynthetic pathway to epikingiside and secologanoside On the other hand, in a biosynthetic study by Damtoft and col. [106], deuterium-traced analogous were administered to three species of the family Oleaceae . In the first group of experiments, the incorporation of iridoidal, and the aglycone of the deoxyloganic acid in Fraxinus excelsior was studied. Iridoidal renders detectable incorporation in 7-ketologanin; while, iridotrial, in addition to the incorporation in 7-ketologanin, is also incorporated in secoiridoids of this species. So, it seems to be feasible that 7-ketologanin may be an intermediate, provided that, the three compounds administered are intermediates as well. In a second group of experiments, deoxyloganic acid and 7-ketologanic acid were administered, giving both significant incorporation in the secoiridoids of Fraxinus excelsior . This confirms that 7-ketologanic acid is involved in
343
the biosynthesis of oleosides. But there is something to be determined in a path between deoxyloganic acid and ketologanic acid or its methyl ester. In the last series of experiments, deoxyloganic acid was administered along with 7-epiloganin, loganin and 7-ketologanin. Loganin failed in being incorporated, while the other three compounds give significant incorporation in the oleosides. These results show that the biosynthetic route for the oleosides involves the 7-a-hydroxylation of the deoxyloganic acid, followed by methylation and oxidation to 7ketologanin (Scheme 6). COOH
COOH
OGIc
OGIc
COOH
OGIc 7-ketologanin
7-epi-Ioganin
Deoxyloganic acid
COOH
COOR
OG|C
COOH
COOH
OGIc
R=H, Oleoside R=Me,Oleoside 11-methylester
10-OH-oleoside
Scheme 6.- Biosynthetic pathway to oleoside derivatives Even though it seems to be clear the involvement of 7-ketologanin in the biosynthesis of oleosides, on one hand and, on the other hand, the corresponding ketologanic acid in that of epikingisidic and secologanoside, however this last conversion is not clear. Damtoft [105] has proposed a conversion in only one path (Scheme 7) via a Baeyer-Villiger-type intermediate to give rise to three feasible routes: Route I.- A reaction initiated by breaking of both the peroxide and the 7-8 link with a simultaneous lost of H-9.
344
Route II.- Similar to the precedent with elimination of a proton at C10 to originate secoxyloganin () and its derivatives. Route III.- Via attack at C-8 to give 8-epikingiside ( ) and related compounds: HOOC
COOR
COOR H
Route I
O
Fro OGIu(OH)4 OGIu(OH)4 COOR
HOOC
COOR
R'O
Scheme 7.- Routes to oleoside, secoxyloganin and epikingiside derivatives
345
If this hypothesis is true, it would explain the presence of the three types of compounds in Oleaceae. In any case, it is possible to design the schemes 8 and 9, that with the data available, distribute the five described routes among the five tribes. COOH
Tribu Forsythiae
,0 H3C
OH
T
HO"
OGIc
HOOC
OGIc
COOH COOH
Tribu Fontanesiae H,C
OGIc
CH 2
OGIc
CH,
COOMe
OGI
COOMe
OGIc
Tribu Myxopyreae
COOMe COOMe
HO"
OGIc
rr
HOOC
OGIc
Scheme 8.- Biosynthetic pathways in Forsythieae, Fontanesieae and Myxopyreae
346
H3C
o
X
COOR
cOOMe
OGIc
COOH
COOH
CH,
Tribu Jasmineae
COOMe
OGIc
Tribu Oleae COOR
COOMe
COOR
COOMe
OGIc
Scheme 9.- Biosynthetic pathways in Jasmineae and Oleae Inouye et al. [107] and Kuwajima et al [108] consider secologanin as an intermediate in the biosynthesis of the oleoside-type glucosides present in Jasminum, Olea, Osmanthus and Ligustrum, However, the incorporation in all cases are lower than 0.4%. Oleoside 11-methyl ester is present in the genus Fraxinus, Syringa and Jasminum, being the origin of a high number of derivatives esterified at the C-7 position. It seems to be reasonable that the compound 11methyl glucoside is an intermediate between the oleoside 11 -methyl ester and the esterified oleosides. The glucosilation must be necessary for it to be transported to the interior of the cell, where this compound is accumulated.
347
This compound, has a carboxylic group at C-7 which is susceptible as being esterified giving rise to a variety of metabolic possibilities, and the result depending very much on the surrounding where the process is developed. The structural features of these derivatives go from compounds so simple as oleoside dimethyl ester up to that of sambacolignoside (182), which is spectacular. There compounds which are esterified with acteoside and similar products, very common also in these species. Jaspofoliamoside A and B (56) and (57) and jaspolinaloside(58), derivatives esterified with lineal monoterpenes have been isolated from./. polyanthum. But, without any doubt, the most habitual esterifications are those carried out with tyrosil to originate ligustroside (82) and with dopalol to give oleuropein, (81) which are common in species of the sub tribes Ligustrinae, Fraxinae and Oleinae, and they even could be extended to the whole tribe Oleae. These compounds may be glucosilated with the aim of new esterifications that originate polymers, either through linkage to the molecule of glucose or directly with hydroxyl groups from its own structure. Esterification with iridane at C-7 of the oleoside 11-methyl ester is quite common in some species of the genus Jasminum, Which may give rise to a series of derivative compounds. Inoue and col [82] explain the biosynthetic rout as due to the existence of an intermediate that originate these secoiridoids by the linkage of 11-methyloleoside and dihydroxyiridane. Several units of oleosides or even secologanoside may be added to this linkage to form dimer- or trimer-secoiridoids, probably from jasminoside or its 9"hydroxy derivative, both present in J. mensyi. 10-hydroxy-oleosides derive probably by hydroxylation of the corresponding oleosides at C-10, as it may be seen by the coexistence of products such as oleuropein (81) and 10-hydroxyoleuropein (80) in Ligustrum ssp. and Osmanthus ssp. and jasmultiside (53) and multifloroside (59) in J. multiflorum . Later on, this hydroxyl group can be oxidized to acid or esterified with an ample variety of acyl groups, as the acetyl group or even the formation of dimer- or trimer-oleosides. The study of the last stages of the iridoid biosynthesis in Oleaceae, can be observed that oleoside and 10-hydroxy derivatives are common to the tribes Jasmineae and Oleae.
348
Taylor [109] considers that Forsythia, Abeliophyllum and Fontanesia are the most ancient genus, provided that oleoside are not present in them, While Chionanthus, Fraxinus, Ligustrum, Olea, Osmanthus, Phillyrea and Syringa That produces oleosides are considered as genus of a more advanced evolution. Taylor includes Jasminum among the most primitive taxons, even though this genus presents an enormous number of these compounds, as it has already been seen, the C-ll derivatives are particularly abundant, mainly those esterified with tirosil and dopalol. This feature parallels from the phytochemical point of view, the tribes Jasmineae and Oleaceae and agree with the phylogenetic results of Wallander and Albert [1] for this family. The esterification with iridane and its derivatives are particularly interesting due to the fact that, they are exclusive to the genus Jasminum , more exactly, to a group of species that has shown to be a potent taxonomic indicator. With the data available, the 10-hydroxyoleoside derivatives originated from those above by hydroxylation, are also common in the tribes Jasmineae and Oleae (subtribes Ligustrianae, Fraxinae Oleinae). Another efficient indicator may be the presence of jasminoside and related compounds also exclusive to a group of species of Jasminum and recently found in Menodora (see table 8). Table 8 7 derivatives 11 methyl ester oleoside
ssp.
Jasminum multiflorum Jasminum polyanthum Jasminum sambac
10-OHoleosides Jasminoside type
•
Jasminum odorutissimum Menodora robusta
•
Jasminum
Jasmineae
Jasminum hemsleyi Jasminum giraldii
7-iridane oleosides
•
Abicoium
Jasminum lanceolarium Jasminum amplexicaule Jasminum humile
10-OHolcosides
•
•
349 Jasmimim urophyllum
•
•
Jasmimim cizoricum
•
•
Jasmimim gram/iflorum Jiisminum nudiflorum
•
Syringa sp.
•
Jiisminum mensyi
•
Ligustrum sp. Comorantlius sp. Schrebera sp. Fraximis sp.
•
Schrebera sp. leea
Chionanthus sp. Haenianthus sp. Forestiera sp. Priogymnanthus sp Olea sp. Nestegis sp. Osmunthus sp.
•
Phi/lyrea sp.
•
Piccania sp.
Therefore, even though the number of species studied is small, two phytochemical groups can be distinguished in Jasminum. A metabolic environment in Jasminum with the presence of 7-methyl oleosides derivatives, that can be esterified at C-l 1 and hydroxylated at C-10, in the same way that it occurs in the tribe Oleae, but with the difference of the characteristic iridane derivatives. There is another environment, that is known as Abicorum which propitiates the origin of jasminoside and similar compounds. This data coincide with the molecular data obtained by Jensen [12] who finds two groups of species in the genus with an important divergence in their phylogeny. Jasminoside can be considered as a derivative of 10-hydroxyoleoside7-methyl ester by esterification at C-10. But this product seems to be far from the proposed biosynthetic routes. It can be originated from 10hydroxyoleoside dimethyl ester very frequent in this group of species by previous or subsequent deniethylation of the esterification at C-10. But, if the origin of mono- and dimethyl esters of 10-hydroxy oleosides are supposed to be the corresponding oleosides, the absence of compounds of
350
the oleoside-type in Abicorum is difficult to explain. They can be consumed up to 10-hydroxyoleoside so efficiently that no trace is left, or perhaps a different route from that of the Jasminum group may be followed. The common point to both routes would be at 10hydroxyoleoside dimethyl ester, which appears in both groups. In the group Abicorum, it is also frequent the presence of epikingisidic acid and its methyl ester, along with the secologanic acid and secoxyloganin. Up to now, all the species of the tribe Oleaceae contain oleoside-type iridoids, which are also present in the group of species Jasminum of Jasmineae. In both tribes, these oleosides may be hydroxylated at C-10, even though, in genus as Syringa and many species of the Jasminum group, this transformation is not present. The esterification of jasminoside is carried out by the cinnamic acid, which may be considered a marker of the group Abicorum, as it appears in all of its species, including Menodora robusta. A group of compounds closely related to those of Abicorum are also present in J. lanceolarium. These compounds could have been formed from them or by esterification with acids such as coumaric, ferulic and cafeic. All of them appear as cis-, trans-mixtures in variable proportions in equilibrium. Even though the authors have been unable to purify the cis-cafeic derivative, its existence in the plant is well established. Both jasminoside and 10-hydroxyoleoside dimethyl ester are present in J. odoratissimum, which may be the origin of other dimethyl derivatives in the plant, and in the same way as it is in J. lanceolarium. The authors have also obtained the cis-jasminoside, though in small amounts in relation to the trans-derivative and to the whole amount of secoiridoids in the plant. The non-glicosidic secoiridoids of the type of jasmolactone would derive from the glucoside of the type of 10-hydroxyoleuropein by glucosidase-catalysed hydrolysis. In fact, Shen and Chan [109] carry out this process by treating the secoiridoids present in J. multiflorum with Pglucosidase. The reaction takes place through a stereoespecific rearrangement of the carbon skeleton in two steps. Lactonization, stereoespecific transference of an alcoxyl group from C-7 to C-8 and a trans- addition to a double bond, where two new chiral centres are generated ( see scheme 10). It is necessary that the C-7 be esterified, since there is no reaction for the corresponding acid. The ligstral-type secoiridoids would be formed from 10hydroxyoleosides esterified at C-10. After the scheme proposed by
351
Gariboldi [46], it is expected that the hydrolisis gives rise to the formation of an aldehyde which rotates along the bond 5-9 to give the cis- and transderivatives (scheme 10). COOR2
COOR, COORj
COOR 2
COOR,
COOR,
OGlu(OH)4
COOR
O-H OR 2
H
H
352
COOR 2
COOR,
OHC'
Scheme 10.- Formation of jasmolactones and jasmoaldehides by the action of glucosidases BIOLOGICAL ACTIVITIES Iridoids and secoiridoids show a wide variety of biological and pharmacological activities which have been reviewed by Ghisalbert [6] up to 1998. We report here a review of the most relevant compounds of this type isolated from Oleaceae. Cardiovascular Activity Oleuropein (81), which may be isolated from several species of Oleaceae, has a role in the increase of a 50% of the coronary blood flew, and shows spasmolitic and antiarritmic effects [110]. On the other hand, it is known that elenolic acid, obtained by hydrolysis of the extracts of olive-tree leaves, has hypertensive properties. All this suggests that the glucoside ring and the 3,4-dihydroxyphenylethanol moieties, both taking part of the oleuropein structure, but absent in the elenolic acid, are not responsible for the hypertensive activity. Oleacin (187) is a strong inhibitor (IC50 = 26 uM) of angiotensin converting enzyme (ACE) [111]. This enzyme catalyses the conversion of angiotensin I in angiotensin II which participates in the increase of the blood tension, by widening the arterioles. Oleuropein and other
353
secoiridoids have inhibitory activity, but the aglycone moieties, obtained by hydrolysis, show similar activities than that of oleacein. In addition to these secoiridoids with cardiovascular activity, p-dihydroxyphenylethanol, has also been isolated from olive tree leaves, which has shown calcium antagonistic activity. Thus, oleacin, oleuropein and Pdihydroxyphenylethanol act through a different mechanism, which would explain the beneficial effects of the olive-tree leave extracts in the treatment of hypertension. Furthermore, the "in vitro" toxicity of oleuropein and its aglycone in human cells is only observed at concentrations higher then those available after the habitual use [112], which indicates that the ingest of these extracts is secure. Oleacein has also been isolated from two species of Jasminum (J. azoricum and J. grandiflorum), along with sambaceine (I-III) (157) (158) (159) [113]. These compounds have shown in vitro ACE inhibition (IC50 = 26 uM-36 uM). The authors suggest that these compounds may derive from their glucosides, sambacosides (A, E, F) (154),(155),(156), by enzymatic hydrolysis, followed by opening of the lactol ring, ketonisation, hydrolysis of 11-methyl ester and decarboxylation. In any case, their IC50 values make them to be among the strongest inhibitors of ACE isolated from plants. Jasmolactone B(189) and D (191) induced on isolated guinea pig heart coronary dilation (MEC 1.3 x 10"s and 4.8 x 10"6 M) and negative chronotropic and inotropic effects (MEC 2.5 x lO"5 and 9.7 x 10 *6 M ) [94]. Antiinflamatory activity Recio et al [114]. have studied the anti-inflammatory activity of loganic acid and loganin, along with those of a series of other iridoid glucosides. They found that loganic acid is the most active (44% edema inhibitory) in the carrageenan-induced paw edema test. In the tetradecanoylphorbol acetate (TPA)-induced mouse ear edema test, loganin showed the highest activity (72-80% inhibition). Secologanin shows anti-inflammatory activity in mice and rats similar to that of aspirin [115]. Secoiridoids of Fraxinus, were assayed for their anticomplement action as well as for their ability for preventing Cobra Venus induced complement activation in normal human serum [116]. The results indicate
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that most of the secoiridoids so far studied are able to eliminate the CP activities (classical pathway) and AP activities (alternative pathway). However the concentration used for the AP assay (lmg/ml) were much higher than that for the CP assay (250(.ig/ml). Then the major effect is produced in the CP activity. The most effective CP inhibitors in guinea pig serum were ligustroside (82) (IC50 = 30(j,g/ml) and insularoside (112) (IC50 = 62ng/ml). Oleuropeoside and ligustroside (82) isolated from Phillyrea [117] were tested for interactions with cyclooxigenase (COX) and 5lipooxigenase (5-LOX) pathways of arachidonate metabolism in calcium ionophore-stimulated mouse peritoneal macrophages and human platelets. These compounds showed a significant effect on prostaglandin E; (PGE2)-release, with inhibition percentages similar to the reference drug indomethacin (IC50 - 0.95 uM). The IC50 values of oleuropeoside and ligustroside were 47pM and 48.53uM, respectively. Ligustroside also showed action on tromboxano B2 (TXB2)-release (IC50 = 122.63 uM) although by far under from the reference compound ibuprofen (IC50 — 1.27uM) Oleuropein has been assayed for it anti-inflammatory properties along with other phenolic compounds of the polar fraction of olive oil [118]. It gave rise to a 30% on the AA (arachidonic acid) inhibition. • • )
Antioxidant activity The antioxidant properties of the components of extracts of olive leaves and the oils of different origins are well known. These properties have been associated with the Mediterranean diet, an eating habit which at the same time has been related to the low frequency of diseases such as cardiovascular and neurological disorders [119-121]. Thus, oleuropein (81) isolated from olive cake butanol extract showed a scavenging effect (IC50 = 25ug/ml on DPPH (l,l-diphenyl-2-picrylhidrazyl)). free radical [122]. This method was used [123] to quantify the anti-free radical activity of 3,4-dihydroxyphenylethyl-4-formylmethyl-4-hexenoate (3,4-DHPEA ). This product which has been isolated from the leaves of Olea europea is formed from oleuropein during the period of storage of the leaves. Oleuropein derivatives from different extra virgin olive oils (EVOOs), have also been studied as anti-oxidants using the following systems: a)
355
xantine oxydase (XOD) / xantine system, that generates supeoxide radicals and hydrogen peroxide, b) diaphorase (DIA/NADH/juglone that generates superoxide radicals and semiquinonic radicals and c) the above mentioned DPPH test (see Lavelli [124] and references cited). The free radical-scavenging properties have been communicated by Visioli et al [125]. In this case, the VDPPH test, gives a value of (EC50 = 3,63 x 10"5 M), close to that of Vitamin C. The value (EC50 = 14.3 uM) for the inhibition of the superoxide anion is also significant. Manna et al [126] reported of the protective effect of the phenolic fraction of extra olive oils against the citotoxic effect of reactive oxygen species in human erytrocites and Caco-2 cells, used as model system. In these fractions oleuropein is included. Oleuropein showed medium antioxidant strength in the Rancimat test, used by Ranally et al [127]. The same compound exhibited moderate antioxidant activity in comparison to other phenolic natural compounds using the Briggs-Rauscher method [128]. Coni et al [129] described the protective effect of this compound on the oxidation of low density lipoprotein in rabbits and of its capacity to diminish the level of total, free and esterified cholesterol in plasma (by 15, 12 and 17%, respectively). It has been described that oleuropein is responsible for the increase in the production of nitric oxide Mouse macrophages [130]. Antioxidant activity have also been found in components of other genus of Oleaceae, more exactly Ligustrum lucidum [53]. In addition to oleuropein, (81) ligustroside (82), isonuezhenide (123), lucidumoside B (94) and lucimoside C (91) exhibited strong anti-oxidant effect against haemolysis and red blood cells induced by free radicals, being lucidumoside C (91) the strongest compound (IC50 = 9.3 uM). Antiviral activity Arbortristoside A (4) and C (6) showed activity against the virus of encephalomiocarditis (EMCV) and of Semilko forest (SFV) [131]. Doses of 0.5 mg-mouse"1 increase the survival time in about 5 to 6 days. Ma et al. [132] evaluated the antiviral activity in vitro of five glucoside secoiridoids, lucidumoside C (91), oleoside (78), oleuropein (81), ligustroside (82), and lucidumoside A (93) of Ligustrum lucidum to see if there were any correlation with the antioxidant activity above cited [53].Four strains of pathogenic viruses were used, simple herpes type 1 virus (HSV-1), influence type A virus (FluA), respiratory syncytial virus
356
(R.SV) and Para influence type 3 virus (Para 3). None of the iridoids had significant activity against HSV-1 and FluA. Oleuropein, however, showed activity against RSV and Para 3 with values of IC50 23.4 and 11.7 ug/ml, respectively. Lucidumoside C, oleoside dimethyl ester and ligustroside showed high or moderate activity against Para 3 with values of IC50 15.6-20.8 ug/ml. Correlations between the antiviral and antioxidant activities were not found. The activity anti HIV of olive leaves extract (OLE), as it is known, possesses a high content of oleuropein, has been studied by Lee-Huang et al. [133]. Antihepathotoxic activity Loganin and some of its derivatives showed promising hepathotoxic activity [134]. Hypoglycemic activity Oleuropien showed hypoglucemic activity and tolerance to glucose by oral administration in normal and alloxan-diabetes rats [135]. Antimicrobial activity Ligustroside (82) and ornoside (114) of F. ornus inhibited the growing of S. aureus and E. coli with the MIC of 500 u.g/ml in all cases [136]. Bissignano et al. [137] reported the antimicrobial activity in vitro of oleuropein against five standards bacterial strains and 44 fresh clinical isolates, all of them agents of infection in the intestinal tract or in the respiratory apparatus in human beings. The values of MIC were between 62.5 and 500 u.g/ml for the standard strain and between 0.97 and 39.25 jig/ml for clinical isolates. I mmunomodulator activity Arbortristoside A (4) and C (6) showed activity against passive cutaneous anaphylaxis and mast cells stabilizing activity [138]. This compound provided protection to mice against systemic infection of Candida albicans [139].
357
Antileishmaiiial activity These same compounds above cited, showed antileishmanial activity in vitro against Leishmania donovani amastigotes in macrophages cultures in vivo using a hamster test system [34]. Comments At the time of the bioactivity of iridoids not only it is necessary to have in mind that their acetyl ester are substrates for acids and acylases, but also for acids and glucosidases. In the first review carried out by Sticher [140] about the pharmacology of iridoids, this lability and predisposition to form polymers are considered as inefficient characteristics for pharmacological agents. The observation that the aglycones behave as more active agents than their corresponding glucosides is very common [6]. The iridoids are considered in many cases as a pro-drug [141], due to the fact that the glucoside iridoids are hydrolysated in the intestinal tract. Geniposide is metabolized to its aglycone genipine, which is found in the whole mouse intestine, mainly in the blind and in the colon. This aglycone does not appear in the homogenised of rats which does show [3glucosidase and esterase activity, being necessary the bacterial (3glucosidase. Human bacteria (Peptostreptococcus anaerobius, Klebsiella pneumoniae) also transform geniposide, gardenioside [142], swertiamarine [143] and acubine [144] and their aglycones. Acylase activities are shown as well [142]. Eubacterium sp. A44 hydrolyses geniposide to genipinine and then to aglycone of geniposidic acid [145]. The formation of pyridin monoterpenoid alcaloids (APMT) is well studied. It proceeds by treatment of the glucoside iridoids with acids and glucosidases in presence of a source of ammonia, frequently ammonium ion [146]. Human intestinal bacteria transform aucubine into two alcaloids, geniposide and gardenoside into genipinine and gardenine and swertiamarine into gentianine [142]. Some of these reactions are reproducible by treatment with glucosidase and ammonium acetate giving rise to rearrangements and dimers of APMT.
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The relevance of these compounds is in their properties, namely antiinflamatory, muscular relaxing, sedative, antihistaminic hypoglucemical and hypotensive, along with those attributed to iridoids. Therefore, the hydrolysis reaction does not mean the loss of pharmacological activities but it originates a dialdehylde-hemiketal intermediate with much more bioactive strength. The aldehyde may be considered as an amine acceptor in the formation of a Shiff base. So in the study of bioactivity of iridoids the glucoside, the hemiketal or aldehyde and a possible APMT must be considered. REFERENCES [I] [2] [3] [4]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
365
PHARMACOLOGICAL ACTIVITIES OF IRIDOIDS BIOSYNTHESIZED BY ROUTE II MARINA GALVEZ, CARMEN MARTIN-CORDERO, MARIA JESUS AYUSO. Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain ABSTRACT: The pharmacological assays and activities of natural iridoids biosynthesized by route II, extracted from a variety of plants are summarized in this review, such as antiinflammatory, antitumoral-chemopreventive, hepatoprotective and healing. Structureactivity relationships are also discussed.
INTRODUCTION The iridoids are a group of monoterpenoids with the methylcyclopentane skeleton, based on the structure of iridane. In the widest sense, iridoids also can include secoiridoids, where the cyclopentane ring is opened between the positions C-7 and C-8. Fig. (1). The "iridoid" term comes from the genus where the first iridoid was isolated, Iridomyrmex ants, that synthesize iridodial, Fig. (1) with a defensive finality [1]. In plants, the study of iridoids started in the middle of the XIX century, with the isolation of asperuloside [2] or gentiopicroside [3], but is not till 1960 decade when the iridoids have reached the highest interest for Scientifics.
10
IRIDANE
SECOIRIDOID
IRIDODIAL
Fig. (1). Structure of iridane, secoiridoid and iridodial.
Actually, it has been identified more than 500 iridoids, where, more than 300 are glycosides, and about 100 are secoiridoids. These compounds are interesting because of different reasons: they are related to indol
366
alkaloids, they have different pharmacological activities and they are used as chemotaxonomic markers [4-6]. Many iridoids are constituents of crude drugs, which have been used in traditional medicine. Among these, the bitter glucosides of gentianaceous plants are used as bitter tonics, valepotriates from Valerianaceae, have a weak sedative effect, geniposide and genipin, from Gardenia has mild purgative and significant choleretic effects, or harpagoside from Harpagophytum procumbes with analgesic and antiphogistic activities [4] Roles in Plant Physiology The iridoids have an ecological interest, because, they often serve as a defense mechanism against herbivores [7]. The glycosides, when are hydrolyzed, have denaturalizing properties of proteins, mainly of lysina amino acid, decreasing the nutritional quality of the plant. This fact joined with their bitter taste that bring to the plant, make that they serve as feeding deterrents for herbivores [8]. Besides, some larvae of butterflies and bees species have learned to sequester some of these iridoids in a selective way, like aucubin, acting, also on them, as defense against their predators. It has been found a relationship between the concentration and the kind of iridoids in some plants rich on iridoids with the oviposition and larvae cycle of some species of the Lepidopterae, Nymphalidae and Chrysomelidae families, like Euphydryas [9], Junonia [10, 11], Melitaea [12], Longitarsus [13] genus. Other insects and pathogenic microorganisms, can induce an iridoids biosynthesis increase, helping to the plants, even more, to protect themselves against hervibores [14, 15]. On the other hand, some iridoids are able to attract certain felines, like iridolactones from Actinidia polygama [16] o Nepeta cataria [17]. The iridoids have antigerminative properties too, as aucubin, harpagoside or lateroside, inhibiting the Hordeum vulgare seeds germination, or even, decreasing the germinated seeds roots length [18]. The enviromental changes can also affect to the iridoids synthesis. In vitro studies with Plantago lanceolata, the levels of iridoids were increased with warm temperatures, between 18-20 °C, and were decreased by low light intensity or high nitrogen levels [19-21]. The UV-B radiation did not affect to the iridoids production [22].
367
Biosynthesis The biosynthesis of plant iridoids have been noteworthy studied. Jensen, has published an useful review discussing their different biosynthetic routes: I, Ila, 116, and III [23]. Our revision is focus on iridoids biosynthesized by route //, which comprises the epz'-series, and 8astereochemistry, divided into two subroutes: Ila, which starts with 10hydroxygeraniol and goes via ep?-iridodial, and -trial, which, with scrambling, gives epz-deoxyloganic acid. But, after elaboration of the acid, decarboxylation of C-11 often takes place. The other, (lib), is identical up to e/>z-iridotrial, where glucosylation takes place, followed by further oxidation of C-11 to the carboxyl stage. No decarboxylation of C-11 is known [23]. Fig. (2). COOH
CHO
OH MEVALONIC ACID
OH 10-HYDROXYGERANIOL
OH £P/-IRIDODIAL
OH £W-IRIDOTRIAL
O-Glc £7>/-DEOXYLOGANIC ACID
Fig. (2). Route II biosynthetic pathway of iridoids.
The iridoids biosynthesized by this route are shown on Fig. (3) and (4). COOCH3
CH3 O-Glc OGIc HO— O.G|C Asperuloside Aucubin
8-Acetilharpagoside
COOCH3
HO
O-Glc
Bartsioside
HO'"
°- G l c HO
Catalpol
Deacetyl-asperulosidic acid
Gardenoside
Fig. (3). Chemical structure of iridoids biosynthesized by route II.
368 H
COOCH3
OH
HO-
1
H
HO-
O-Glc Geniposide
Genipin
HCi
c/}T° Ict r° \
V4..0
COOH
H
J,
H
<
i
i. H A
HO'
CH3
H
O-Glc
Harpagide
Geniposidic OH acid
COOCH,
Harpagosid'"3
COOCH3 HO
^ - l l c , , / " °"° l c Kutkoside
H
HO
^
^ Ipolamiide
COOH
HO HO' ' C H = O-GIC Lamiide
H3C
°-G'C Logamn
H
HO
H3CSCOO—
^C O-GIC Loganic acid
COOH
OGIc
Paederosido
HO
H
COOCH 3 Q
COOCH3
o-^ = H
O-Glc-Cinammoyl OH
Picroside I
Shanzhiside
~
O-Gluc Verbalin
Scandoside
O-Glc Scorodioside O-Glc Scropolioside B
Fig. (4). Chemical structure of iridoids biosynthesized by route II.
The distribution of the iridoids is shown in the Dahlgrenogram in Fig.(5). The concentration of iridoid-producing plants in closely connected superorders may indicate that the ability to biosynthesize the compounds has only arisen a few times. The iridoids are synthesized by dicotyledonous plants (Simpletalae) and by insects. The most important plant orders were iridoids are present are: Dipsacales, Gentianales and Lamiales.
369
Fig. (5). Distribution of iridoids decarboxylated at C-l 1 of Route Ila (dots)[23].
The iridoids belonging to route Ila are concentrated in Lamianae with outlying occurrences in Eucommiaceae, Aucubaceae, and Garryaceae, as well as in Ericaceae. There are some compounds, like geniposidic acid, that it is an intermediate in Route I and II.
METHODOLOGY The data have been obtained after a revision of the literature available by different data basis, such as MedLine or Chemical Abstracts, and by the references in the literature. The order of the tables is based on the alphabetical order of the family, following "The International Plant Name Index", by The Royal Botanic Gardens, Kew, The Harvard University Herbaria and Australian National Herbarium (www.ipni.org/index.htm 1), for the nomenclature. RESULTS The results are presented in tables divided in five columns that include the family of the plant specie where the iridoid has been isolated, the specie, some of the popular uses of the plant, the trivial name of the iridoid, the assayed activity with the result, also, including the negative results, and the reference.
Table 1: Pharmacological assays developed on iridoids biosynthesized by route Ha. Family Species Popular use Iridoid Assayed activity Aucubaceae Aucuba japonica Aucubin An ti-Hepato toxic: • Antidotal effects on Amanita virosa poisoning in beagle dogs. • Against carbon tetrachloride-induced hepatic damage in mice. • Mechanism of hepatoprotection: Stimulating effect on toxic excretion. Anti-inflammatory: • Inhibitor of antigens-induced TNF-a and IL-6 production. • Strongly active (p.t.) against TPA-induced mouse ear edema. ' Active (p.o.) against carrageenan-induced mouse paw edema. Antitumoral: • DNA and RNA polimerases inhibitor (by aucubigenin). • DNA interaction. • Inhibition of NF-kB activation. • Weak cytotoxicity against leukemic and lymphoid human cells. Antiviral (Hepatitis B) Cancer chemopreventive: * Citochrome P-450 inhibition by aucubigenin. Choleretic Hemodynamic Neuritogenic: Induction of neuronal differentiation on PC12h cells • No active, but active the genine Toxicity: • The open-chain aglycone can form an irreversible imine bond with a nucleophilic site of the proteins. Bignoniaceae Catalpa ovata Infections. Catalposide Antibacterial: Inflammation. . Inhibits the binding of FITC-conjugated LPS to CD 14 on the Cancer surface of cells. Antiinflamatory-chemopreventive: • Inhibits TNF-a, IL-ip and IL-6 and the activation of NF-kB in RAW 264-7 macrophages activated with LPS.
References
J24L [25, 26] [27] [28]
[29] [30] [30] [31] [28] [28] [29] [32]
J27L [33] [341
[35.1
[36, 37]
[38]
[39]
[39]
Family Species Bignoniaceae (cont.) C. ovata (cont.)
Popular use
Kigeliapinnata
Skin infections Cancer
Lonicera implexa
Inflammation
Iridoid
Assayed activity
Catalposide (cont.)
Antiinflamatory-chemopreventive(cont.): • Inhibits TNF-a, IL-lp and IL-6 expressions and the nuclear translocation of p65 subunit of NF-kB in RAW 264-7 macrophages activated with LPS. • Inhibits NO production in LPS-stimulated RAW 264-7 macrophages (by suppresion of iNOS expression and iNOS protein) Chemoprevention: Protection against the oxidative injury of Neuro A2 cells. • Active through protection of hydrogen peroxide-induced cell death by zinc protoporphyrin IX. ^_^^^^^^^^^^^^^ Antibacterial and antimycotic Antitumoral: • Cytotoxic on skin tumoral cell lines.
Norviburtinal
References
[39]
[40]
[41]
[42] [42]
Caprifoliaceae
Loganicacid Loganin
Anti-inflammatory: (p.o.) against carrageenan-induced mouse paw edema and (p.t.) against TPA-induced mouse ear edema • Active • Active
[30]
Lamiaceae Ajuga decumbens
A. remota
Inflammation Cough. Respiratory diseases.
8-O-acetylharpagide
Malaria 8-O-acetylharpagide
8-O-acetylharpagide
Cancer chemopreventive: • Strong inhibitory effects on Epstein-Barr virus early agntigen (EBV-EA) induction. • Active as inhibitor on two-stage carcinogenesis test for mouse pulmonary tumors promoted by glycerol. • Active as inhibitor on two stage carcinogenesis test of mouse skin tumors induced by nitric oxide donor (NOR 1) and TPA. • Active as anti-tumor promoter on two-stage carcinogenesis test of mouse hepatic tumor using N-nitrosodiethylamine and phenobarbital. Antiplasmodial against Plasmodium falciparum: • Inactive. Dose 500 (xM. Antitumoral: Cytotoxicity against A431 • Weak activity
[43]
[44]
[45]
[45]
1
Family Species Lamiaceae (cont.) Phlomis armienica P. crinita
Popular use
Inflammation
Iridoid
Assayed activity
References
Ipolamiide Lamiide
Antitumoral (Cytotoxicity, cell lines not showed on the paper) Anti-inflammatory: • Active (p.t.) against TPA-induced mouse ear edema. Cancer chemopreventive: Antioxidant (radical scavenging against DPPH): • Inactive Vasocontracting: against free radical-induced impairment of endothelium-dependent relaxation. " Inactive Antitumoral (Cytotoxicity, cell lines not showed on the paper)
[461 [30]
P. physocalyx Lamiide P. pungens Lamiide Ipolamiide
Scutellaria salviifolia
[47]
[48]
[461
Loganiaceae Buddleja spp
Liver ailments. Aucubin Catalpol Catalposide 7-methoxycinnamoylaucubin 7-p-metoxycinnamoylcatalposide
Anti-Hepatotoxic: Against carbon tetrachloride and galactosamine-induced hepatic damage in cultured hepatocites. • Inactive. • Weak activity. • Inactive • Inactive
[49]
• Weak activity.
Magnoliaceae Eucommia ulmoides
To strengthen tendons and bones, muscle benefit liver and kidney, prevent miscarriage, hypertension, antiaging
Asperuloside Asperulosidic acid Aucubin Deacetyl asperulosidic acid Eucommiol Geniposidic acid
Cancer-chemopreventive: Anticlastogenic effects in CHO cells and mice. • Strong activity. • Strong activity • Inactive. • Weak activity • Inactive. • Strong activity
[50,51]
Family Species Magnoliaceae (cont.) E. ulmoides (cont.)
Popular use
Iridoid
Assayed activity
References [52-54]
Aucubin Geniposide Geniposidic acid
Healing effect: Promoting effect on collagen synthesis and turnover rate in the stratum corneum. • Active " Active • Active Cardioactive effect: • Negative chronotropism, negative inotropism and coronoary perfusion rate.
[55]
Leishmanicidal: In vitro (against amastigotes in macrophage cultures), and in vivo, (in hamsters). • Active. • Active. • Active. • Active.
[56]
Myoporaceae Eremophila alternifolia
For headaches, reduce fever and analgesic effect, eye, skin and body washes
Geniposidic acid
Oleaceae Nyctanthes tristis
arbor- Leishmaniosis Arbortristoside A Arbortristoside B Arbortristoside C 6-P-hydroxy-loganin
Pedaliaceae Harpagophytum procumbens
Pain Arthritis Inflammation Degenerative disease of the skeletal system
Anti-inflammatory: 8-pcoumaroylharpagide Harpagide Harpagoside
• Active as neutrophile elastase inhibitor • No effect on LPS-induced TNF-a release in stimulated primary human monocytes. • Inactive as neutrophile elastase inhibitor. • Inhibitor of leukotriene biosynthesis • No effect on LPS-induced TNF-a release in stimulated primary human monocytes. • No active against carrageenan inflammatory effects (5 and 10 mg/kg) • Analgesic on writhing test (acetic acid-induced pain in mice)
[57] [58] [57] [59] [58] [60] [60]
Family Species Pedaliaceae (cont.) H. procumbens (cont.)
Popular use
Iridoid
Harpagide Harpagoside
Assayed activity
References
Cardiovascular effect: • Negative chronotropic and negative inotropic effects • Negative chronotropic and positive inotropic effects • Protector toward hyperkinetic ventricular arrhythmias induced by reperfusion
[35] [35] [61]
Plantaginaceae Plantago major
Skin and respiratory ailments. Cancer.
Aucubin
Inflammation Hypertension Fever Edemas Hemostatic effects Sedative Liver ailments
Geniposide and genipin
• Antiviral: Suppress hepatitis B virus DNA replication in vitro but, only when is incubated with p-glucosidase (The activity is due to aucubigenin) • Immunomodulatory: Enhances the activity of human lymphocyte proliferation and secretion of IFN-gamma.
[62]
Antithrombotic:
[64]
[63]
Rubiaceae Gardenia jasminoides
Geniposide, genipin, geniposidic acid.
• Active. Prolonge the time required for thrombotic occlusion in the mouse femoral artery. • Both are inhibitors of collagen-induced mouse platelet aggregation. • Inactive as inhibitors of arachidonate-induced, mouse platelet aggregation. • PLA(2) inhibition. Cancer chemopreventive: Active as inhibitor of antitumor promoting induced by EBV and TPA. Choleretic
[65] [34]
• Active
Crosslinking activity • Active Hepatotoxicity: • Active
[66]
[67]
4-
Family Rubiaceae (cont.)
Species
Popular use
G. jasminoides (cont.) Deacetylasperulosidic acid methyl ester. Gardenoside Geniposide Scandoside methyl ester Gardenoside Geniposide, genipin Geniposide, genipin Genipin combined with proteins (blue pigment) Geniposide Genipin and Geniposidic acid
G. americana
Hedyotis corymbosa. H. diffusa
Antineoplastic, anti-toxic and immunoproperties.
Assayed activity
Iridoid
Geniposide
Geniposidic acid
Hypoglycemic: • Active
References [68, 69]
• Inactive • Active • Inactive Neuritogenic: Induction of neuronal differentiation on PC12h cells. • Active. • Active. (PKA stimulation) Neuronal protective: Prevent the toxicity of amyloid beta-protein. Procarcinogenic: • No carcinogenic effect on F344 rats. Purgative Cancer chemopreventive
Antitumoral: • Decreases the growth of the implanted tumor by ascitic cells. • Increase the tumor growth inhibition when is combined with the X-irradiation. • Inhibit the hematological and blastogenic damage by radiotherapy. Antitumoral:. • Decreases the growth of the implanted tumor by ascitic cells. • Increase the tumor growth inhibition when is combined with the X-irradiation. • Inhibit the hematological and blastogenic damage by radiotherapy.
[36]
[70] [71]
[65]
[73]
[73]
1
Family Rubiaceae (cont.)
Species Morinda citrifolia
Popular use Different diseases. Cancer
Asperulosidic acid
Citrifolinoside
Paederia scandends
Saprosma scortechhinii
Assayed activity
Iridoid
Paederoside
Fever Asperuloside Asperulosidic acid 6-epi-paederosidic acid Gardenogenine A Gardenogenine B 6-a-hydroxygeniposide Paederosidic acid Macrophylloside Methylpaederosidate Paederoside Saprosmoside A Saprosmoside D Saprosmoside E Saprosmoside G
Cancer chemopreventive: • Active as inhibitor of TPA and EGF-induced AP-1 transactivation and cell transformation mouse epidermal JB6 cells. • Blocked c-Jun phosphorylation. • Active as UVB-induced Activator Protein (AP-1) activity inhibitor in cell cultures. • Active as inhibitor of TPA and EGF-induced AP-1 transactivation and cell transformation mouse epidermal JB6 cells.. * Blocked c-Jun phosphorylation. Cancer chemopreventive: • Active as inhibitor of Epstein-Barr virus activation and antitumour promoting activity. Anti-inflammatory-Cancer chemopreventive: Hyaluronidase and lipoxygenase in vitro inhibition • Inactive against both. • Inactive against both. • Inactive against both. • Inactive against both. • Inactive against both. • Weak activity against lipoxygenase only. • • • • • • • •
Inactive against both. Inactive against both. Weak activity against lipoxygenase only. Inactive against both. Active against lipoxygenase inhibition Active against lipoxygenase inhibition Active against lipoxygenase inhibition Active against lipoxygenase inhibition
References
[74] [74] [75] [74] [74] [76]
[77]
Family Scrophulariaceae
Species
Popular use
Iridoid
Parentucellia latifolia Peracetylated aucubin Peracetylated catalpol Peracetylated penstemonoside Penststemon serrulatus Penstemide Penstemoside Serrulatoside
Picrorhiza kurroa
Hepatitis and jaundice. Disorders of upper respiratory tract. Fever Dyspepsia Chronic diarrhea Scorpion sting
Penstemoside Picroliv(picroside-1: kutloside)(l:l,5)
Assayed activity
References
Antispasmodic activity: Antagonist non-competitive against acetylcholine. • Active • Active • Active
[78]
Antitumoral: Antiproliferative activity on mouse spleen lymphocytes and hepatoma cells in the Syrian hamster. • Active • Active. • Active Antitumoral: Citostatic activity against P-388 cell line. • Active Anti-Hepatotoxic: Active against aflatoxin B! and Amanita phalloides. through prevention of the biochemical changes induced by aflatoxin Bi • Active against carbon tetrachloride, oxytetracycline and ethanol, galactosamine, thioacetamide or paracetamol- induced hepatic injury in rats • Active induced hepatic injury in rats. • Curative effect on hepatocytes against toxicity induced by thioacetamide, galactosamine, carbon tetrachloride • Active in viral hepatitis and hepatoprotection Antileishmanial: • Active against Leishmania donovani, and enhances the efficacy of other antileishmanials. Antitumoral • Inhibition of topoisomerases I and II. • Inhibition of sarcoma develoopment • Anti-tumor-promoting activity on two-stage carcinogenesis test on mouse skin using DMBA and TPA. • Increase of the life span of transplanted ascites carcinoma. • Reduction of transplanted solid tumors.
[79]
[80] [81-87]
[88-97]
[89] [98] [99] [100]
[101]
^
,
Family Species Scrophulariaceae (cont.) P. kurroa (cont.)
,
Popular use
f^J
Iridoid
Assayed activity
Picroliv (cont.)
Antitumoral (cont.): • Reduction of PKC synthesis. • Selective inhibition of TK Antiviral Cancer chemopreventive: • Antioxidant. • Inhibition of VEGF and HIF-1 expression in glioma cells. • Inhibition of the hepaotcarcinogenesis induced by NNitrosodiethylamine (NDEA) in rats. • Inhibition of enzymatic and non enzymatic lipid peroxidation in microsomes, rat liver and kidney, and induced by different agents •Inhibition (p.o.) of 20-methylcholanthrene (20-MC)-induced sarcoma model and DMBA (7,12-dimethylbenz(a)anthraceneinitiated papilloma formation in BALB/c mice. • Anti-tumor promoting activity on a two-stage carcinogenesis test on mouse skin using DMBA and croton oil. • Increase the life span of transplanted DLA and EAC harboring mice and reduces the volume of transplanted solid tumors • Inhibition of DMG-induced hepatic carcinogenic response and necrosis. • Reduction of gamma-glutamyl transpeptidase • Restoration of catalase and superoxide dismutase levels. • Down-regulation of API factor • Decreased the level of c-fos mRNA and c-jun and c-fos proteins in liver tissue Immuno-modulatory: • Increase T cell response against Mycobacteria. • Anti-allergic through the inhibition of passive cutaneous anaphylasis in mice and protection of mast cells from degranulation. • Absence of a direct post-synaptic histamine receptor blocking activity Preventive of renal injury
References
[102] [103] [104, 105] [102] [105-107] [87, 108113] [101]
['01]
[HI]
[H2]
[114] [115]
[116]
~4
Species Family Scrophulariaceae (cont.) P. kurroa (cont.)
Popular use
Iridoid
Assayed activity
References
Picroliv (cont.)
Protective against hypoxia. • Reduction of the cellular damage caused by hypoxia on Hep 3B and glioma cells. Active against hemorrhage-resuscitation injury • Increased levels of IGF-I and IGF-IR implicated in tissue repair and regeneration after hypoxicischemic injury • Active as protector against hepatic ischemia-reperfusion injury in vivo. • Active as protector of ischemia-reperfusion injury on rat kidneys Neuritogenic: Induction of neuronal differentiation on PC12h cells. • Active. Anti-inflammatory: against ethyl-phenylpropionate-induced edema • Active. • Active.
[117] [ 102]
Rehmannia glutinosa Catalpol Scrophularia auriculata Scropolioside A Scrovalentinoside S. buergeniana 8-O-E-pmethoxycinnamoylharpagide 8-O-Z-pmethoxycinnamoylharpagide 6'-O-E-pmethoxycinnamoylharpagide 6'-O-Z-pmethoxyc innamoylharpagide Harpagide E-harpagoside Z-harpagoside
Neuroprotective: Attenuation of glutamate-induced neurotoxicity on rat cortical neurons. • Active. 1
[112] [118] [119] [HO] [36]
[120]
[121]
Active. Active. Active. Active. Active. Active.
1
Family Species Scrophulariaceae (cont.) S. deserti
Popular use Fever, Kidney diseases, cardiotonic, diuretic, cancer, hypoglycemic
Iridoid
8-O-acetylharpagide Harpagoside Koelzioside Scropolioside D2 Scropolioside D 8-O-acetylharpagide Harpagoside Koelzioside Scropolioside D2 Scropolioside D
S.frutescens
Inflammation Harpagoside
S. ningpoensis Harpagide Harpagoside S. scorodonia
Inflammation
8-O-acetylharpagide Aucubin Bartsioside Harpagide Harpagoside i-O-acetylharpagide Aucubin Bartsioside Harpagide Harpagoside Scorodioside Scropolioside B
Assayed activity
References
Anti-inflammatory: On paw swelling induced by carrageenan • Active • Active, but not significant. • Active • Inactive • Inactive Hypoglycemic: • Strong activity • Inactive • Active • Active, but not significant • Strong activity Anti-inflammatory: On carrageenan-induced edema in rat.. • Weak activity Chemopreventive: Reparing of oxidized OH radical adducts • Inactive • Inactive Anti-inflammatory: Inhibition of PGE2, LTC4 and TXB2 release in calcium ionophore-stimulated mouse peritoneal macrophages and human platelets. • Active as inhibitor of PGE2 and TXB2 release • Active as inhibitor of LTC4 and TXB2 release • Active as inhibitor of TXB2 release (more active than ibuprofen). • Active as inhibitor of LTC4 release • Active as inhibitor of LTC4 and TXB2 release Antiviral: Against HSV-1 and VSV • Weak activity against VSV • Inactive • Inactive • Inactive • Active against VSV • Active against VSV and HSV-1 • Inactive.
[122]
[122]
[123] [124]
[125]
[126]
Family Selaginaceae
Species Lagotis brevituba
Popular use Fever. High blood pressure, Hepatitis
Iridoid
Assayed activity
References [127]
Aucubin
Antitumoral: Citotoxicity against a A-431, BC-1, HT, Lu-1, Mel2, U-373,Raji human Burkitt lymphoma, Hep-3B, E-367, RN-6, S180, B-16, L-929.3T3. • Inactive (EC50 > 20 (xg/mL): " Inactive (EC50 > 20 ng/mL):
Catalpol Verbenaceae Bouchea fluminensis
Stimulating. Regulator of digestive functions. Antiinflammatory
Lamiide
Stachytarpheta cayennensis
Ipolamiide
Verbena officinalis
Hastatoside Verbalin
Anti-inflammatory: • Active (p.o.) in carrageenin-induced paw edema test. Cancer chemopreventive: • Strong inhibitor of rat-brain phospholipid peroxidation. Anti-inflammatory: • Active (p.o.) against carrageenan-induced mouse paw edema. • Active as inhibitor on histamine and bradykinin induced contractions of guinea-pig ileum. Anti-inflammatory: • Active (v.o.) against carrageenan-induced mouse paw edema. Anti-inflammatory: • Active (v.o.) against carrageenan-induced mouse paw edema. • Active (v.t.) against TPA-induced mouse ear edema
[128] [128] [129]
[130] [130] [30]
OS
382
DISCUSSION Many reviews have dealt with the distribution, structure, properties and biosynthesis of iridoids and secoiridoids [131-137] without distinguish between the biosynthetic pathway route. In our case, some of the most studied activities, as anti-inflammatory, antitumoral-chemopreventive, and protective by iridoids biosynthesized by route lla, have been discussed. Anti-inflammatory and analgesic activities: Different assays related with inflammation have been developed on different iridoids. Some iridoids appear to be efficient on subacute processes but not or only slightly active in acute processes. On paw and ear edema, has been established different relationships: Recio and cols.[30] have published the modest relevance of iridoids as systemic antiinflammatory agents. However, some of them, are very active when were locally administrated. This could be due to the weak acidic nature of the iridoids, that makes them more lipophilic in acidic environments such as inflamed tissues. About the structure-activity relationship, they conclude that, the double bond between C-7 and C-8 is responsible for the activity and an OH group at C-5 may increase the activity. The absence or presence of an extraannular carboxyl or carboxymethyl group at C-4 is not relevant, but the conversion of COOH to COOMe increased the topical activity. On the other hand, the facts that can reduce the anti-inflammatory activity could be the oxidation of the double bond to an epoxy group, or an OH-group at C-8. We have realized that other iridoids biosynthesized by route II have not been studied on inflammation tests, and it should be interesting in order to confirm this structure activity relationship. These iridoids might be geniposide, which is similar to bartsioside but with a COOMe group on C4. This assay could find out if COOMe really increase the topical antiinflammatory activity. Testing the activity of ipolamiide, which have an OH-group on 5 and 8, and comparing with lamiide which have OH on 5, 7 and 8 could help to know the real influence of OH radicals on the activity. Besides, only few of these iridoids have been studied further to find their mechanism of action. For example, Bermejo and cols.[125] have
383
studied the effect of some of these iridoids on the arachidonic acid metabolism. Aucubin showed a significant effect as LTC-4 inhibitor, higher than harpagoside and harpagide. Harpagoside and 8-acetylharpagide inhibited PGE-2-release on macrophages. Aucubin, bartsioside, 8-acetylharpagide and harpagoside have shown also TXB-2 inhibition. These results confirm the hypothesis that the anti-inflammatory activity is due to the double bond between C-7 and C-8 with additional OH-radicals in the body of the molecule. Moreover, the substitution with an additional moiety at C-8 is a positive chemical feature for inhibition of TX-synthase activity, conclusion opposite to Recio and cols. [30] where the OH on C-8 reduced the topical activity. Despite of what Bermejo and cols. [125] have published in relation to the unfavorable effect that the presence of a foreign moiety at C-6 have on the anti-inflammatory effect, we think that this conclusion is not clear, since the compounds that they compare do not only have that modification, but also they have oxidized the double bond between C-7 and C-8 as an epoxy group, fact that makes it to loose the activity, as have been described previously [30]. For all these reasons, we also suggest to investigate the inhibitory activity of TX-synthase of lamiide, ipolamiide, loganin and loganic acid, as well as geniposide and derivatives to confirm the structure-activity relationship. In addition, this test could justify the antithrombotic activity showed by geniposide and derivatives [64]. Anti-tumoral and chemopreventive activities: Only few of the iridoids biosynthesized by route II have been assayed for antitumoral or chemopreventive activities. These compounds showed higher cancer chemopreventive than antitumoral activity. The iridoids that stand out against the rest are aucubin, geniposide and derivatives, and picroliv. Aucubin is chemopreventive through different mechanisms: first, it inhibited phase I enzymes, as P-450 [33], avoiding the activation and transformation of procarcinogens to carcinogens agents. It has been proved that the responsible of this activity is its genine, aucubigenin. Secondly, it activated apoptosis, increasing IkB levels, through IkB degradation inhibition and therefore non active NF-kB cannot translocate to the nucleus [29]. It also inhibited antigens-induced TNF-a and IL-6
384
production [29]. This compound has not shown a cytotoxic activity on different tumor cell lines as A-431, BC-1, HT, Lu-1, Mel-2, U-373, Raji human Burkitt lymphoma, Hep-3B, E-367, RN-6, S-180, B-16, L-929 and 3T3 [127], except for Hep G2 cells [28] and a weak activity against leukemic cells [32]. Aucubigenin has shown DNA- and RNA-polimerase inhibition activity [28]. The low cytotoxicity of aucubin is interesting for the chemoprevention point of view. Geniposide and derivatives have also shown very interesting antitumoral and chemopreventive properties: In vivo studies have shown that geniposide with its derivatives, geniposidic acid and genipine decrease the growth of the implanted tumor by ascitic cells, and have a synergic effect with X-irradiation. Besides, geniposide and geniposidic acid are able to decrease undesirable radiation damage to the hematological tissue after radiotherapy [73]. Geniposide inhibits the tumorigenesis induced by different carcinogens, as TPA, benzo-a-pyrene, or Epstein-Barr virus [65, 138]. It can induce phase II enzymes like glutathion-S-transferase (GST) or glutamil-cistein synthetase, helping to the carcinogens detoxification [139]. At the same time, they inhibit phase I enzymes (e.g. cytochrome P-450 and others microsomal monooxygenases), that can activate the transformation of procarcinogens [140]. Besides, it showed an antioxidant activity able to inhibit the lipidic peroxidation, and antimutagenic activity [51], due to the capacity to protect the DNA from the carcinogens-adduct formation, as aflatoxin Bi [139, 141]. Geniposide inhibits enzymes like mieloperoxidase (MPO) or ornithine-decarboxylase (ODC) that are involved on oxidative and tumoral processes [138]. Recently, it has been published the capacity of geniposide to inhibit the angiogenesis, another step of the tumor formation process [142]. Genipin showed a higher chemopreventive activity as inhibitor of induced-formation of carcinogens than geniposide [65]. However,genipin also elicit negative effects like anti-apoptotic [143], mutagenic and genotoxic activities that induce cellular death [144]. A synthetic derivative of geniposide obtained by acetilation, pentaacetyl-geniposide, has also shown cancer chemopreventive properties against some carcinogens [65, 145, 146] and antitumoral activity due to its ability to inhibit DNA and RNA synthesis decreasing therefore the C6 glioma cell line growth rate in vitro and in vivo [147, 148]. This compound also induces apoptosis by different pathways, activating pro-apoptotic
385
signals as p-53 and c-myc, inhibiting Bcl-2 [149] or through the activation of protein kinase C-delta (PKC-6) [150]. Since there are not references about apoptosis and geniposide, we could think that the acetylation of geniposide exert apoptosis activation. We suggest future studies of apoptosis with geniposide. Picroliv, a mixture of the iridoids picroside I and kutloside, has been extensively studied as a chemopreventive agent. It showed antioxidant activity [104, 105], inhibition of lipid peroxidation [87, 108-113], inhibition of VEGF and HIF-1 expression in glioma cells [102], (proteins related to the cell cycle that are over-expressed on tumors), and anti-tumor promoting against different carcinogenesis models [101, 111]. It also down-regulated AP-1 and oncogenes [112]. Picroliv was also able to inhibit sarcoma development, and to reduce transplanted solid tumors, inhibited topoisomerases I and II and different enzymes implicated on the regulation of the cell cycle as, PKC or TK [101, 102]. Picroside and kutloside structures are very similar to catalpol, except for a cinammoyl or vanilloyl radical. Catalpol has not been deeply studied from the antitumoral point of view, and we think that it could be convenient to do it. Catalpol is very close to aucubin with the only difference of the oxidized double bond to an epoxy group. Comparing both of them, we could know the influence of this group on the antitumoral activity. There are others iridoids, as 8-acetylharpagoside that only showed a weak cytotoxicity on A431 [45]. The anti-inflammatory activity also is positive for the chemoprevention, because of the capacity of inhibiting different interleukines release contributes to the chemopreventive activity. Despite most of the compounds did not shown antioxidant activity against free radicals, they were able to inhibit the lipid peroxidation induced by different carcinogens and toxics, also related to thier, also hepatoprotective effect [87, 108-113, 128]. Hepatoprotective and injury protective activities: Some of the iridoids have been tested in vitro and in vivo against different hepatic toxics as a-amanitin or carbon tetrachloride. Aucubin and picroliv are the only iridoids assayed against a-amanitin [24-28], the toxic
386
compound of Amanita sp being both of them active. As seeing on the formula, one of the differences between them is that the C-7-C-8 double bond of aucubin, is oxidized as an epoxy group in picroliv iridoids. Therefore, the activity may be due to the radical OH in C-6 and CH2-OH on C-8. Related to carbon tetrachloride hepatotoxicity, aucubin is active only on in vivo assays [27], but not in vitro [49]. This fact also is observed in other iridoids as loganin or catalposide [49], suggesting that the active agent could be a metabolite. The iridoid that has shown most important hepatoprotective activity is picroliv [88-98], and we emphasize that it could be interesting to assay catalpol in the showed assays for picroliv, and study the influence of these radicals on the hepatoprotective activity. Picroliv has been also assayed against aflatoxin Bi [81-87]. There is some controversy about the effect that geniposide has at the hepatic level. Some authors showed that its hepatotoxicity is due to the increase of transaminase levels [67, 151], whereas recent references defend the protective effect on the liver of this compound [139-141, 152, 153]. Despite genipin is hepatotoxic [67, 151], it is able to increase the glutathion-transferase (GST) levels [139]. This data is surprising, since some of the plants, rich on geniposide are used in popular medicine for treating hepatic diseases. Recently, it has been published that genipin may enhance the bile acid-independent secretory capacity of hepatocytes, effect that make consider genipin as a potent therapeutic agent for a number of cholestatic liver diseases [154]. All these results suggest to work deeply on the hepatic effect of geniposide and derivatives. The protective effects of iridoids are not only at the hepatic or antitumoral levels, but also, may act on neurons or on other tissues against hypoxia. Aucubigenin, catalpol, gardenoside, geniposide and genipin have shown neuritogenic properties, since they are able to induce the neuronal differentiation on PC12h cells through activation of cellular cycle pathways activation as protein-kinase A or cAMP [36, 37]. In addition, genipine is active against Alzheimer's disease, because it can protects the hippocampal neurons from amieloid beta-protein toxicity. [70]. Other iridoids as harpagoside and harpagide and derivatives have attenuated the glutamate-induced neurotoxicity on rat cortical neurons [121]. Catalposide also has been tested as a protector against oxidative injury of Neuro A2 cells, being active by protection of hydrogen peroxide-induced cell death by zinc protophrphyrin IX [41].
387
Picroliv has also shown also a protective effect on different cells and tissues damaged by ischemic injury [102, 110, 112, 117-119], although this compound has not been studied as a neuritogenic agent. Other activities: It is also interesting to comment other activities as the immunoenhancing, anti-allergic, antiasthmatic or healing properties of some of the iridoids biosynthesized by route II. Aucubin, at concentrations lower than 5 \ig/mL significantly stimulated the proliferation of human peripheral blood mononuclear cells and enhanced the secretion of IFN-gamma [63], able to activate non-specific cytocidal activity in macrophages towards a variety of intracellular parasites and neoplastic cells, and also has a direct antiviral action. Geniposide has anti-asthmatic properties, because its inhalation is able to decrease the tight junction permeability of trachea and to produce the reestablishment of the integrity of the tissue affected by the antigens exposure [155]. Picroliv has shown anti-allergic properties, through the inhibition of passive cutaneous anaphylasis in mice and protection of mast cells from degranulation, although, this effect is not due to a direct blocking of the histamine receptor [115]. Picroliv is also able to increase T cell response against infections by Mycobacteria sp. [114] Aucubin, geniposide and geniposidic acid have promoting effects on collagen synthesis and turnover rate in the stratum corneum [52-54]. This activity has made geniposide subject of clinical investigation as a crosslinking agent [66]. In conclusion, we could say that on a wide sense, the dialdehydes generated by the metabolism and hydrolysis of the aglycones of iridoids, could be in part responsible for the described activities. The non-saturated carbonyl group can react with the SH groups, from amino acids and nucleic acids, causing different effects as the cellular growth inhibition [38, 77]. It seems that the pharmacological activities showed by different iridoids are due to their genine, like aucubin. The structural similarity between the aglycone and glutaraldehyde suggests a similar mechanism of enzyme inhibition through protein cross-linking by Schiff reactions.
388
ABBREVIATIONS p.o. p.t. TNF-a IL-6 NF-kB LPS iNOS NO EBV-EA TPA A431 DPPH PLA(2) PKA EGF 20-MC INF DMBA NDEA IGF-I IGF-IR PGE2 LTC4 TXB2 HSV-1 VSV BC-1 HT Lu-1 Mel-2 U-373 Hep-3B E-367 RN-6 S-180 B-16 L-929
Administrated orally. Administrated topically. Tumoral necrosis factor Interleukine Nuclear factor k-B Lipopolysaccharide inducible Nitric oxide synthase. Nitric oxide. Epstein-Barr virus early antigen 12-0-tetradecanoylphorbol-13 -acetate Human epidermoid carcinoma 1,1 ,-diphenyl-2-picrylhydrazyl radical Phospholipase A-2 Protein-kinase A Epithelial growth factor. 20-methylcholanthrene Interferon 7,12-dimethylbenz(a)anthracene N-Nitrosodiethylamine Insuline growth factor Insuline growth factor receptor Prostaglandin E-2 Leukotriene C-4 Thromboxane B-2 Herpes simplex type 1 Vesicular stomatitis virus Human breast cancer Human fibrosarcoma Human lung cancer Human melanoma Human glioma Human hepatocarcinoma Rat neuroblastoma Rat neurinoma Mouse sarcoma Mouse melanoma Mouse conective tissue
389
3T3 HeLa
= =
Mouse fibroblast Human epitheloid cervical carcinoma
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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CHEMISTRY AND NEUROTROPHIC ACTIVITY OF S£C0-PREZIZAANE- AND ANISL AC TONE-TYPE SESQUITERPENES FROM ILLICIUM SPECIES YOSHIYASU FUKUYAMA3 and JIAN-MEI HUANGb "Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan and bBeijing University of Chinese Medicine, Beijing 100029, China ABSTRACT: This review focuses mainly on our findings of chemistry and neurotrophic activity of the seco-prezizaane- and anislactone-type sesquiterpenes. The genus lllicium is the only member of the family Illiciaceae. From a chemical and biological point of view, the lllicium species are known to be rich in biosynthetically unique seco-prezizaane-type sesquiterpenes, some of which have been known to exhibit neurotoxic activity. Our continuing chemical studies of the lllicium species such as lllicium anisatum, I. tashiroi, I. merrillianum and /. jiadifengpi have resulted in the isolation of a number of sesquiterpenes, most of which belong to both of secoprezizaane- and anislactone-type sesquiterpenes. Chemical studies on unique sesquiterpenes, such as illicinolide, tashironin, merrilactone A, and jiadifenin isolated from the above plants, allow us to further categorize .seco-prezizaane-type sesquiterpenes into six sub-groups according to their carbon skeletons: anisatin-subtype, pseudoanisatin-subtype, majucin-subtype, minwanensin-subtype, cycloparvifloralonesubtype, and a//o-cedrane-type. On the basis of chemical correlation of these subtypes, plausible biosynthetic pathway for all the subtypes of seco-prezizaane-type and anislactone-type sesquiterpenes is discussed. In addition, some sesquiterpenes such as merrilactone A, 11-debenzoyltashironin and jiadifenin are shown to have potent neurite outgrowth promoting activity in primary cultured rat cortical neurons.
INTRODUCTION The genus lllicium belongs to the family Illiciaceae and is an evergreen shrub or tree. About 40 species have been disjunctively distributed in the eastern North America, Mexico, the West Indies and the eastern Asia. The highest concentration of species is in the northern Myanmar and the southern China where nearly 35 species have been described [1]. The genus is comparatively primitive and has close affinities with the Magnoliaceae. Therefore, lllicium was classified into the family Magnoliaceae in much of the early taxonomic literature [2] but was subsequently excluded from the Magnoliaceae by Smith on the basis of floral morphology and vegetative anatomy [3] and assigned familial rank as the Illiciaceae.
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The fruits of the Illicium species are distinctive star-shaped follicles which emit a characteristic refreshing flavor. The fruits of /. vernum Hook, in particular, are the source of the only economically important product derived from the genus Chinese star anise, which is widely used as a spice for flavoring food and beverages. Hence, essential oils have been the primary subject of chemical research on Illicium species and the presence of volatile phenols have been reported as constituents of various parts of all Illicium studied so far. On the other hand, the fruits of /. anisatum, Japanese star anise, have been known to be very toxic for several centuries. Many researchers had been involved in the attempt to isolate the toxic substance since the middle of the 19th century. In 1952, Lane et al. succeeded in the isolation of the pure toxic principle named anisatin (1) [4] for the first time. Its complete structure was later established by Yamada and Hirata [5]. Since anistatin (1) and neoanisatin (la) were recognized as the unprecedented sesquiterpene dilactone containing an unusual P-lactone, several other members of the toxic Illicium species have also been the subject of chemical investigation mainly for Fig. (1). Structure of anisatm(l) ^ P Ur P 0Se ° f clarifying relationship and neoanisatin (la) between structure and toxicity, thus resulting in the isolation of a number of unusual sesquiterpenes and neolignans. Kawano et al., who had continued to investigate the toxic substance in /. anisatum [6], succeeded in systematic studies on chemical components in Illicium plants. Later on, Schmidt and our group have joined in chemical and biological studies on Illicium plants. A number of unique seco-prezizaane-type sesquiterpenes or so called Illicium sesquiterpenes have been reported exclusively by the above three groups, and the occurrence of Illicium sesquiterpenes has been found limited to the genus Illicium. The results of previous research have culminated in classifying them into further subgroups on the basis of a lactone-type as follows: anisatin-subtype, pseudoanisatin-subtype, minwanensin-subtype, majucin-subtype, pseudomajucin-subtype, and cycloparvifloralonesubtype. Also classification of two main types, anislactone-type and a//o-cedrane-type, which consist of rare carbon skeletons, differs from seco-prezizaane-type. This review surveys first the reported secoprezizaane-type sesquiterpenes in terms of the formation of a lactone, and secondly focuses particularly on our chemical studies and lastly on the neurotrophic activities of the sesquiterpenes isolated from /. tashiroi, I. minwanense, I. merrillianum, and /. jiadifengpi.
397
Seco-Prezizaane-Type Sesquiterpenes Anisatin-Subtype
The presence of a very toxic anisatin is noticed in many Illicium plants, such as /. majus [7], /. floridanum [8], /. minwanense [9] and /. merrillianum [10]. Anisatin-subtype sesquiterpenes are characterized by having two types of a 13,14-|3-lactone and a 11,7-6-lactone. The previously reported anisatin-type sesquiterpenes are shown in Fig. (2). All of them except for 5 and 8 belong to neoanisatin derivatives lacking the hydroxyl group at the C-3 position. 1-Hydroxyneoanisatin (2), 6deoxy-1-hydroxyneoanisatin (3), 2-oxo-6-deoxyneoanisatin (9), and an epimeric mixture of 10 and 11 were isolated from /. majus [11, 12], whereas 2a-hydroxyneoanisatin (4) and 2a-hydroxyanisatin (5) were isolated from the pericarps of/, anisatum [13] and /. merrillianum [14], respectively. It should be noted that veranisatins A (6), B (7) and C (8) were isolated as new convulsants in a small amount from the non-toxic Chinese star anise (/. verum Hook, f.) which has been used as a basic spice and also in traditional Chinese and Japanese medicines. They have caused severe convulsions and death at 3 mg/kg (p. o.) in mice [15, 16]. Compound 9 also exhibited picrotoxin-like convulsion and its LD50 was 1.46 mg/kg in mice (i. p.) [11]. HO
HO
RN, H O O 2 R, = R4= OH, R2 = R3 = H 3 R 1 = O H > R 2 = R3 = R4 = H 4 R! = R3 = H, R2 = R4 = OH 5 Rt = H, R2 = R3 = R4 = OH
6 R, = H , R2 = CH2OCH3 7 R, = H, R2 = CO2CH3 8 R = OH R = l > 2 CO2CH3
10 R, = H, R2 = CH3 11 R! = CH3, R2 = H
Fig. (2). Anisatin-type sesquiterpenes 2 - 1 1
Pseudoanisatin-Subtype
Pseudoanisatin was first isolated as a nontoxic compound from /. anisatum by Lane et al [4]. The wrong structure of pseudoanisatin was first proposed on the basis of the spectral data [17], but later it was revised as 12 with a 7-membered 11,14-lactone by an X-ray crystal
398
HO. HO
R, HO 12 R, 13 R, 14 R, 1SR, 16 R, 17 R,
= H, R2 = R3 =OH = R2 = H, R3 = OH = OH, R2 = R3 = H = R2 = OH, R3 = H = R3 = H, R2 = OH =H, R2 = =O, R3 = OH
18 R, = R2 = OH 19 R, = H, R2 = OH 20 R, = OH, R2 = H
23
Fig. (3). Pseudoanisatin-type sesquiterpenes 12-24
26a
27 R, = R3 = OH, R2 = R4 = H
29
Fig. (4). Ketone and acetal equilibrium of pseudoanisatin-type sesquiterpenes 12 and 25 - 26, and acetal sesquiterpenes 27 - 29
399
structure determination [18]. Another pseudoanisatin-type sesquiterpenes, 6-deoxypseudoanisatin (16) [19, 22] and l a hydroxypseudoanisatin (18) [20] occurred in the seeds, fruits and leaves of /. anisatum. Recently, another deoxypseudoanisatins, 3dexoxypseudoanisatin (13), 2|3-hydroxy-3,6-dideoxypseudoanisatin (14) and la-hydroxy-3-deoxypseudoanisatin (19) were isolated from /. merrillianum [21], and (2iS)-hydroxy-6-deoxypseudoanisatin (15), 3oxopseudoanisatin (17) and la-hydroxy-6-deoxypseudoanisatin (20) were isolated from /. miwanense [22]. 3,6-Dideoxy-10hydroxypseudoanisatin (21) bearing a hydroxy group at the C-10 position was first found in the fruits of /. merrillianum [24] along with 2,10-epxoy-3-dehydroxypseudoanisatin (22) having an ether linkage between C-2 and C-10. The unusual structure of 22 was established by X-ray crystallographic analysis [25]. Another group of unusual sesquiterpenes having a 1,4 ether linkage and a 14,15-6-lactone named 1,4-epoxy-6-deoxypseudoanisatin (23) and neodunnianin (24) were isolated from /. dunnianum [30]. Later, the structure of 23 was revised to be the same as that of 20 by comparing the spectral data of both. Some pseudoanisatin-type sesquiterpenes were lately found to coexist as ketone and acetal equilibrium such as pseudoanisatin (12) and cyclopseudoanisatin (12a), parviflorolide (25) and cycloparviflorolide (25a) [26], merrillianolide (26) and cyclomerrillianolide (26a) [10] as shown in Fig. (4). It is interesting that 8a-hydroxy-10deoxycyclomerrillianolide (27) [21], 2a-hydroxycycloparviflorolide (28) [14] and an acetal form of 6-deoxypsudoanisatin (29) [23] exist exclusively even in methanol-G?4. However, we have no evidence to rationalize what kind of factors play key role in a favorite equilibrium between a keto and an acetal form in pseudoanisatin-subtype on the basis of the global minimum energy calculated by MM2. The structures of the sesquiterpenes, which belong to pseudoanisatin-subtype having a hydroxyl group at the C-7 position, seem to be confused. The structure of 7-deoxy-7(3-
HO
R,Cf
HO
OR, 30 R, = R 3 == H, 31 R, = Bz, R2 = 32 R, = Ac, R2 = 33 R, = Bz, R2 -
R2 = O H OH, R3 = H OH, R3 = Bz K3 - M
30a R, = R3 = H,R 2 = OH 31a Rj = Bz, R 2 == OH.R3 = H 32a R, = Ac, R 2 == OH =Bz 33a Ri = DZ, K.7= = R 3: = H
Fig. (5). Revised structures of pseudoanisatin-type sesquiterpenes 30-33
OH 30b
400
hydroxypseudoanisatin [13] from the seeds of /. anisatum was first elucidated as pseudoanisatin-type sesquiterpene 30, then revised as an 11,3-6-lactone 30a and finally corrected as an 11,7-5-lactone 30b [Fig. (5)] [8], which belongs to the minwanensin-type. Other Illicium plants, /. dunnianum, I. tashiroi and /. merrillianum elaborated dunnianin (31), 6deoxydunnianin (33) [27], and isodunnianin (32) [28], respectively. The structures of these compounds were assigned on the basis of NMR spectral data and by comparison with spectral data of pseudoanisatin (12) and dunnianin (31). Schmidt et al. [29] reported that dunnianin (31) and 7-deoxy-7|3-hydroxypseudoanisatin (30) were isolated from the American star anise, /. floridanum, and their structures were reinvestigated by NMR spectroscopic analyses as well as X-ray crystallographic analysis of dunnianin. As a result, the structures of 31 and 30 were revised as 31a and 30a, which consist of an 11, 3-6-lactone instead of an 11, 14-e-lactone. In comparison with their spectral data, the occurrence of the very large geminal coupling constant (near 20 Hz), accounting for the presence of a 6-lactone ring, and the appearance of H3 as a doublet (about 5Hz) with essentially no coupling with H-2|3 were found to be characteristics of sesquiterpenes having an 11, 3-6-lactone. In light of the spectral data of the other pseudoanisatin-type sesquiterpenes 32 and 33, the structure of 32 and 33 may be revised as 32a and 33a. However, the structure of 30a was revised one more time as 30b in 1999. The 11, 3-6-lactone ring was revised to a 5-lactone closed between C-ll and C-7. However, the vicinal coupling constant of about 0 Hz between H-2|3 and H-3 was not consistent with such a structure. The X-ray crystallographic analysis proved that the structure of 30b was correct and the aforementioned spectral discrepancy could be attributed to a strong intramolecular hydrogen bond between the OH group attached to C-14 and C-3 [8]. Pseudoanisatins derivatives, which have been checked to this point, are non-toxic substances unlike anisatin, but it is worthy to note that isodunnianin (32) not only promote neurite outgrowth in primary cultured fetal rat cortical neurons at 10 uM but also increase the choline acetyltransferase activity [28]. Miwanensin-Subtype
The opening of the spiro (3-lactone of the anisatin-type sesquiterpene leads to the minwanensin-type. The structure of minwanensin (34) from the pericarps of/, minwanense was elucidated [9] and later revised by X-ray crystallographic analysis of its pbromobenzoyl derivative [23], as shown in Fig. (6). This type of compounds, such as 3-acetoxy-14-«-butyryloxy-10-deoxyfloridanolide (35), 14-acetoxy-3-oxofloridanolide (36), 13-acetoxy-14-(«-
401
butyryloxy)floridanolide (37), debenzoyldunnianin (30b) and \A-O-nbutyrylfloridanolide (38), were also found in the fruits of /. floridanum [8] and /. merrillianum [24], respectively. Recently, two C-l epimeric keto forms, (15) and (li?)-miwanenones (39, 40) were isolated from /. miwanense [21]. Additionally 3,4-dehydro-compounds having a hydroxyl group at C-10, 3,4-dehydro-13,14-dihydroxyfloridanolide (41) [31] and 3,4-dehydrofloridanolide (42) [24] also occurred in the above two plants. The structure of minwanensin is considered to be similar to that of anisatin, but it does not show toxicity to mouse at a dose of 50 mg/kg (p. o.). It has no neurotrophic effect either. This suggests that the presence of a (3-lactone in a molecule may be responsible for neurotoxicity.
HO
RiO
34 R, =H, R2 = OH, R3=H 35 R, =Ac, R2 = OCO/iPr, R3= OH
36 R, = = O, R2 = OAc, R3=H 37 R, = H, R2 = OCOnPr, R3= OAc 38 R, = H, R2 = OCOnPr, R3= H
HO Ho.
39 R, = CH3, R2 = H 40 R, = H, R2 = CH3
41 R = OH 42R = H
Fig. (6). Minwanensin-type sesquiterpenes 34-42
Majucin-Subtype
A number of new majucin-type sesquiterpenes having a y-lactone ring as shown in Fig. (7) were found in the pericarps of /. majus, belonging to one of the Chinese Illicium plants. Majucin (43) was the first to assign structure by extensive spectroscopic analysis and
402
comparing its NMR data with tnose of anisatins as well as the data of neomajucin (44) established by an X-ray diffraction method [32, 7]. More majucin-type sesquiterpene lactones, such as (2S*)hydroxyneomajucin (45), 2-oxoneomajucin (48), 2,3-dehydromajucin (49), (2i?*)-hydroxy-3,4-dehydroneomajucin (51), (15*)-2-oxo-3,4dehydroneomajucin (52), (li?*)-2-oxo-3,4-dehydroxyneomajucin (53) and (IR*, 101S'*)-2-oxo-3,4-dehydroneomajucin (54), were reported [7, 34]. Particularly, it should be noted that compound bearing the (10S1*)hydroxyl group is only 54 among the anisatin-like sesquiterpenes. 6Deoxy-neomajucin (46), isolated from the seeds of/, anisatum, is the
R2
43 44 45 46 47
R, R, R, R, R,
= = = = =
H, R2 R3 R3 R2
R 2 = R 3 = OH, R, = H = H, R 3 = OH, R4 = H =OH, R 2 = H, R4 = H = H, R 2 = OH, R4 = H = H, R 3 = H, R 4 = Bz
HO
HO
'.
51
52 R, = CH3, R2 = H 53 R, = H, R2 = CH3
54
CO 2 CH 3
HZ
55
Fig. (7). Majucin-type sesquiterpenes 43-55
first example of the majucin type found in the Japanese star anise [19]. /. angustisepalum also contains the majucin-type sesquiterpene like 10benzoyl ester of neomajucin (47) [33]. In the course of searching for neurotrophic compounds in /. jiadifengpi, we could isolate jiadifenin (55) and 51 as active substances together with a new sesquiterpene, 1,2dehydroneomajucin (50) [35]. In particular, 55 is the first example of a majucin-type sesquiterpene with an oxo-function at the C-10 position. However, since jiadifenin consists of an inseparable equilibrated mixture
403
with regard to the C-10 acetal carbon, its structure elucidation remained ambiguous on the basis of the spectral data. In order to confirm and establish the absolute structure of 55, a synthesis of 55 was attempted starting from 2-hydroxy-3,4-dehydroneomajucin (51), which was the main sesquiterpene isolated from /. jiadifengpi. HO. -0.14 - 0 . 0 ^ MTPAO
( + 0.09
+ 0.13 Fig. (8). A6 (S-R) values (ppm) for MTPA ester derivatives of 51
First, the modified Mosher's method [36] was applied to clarify the absolute configuration of 51 which had never been determined. The A 6 (S-R) value as shown in Fig. (8) enabled us to assign the configuration for C-2 as S. Next, the C-2 hydroxyl group of compound 51 was oxidized with Dess-Martin reagent to give ketone 52 in 81% yield, which previously was obtained as a natural product from Illicium majus [7]. Treatment of 52 with DBU in benzene caused an epimerization on the C-l carbon, resulting in a thermodynamically more stable product 53 [34]. It is noted herein that the chemical conversion of 51 to 52 and 53 can assign the absolute configurations of 52 and 53 as (lS)-2-oxo-3,4-dehydroneomajucin and (li?)-2-oxo-3,4dehydroneomajucin, respectively. It is anticipated to spontaneously produce compound 55a if the C-10 hydroxyl group is oxidized to ketone. Thus, 53 was subjected to Swern oxidation, giving rise to unexpected oxetane 56. This unusual reaction was reasonably rationalized based on the generally accepted mechanism of Swern oxidation as indicated by the following. A sulfoxonium intermediate A abstracts more acidic H-l from H-10, and then the formed carbanion attacks on the oxygen of the sulfoxonium species in B, resulting in the formation of 56. However, Dess-Martin reagent could oxidize the C-10 hydroxyl group to ketone without problem and a usual work-up provided the expected acetal 55a in good yield. Adding methanol to the reaction mixture led this oxidation to the direct formation of jiadifenin (55) as an equilibrated mixture, which was identical in all respects with the natural mixture [35]. It should be noted that compound 56 exhibited neurotrophic activity at 1 \iM, compared with that of 51.
404
HO,
O 52
51
53
56 HO.
CO2H
52
55a
55
a
Scheme l. Synthesis of jiadefenin (55) from 51 "Reagents and conditions: (a) Dess-Martin reagent, CH2C12, r. t., 81%; (b) DBU, benzene, 80°C, 74% (c) (COC1)2, DMSO, CH2C12, -78°C; (d) Et3N, -78°C - 0°C, 34%; (e) Dess-Martin reagent, dioxane, r. t , 70%; (f) MeOH, r. t., 90%.
Pseudomajucin-Subtype
Sesquiterpenes belonging to pseudomajucin-subtype as shown in Fig. (9) feature a y-lactone ring closed in a 11,4-manner. Typical ones are pseudomajucin (57) and its 7-O-(3-D-glucoside (58), which were isolated from the pericarps of /. majus [37]. The structure of 57 was established using X-ray crystallographic analysis. The glucoside linkage position in 58 was determined as C-7 by glycosylation shift (2.6 ppm) at C-7 compared with that of 57. Another group of novel
405
sesquiterpene lactones, (6i?)-pseudomajucin (60), (6i?)pseudomajucinone (61) and merrillianin (62), were isolated from the pericarps of/, merrillianum, and their structures were elucidated on the basis of the spectral data [38]. It should be noted that a methanol solution of 60 coexists with (6i?)-pseudomajucinone (61) in keto/acetal equilibrium, whereas the crystals solely consist of keto-type 61. In fact, treatment of 60 with trimethylorthoformate and methanol in the presence of Amberlyst R 15 afforded a sole 7-O-methylated product 60a, which was then reacted with 4-bromophenyl isocyanate and 1,8diazabicyclo[5.4.0]undec-7-ene as a base in toluene to give rise to the/?bromophenylcarbamate 60b as a single crystal, thus suitable for X-ray analysis. These results allowed us to assign the absolute configurations of the chiral centers of 60b as IS, 2R, 4S, 5S, 6R and 9R, and thereby 60 could be determined as (67?)-pseudomajucin. Accordingly, 61 turns out to be (6i?)-pseudomajucinone having the same absolute configuration as that of 60. Moreover, 61 was crystallized from an ethyl acetate solution although 60 and 61 coexisted as an equilibrated mixture in solvent. Thus, the stereochemistry of 61 was unambiguously established by Xray crystallographic analysis. The structure of 61 was obviously identical with the keto-form of 60 as shown in Fig. (9). Although some pseudoanisatin-type sesquiterpenes have been reported to occur as an
10
R,O 12
12 57 R, = R2 = H 58 R, = H, R2 = Glucose 59 R, = COnPr, R2 = H
60 R, = R2 = H 60a R, = H, R2 = Me 60b R, = CONHp-BrPh, R2 = Me
OH
63 Fig. (9). Pseudomajucin-type sesquiterpenes 57 -62
406
60b
acetal/keto equilibrium [10, 26], it is the first example that each absolute structure of pseudomajucin-type sesquiterpenes (60 and 61) coexisting in an acetal/keto equilibrium has been established independently by Xray crystallographic analysis as shown in Fig. (10). In contrast with (6£)-pseudomajucin (57) existing as a sole acetal-form, its 61 6i?-form 60 readily reaches to keto-form 61 in equilibration. Fig. (10). The ORTEP drawings of 60b and 61 Although we attempted MM2 calculations to compare the global minimum energy between 60 and 61, no reasonable explanation could be provided for 60 and 61 coexisting as an acetal/ketal equilibrated mixture, as well as for (6S)pseudomajucin (57) not being so. Merrillianin (62) is a unique seco-prezizaane-type sesquiterpene with an unprecedented dilactone having a seven-membered 14,7-lactone, which may be biosynthesized from pseudomajucin (57) or an unknown pseudomajucin-type sesquiterpene 63 by oxidative cleavage of the C6/C-7 bond. The oxidative cleavage of the C-6/C-7 bond in 63 or 57 should lead to 62. However, the oxidative cleavage of the C-6/C-7 bond of pseudoanisatin-type sesquiterpenes also may afford a similar skeleton, which takes a spatially revised relationship of two lactone rings in comparison with 62. Thus we attempted an oxidative cleavage of a pseudoanisatin-type sesquiterpene, 1 a-hydroxy-3-deoxypseudoanisatin (19), which we previously isolated from /. merrillianum [10]. Oxidative cleavage of 19 with sodium periodate in ether and water
407
,OH
(O)
57
NaIO4
HO \5
OH
ether/H2O r.t. 88%
19 Scheme 2. Oxidative cleavage of the C-6/C-7 bond of 19
nicely provided the product 64 in good yield (Scheme 2). The structure of 64 was elucidated by extensive analysis of spectroscopic data. Especially, the NOESY spectrum of 64 was carefully compared with that of 62. The NOESY spectrum of 64 revealed the presence of an additional correlation between H-8a and H-12, and no correlation between H-8p and H-15. Hence, the y-lactone ring in 64 is fused down onto C-4 and C-9 opposite to that of 62. This chemical result not only confirms the configuration of the lactone rings in 62, but also supports that a plausible biosynthetic precursor of 62 may be either the unknown pseudomajucin 63 or pseudomajucin (57). Merrillianin (62) is the first example of the C-6/C-7 seco-pseudomajucin-type sesquiterpene, which can be regarded as biogenetically significant for a variety oilllicium sesquiterpenes [38]. Cycloparvifloralone-Subtype
In 1999, new cycloparvifloralone-type sesquiterpenes were reported to consist of a unique acetal-hemiacetal and/or ortholactone structure as shown in Fig. (11). Cycloparvifloralone (65) occurred in the leaves of/, parviflorum, and (11)7,14-ortholactone-14-hydroxy-3-
408
65 R, = R2 = H 66 R, = OH, R2 = H 67 R, = H, R2 = OH HO,
68
HO
f HO HO
'-
OH
71 Fig. (11). Cycloparvifloralone-type sesquiterpenes 65-72
oxofloridanolide (70) was isolated from the fruits of /. floridanum [26], whereas /. merrillianum yielded a number of this type of sesquiterpenes, such as 2a-hydroxycycloparvifloralone (66) [14], 3ahydroxycycloparivifloralone (67) [39], merrillianone (68) [10], 1,2dehydrocycloparvifloralone (69) [39], (ll)7,14-ortholctone-3ahydroxyfloridanolide (71) [39] and merrilliortholactone (72) [14]. All of these possess a hitherto rare ring system with a cage-like acetalhemiacetal and/or an orthoester structure. As cycloparvifloralone-type sesquiterpenes contain an acetal-hemiacetal or an orthoester group in a molecule, it can be anticipated that they are probably equilibrated between an acetal-hemiacetal and an aldehyde-ketone or between an orthoester and a lactone. However, neither of the aldehyde-ketone form or the lactone for 65-72 has been detected by the NMR spectra even in a protic solvent such as methanol-6?4. This means that the presence of compounds 65-72 in solution is considerably favored over that of their 11,7- or 11,14-lactone forms in keto/acetal and/or lactone/orthoester equilibrium. It is generally accepted that they should coexist with each corresponding aldehyde-ketone or lactone form. In fact, acetylation of 68 under normal conditions afforded diacetate 68b as a single product. The absolute structure of 68b was unambiguously established by X-ray crystallographic analysis [14]. This result indicates that compound 68 is equilibrated with aldehyde-ketone 68c through hemiacetal-ketone intermediate 68a. It should be noted that recently 8-
409
deoxymerrilliortholactone (73) and its lactone form 74 were found as an inseparable equilibrated mixture in/, merrillianum [21].
CHO
68
HO-.J
HO
'-
OH
73 Fig. (12). Acetal and aldehyde, and ortholactone and lactone equilibrium of cycloparvifloralone-type sesquiterpenes 68, 73 and 74
/4//0-Cedrane-Type Sesquiterpenes New carbon skeletal sesquiterpenes, which are not able to take their place with the known subclasses of seco-prezizaane-type sesquiterpenes typical of Illicium plants, have been found as natural products, as shown in Fig. (13). A new skeletal sesquiterpene named tashironin (75) was isolated first from /. tashiroi [40] and later found with 11-O-debenzoyltashironin (76) in /. merrillianum [39]. The structure of tashironin (75) was elucidated by extensive analysis of spectroscopic data. Tashironin consists of a 2oxatricyclo[4.3.1.04'9]heptane skeleton, which belongs to a unique tetracyclic sesquiterpene possessing the very rare a//o-cedrane skeleton. Following our report on the isolation of tashironin, three tashironin congeners, debenzoyl-7-deoxo-la,7a-dihydroxytashironin (77), debenzoyl-7-deoxo-7a-hydroxytashironin (78) and debenzoyl-7-deoxo7a-hydroxy-3-oxotashironin (79), were isolated from I.floridanum [31]. Although anisatin (1) and its related seco-prezizaane-subtypes such as pseudoanisatin (12) have been postulated to be biosynthesized
410
from a tricarbocyclic precursor, a//o-cedrane [41], there is no evidence to support this plausible biosynthetic route leading to seco-prezizaane skeleton. It has been still obscure because no tricarbocyclic sesquiterpene made up by the same carbon skeleton as a//o-cedrane has been found in the Illicium species. Thus it should be emphasized that tashironin (75) possesses a precise carbon skeleton corresponding to the a//o-cedrane skeleton, which can be regarded as a biogenetic key for an intermediate leading to all the Illicium sesquiterpenes, i.e. anisatin-, pseudoanisatin-, majucin-, miwanesnin-, pseudomajucin- and cycloparvifloralone-subtypes. Other new skeletal sesquiterpenes named illicinolide A (80) and B (81) were isolated from /. tashiroi [44, 45]. The structure of illicinolide A was elucidated on the basis of the spectral data and then its absolute configuration was established by X-ray crystallographic analysis of the />-bromobenzoyl derivative 80a [44]. The structure of Illicinolide B was assigned as 6o>hydroxyillicinolide A by spectral data compared with those of illicinolide A and its absolute structure was determined by applying the CD dibenzoate rule to the /?-bromobenzoyl derivative 81a [45]. While illicinolides A and B are likely to be closely related to the previously reported anisatin (1) and majucin (43), the structural feature containing y-lactone ring closed between C-7 and C-9 is rather similar to noranisatin [46], an oxidatively degraded product of anisatin.
O VOH
75 R = Bz 76R = H
77 R, = OH, R2 = H 78 R, = R2 = H 79 Rx = H, R2 = =O
Fig. (13). /4//o-cedrane-type sesquiterpenes 75-81
80 80a 81 81a
R, R, R, R,
OCH3 = R2 = R3 =H = R2 = /7-BrBz, R3 = H = R2 = H, R3 = OH = R2 =/?-BrBz, R3 = OH
411
It is generally accepted that anisatin could be biosynthesized from an acorane through a tricarbocyclic precursor, a//o-cedrane, after breaking the bond between C-6 and C-ll as shown in Scheme 3 [47]. Tashironin should be derived directly from a//o-cedrane through oxidation of the C-ll in a cationic intermediate A. On the other hand, the highly oxygenated abnormal structure of illicinolides A (80) and B (81) may be rationalized biogenetically by assuming that the C-ll HO
Seco-prezizaane
HO' HO
j
OH
Pseudoanisatin (12)
Acorane
HO Tashironin (75)
HO HO' OCH3 Illicinolide A (80) Scheme 3. Plausible biosynthesis of seco-prezizaanes, tashironins and illicinolides through a common key biosynthetic intermediate, a//o-cedrane
412
carbon would originate from the C-11 in the normal anisatin skeleton by breaking both the C-ll/C-7 and C-ll/C-10 on a tetracyclic intermediate B which could be made up through a bond formation between C-ll and C-14 from a//o-cedrane as outlined in Scheme 3. Thus, the isolation of tashironin, illicinolide, anisatin, and their related sesquiterpenes from the same source is of considerable significance and throws light on the biogenesis oi Illicium sesquiterpenes. Anislactone-Type Sesquiterpenes In 1989, anislactones A (82) was isolated first as a minor component from the fruits of /. anisatum [48]. The structure of anislactone A was established by X-ray crystallographic analysis, whereas anislactone B (83) was determined to be an epimer with regard to the hydroxyl group attached at the C-7 position [49]. Both compounds have a unique carbon-skeleton which has never been recorded as of natural compounds. Anislactones are most likely to be from the majucin-subtype sesquiterpene because they bear a y-lactone ring typical of majucin. According to structural similarity, the biosynthesis of anislactones was proposed to be presumably derived from the majucin-type sesquiterpene as follows: the ring construction occurs between C-7 and C-8 in the majucin-type compound, followed by the bond formation of C-6 and C-8, and then hydroxylation at C-8. In this case, C-7 should become a C-8 methyl group. Although this seems to be a better explanation for converting from the majucin-type compound to anislactones, the inversion of the C-9 configuration as well as the origin of the C-8 methyl group still have remained ambiguous. Herein, we propose a new term "anislactone" for this type of sesquiterpenes since anislactones should take a position independent of the previously known Illicium sesquiterpenes. We found that anislactone B (83) was the main component in Illicium merrillianum. Following the isolation of 83 and 82, other new anislactone-type sesquiterpenes named 7-0- actetylanislactone B (84) [10], merrilactones A (85) [51], B (88) [50] and C (86 and 87) [50] were obtained from the pericarps of /. merrillianum. In particular, merrilactone A (85) possesses an oxetane ring in feature with two kinds of y-lactones and has an interesting neurotrophic property such as promoting neurite outgrowth in primary cultured rat cortical neurons. Its structure has been elucidated first to be a unique sesquiterpene bearing two y-lactones and an oxetane ring by extensive analyses of spectral data and then substantiated by X-ray crystallographic analysis. Further, its absolute configuration has been established by applying the modified Mosher's method.
413
0 82 R, = H, R2 = OH 83 R, = OH, R2 = H 84 R, = OAc, R2 = H
85
OAc
0
HO
86 R, = OH, R2 = H 87 R, = H, R2 = OH 86a R, = OCH3, R2 = H
88
Fig. (14). Anisalactone-type sesquiterpenes 82-88
On the other hand, merrilactone C (86 and 87) was obtained as an inseparable mixture with a ratio of 5 : 1 due to a lactol ring. In fact, this mixture was treated with trimethylsilyl diazomethane in methanol to give a sole methylated product 86a. Fortunately, 86a gave single crystals suitable for X-ray analysis. The ORTEP drawing of 86a as shown in Fig. (15) reflects that the methoxyl group attaches Fig. (15). The ORTP drawing of 86a downward at the C-14 position, making up the same convex-shaped structure as 84.
414
Merrilactones A (85) and B (88) are presumably derived from anislactone B (83) by cross-esterification between the C-l and C-4 hydroxyl groups. Thus intramolecular transesterification was attempted to preliminarily obtain 88 from 83. At first, 83 was subjected to acidic conditions at room temperature, but no reaction occurred. Next, heating a solution of 83 in methanol-water (1:1) in the presence of sodium hydroxide, followed by acidification, afforded a mixture of anislactone A (82) and two diastereomers (89 and 90) of 88 in 26%, 12%, and 14%, respectively (Scheme 4). Contrary to our expectations, none of 85 and 88 was found in the products. The structural assignments for 89 and 90 were unambiguously done by extensive analyses of their 2D NMR data, indicating the plane structures of 89 and 90 being the same as 88.
.OH
l)NaOH MeOH/H 2 O(l:l) reflux, 16h
HO
O OH
HO 90 (14%) yK
83
,o
O
89(12%)
Scheme 4. Chemical conversion of anislactone B (83) into other anislactones
415
88
89
90
Rg. (16). Selected NOES Y of 88, 89 and 90
However, their NMR data were not identical with one another. The 2D NOESY of 89 and 90 as shown in Fig. (16) clarified them to be epimers of 88 with respect to C-l and C-7. It turns out that two hydroxyl groups at the C-l and C-7 positions take ^-configurations in 89, whereas they take a (3- and an a-configuration in 90, respectively. This reaction probably involves a series of retro-aldol reactions and subsequent aldoltype ring construction as shown in Scheme 5. When 83 was treated with base, the (3-hydroxyl ester moiety initiated a retro-aldol mediated C6-C7 bond cleavage to give A, which underwent an intramolecular aldol condensation to yield anislactone A (82). Additional formation of p-hydroxyaldehyde B under basic conditions caused another retro-aldol mediated C1-C9 bond cleavage to give C, which in turn brought about consecutive ring closures (D, E) by an aldol condensation, thereby giving rise to 89 and 90 after acid work-up. Taking this mechanism into consideration, the conversion of 83 to 89 and/or 90 is favorable when the C-l hydroxyl group takes a ^-configuration, whereas the a hydroxyl group at the C-1 position rather leads to anislactones 82 and/or 83 than 88 due to the ring strain of y-lactone [51]. Thus it is concluded that merrilactone B (88) is not an artifact, but a natural product. Next, our attention focused on the preparation of merrilactone A (85), because an available amount of 85 was very limited to further biological studies. We envisioned a way to utilize anislactone B (83), a large amount of which could be easily obtained from lllicium merrillianum. Our synthetic plan for 85 starting from 83 involved three-step procedure i.e. dehydration, epoxidation and ring expansion (Scheme 6) [51]. At first, a solution of 83 in neat trifluoroacetic acid was refluxed to bring about the lactone transformation to the C-4
416
hydroxyl group and the dehydration of the C-l hydroxyl group, giving rise to 91 in 90% yield. Then, epoxidation of 91 with mchloroperoxybenzoic acid afforded a separable mixture of the desirable
A
90 Scheme 5. Possible mechanism for transformation of anislactone B (83) to 82, 89, and 90 upon treatment of base via sequential retro-aldol and aldol reactions
417
• < % < \o
OH 'v% n
mCPBA CH 2 C1 2
92 (64%)
rt, 36h
91
83
93 ( 4 % )
p-TsOH, dry CH 2 C1 2
92
»rt, 24h, 7 8 %
85
Scheme 6. The synthesis of merrilactone A (85) from anisalctone B (83)
a-epoxide 92 and the unnecessary (3-epoxide 93 in 64% and 4% yield, respectively. High stereoselectivity of epoxidation could be rationalized due to a favorable attack of the peroxyacid from less hindered convex face of 91. Finally, 92 was treated with ptoluenesulfonic acid to give 85 in 78% yield, which was identical in all respects with natural merrilactone A. Thus, we have established a practical preparation of merrilactone A from anislactone B, and thereby have been able to prove the absolute stereochemistry of anislactones A (82) and B (83) to be the same as that of merrilactone A (85) [51]. After our paper was published, Danishefsky et al. reported the total synthesis of merrilactone A [52]. The last two steps of their synthesis of 85 essentially utilized our procedures. Anislactone-type sesquiterpenes are composed of a new type of carbon skeleton and their occurrence is limited only to /. anisatum and /. merrillianum. As these rare natural products feature the presence of a y-lactone ring closed between C-5 and C-6, they are most likely to be biogenetically derived from the majucin-type sesquiterpenes having a ylactone at the same positions. Kouno proposed that anislactones came from the majucin-type compound by the ring contraction between C-7
418
and C-8, followed by the bond formation of C-6 and C-8 [49]. However, this biogenetic hypothesis is not able to reasonably explain the inversion of the C-9 configuration and the origin of the C-8 methyl group in the anislactones. As shown in Scheme 7, it is generally accepted that a tricyclic carbon skeleton, a//o-cedrane A, turns into secoprezizaanes such as anisatin, pseudoanisatin, miwanensin, majucin, pseudomajucin and cycloparvifloralone after breaking the C6-C11 bond of A or the C7-C11 bond of the prezizaane B, which is also derived from A. Co-occurrence of tashironin (75) and its congeners suggests that an intermediate A plays an important role in the biosynthesis of secoprezizaane-type sesquiterpenes. Herein, we propose an alternative biosynthetic pathway leading to anislactones from A as shown in Anisatin Pseudoanisatin miwanensin Majucin Pseudomajucin Cycloparvifloralone
jeco-Prezizaane C
t Tashironin Illicinolide
Prezizaane B ^//o-cedrane A
Anislactone Merrilactone
Scheme 7. Plausible biosynthetic route of anislactone-type sesquiterpene via a commnon intermediate, a//o-cedrane
419
Scheme 7. The bond cleavage between C-10 and C-ll in A gives rise to a bicyclical carbon skeleton D, which repeats the breaking of the C6C7 bond and then the five-membered ring construction between C-6 and C-10, resulting in the formation of anislactone-type carbon skeleton E. This biogenetic hypothesis seems to have no contradiction in explaining the inversion of the C-9 configuration and the origin of the C-8 methyl group. Thus, a//o-cedrane A can be regarded as a significant intermediate for the biosynthesis of all of the Illicium sesquiterpenes. Biological Activity Neurotoxic Activity
Anisatin (1) and neoanisatin (la) are convulsive toxic principles in /. anisatum and regarded as picrotoxin-like potent phytotoxins. The neuropharmacological study of anisatin demonstrates that its convulsive toxicity is probably due to a potent non-competitive GABA antagonist [53]: but, at the present time, which structural part of anisatin is of significance to cause convulsive activity has remained equivocal. A systematic study of structure and toxicity-relationship has not been carried out because of the limited available quantity of compounds, although a number of various anisatin-related compounds have been known as natural products. The toxicity of representative compounds to mice (i. p.) was examined, and compared with that of anisatin (1) and neoanisatin (la) [7, 54]. Among anisatin related compounds, veranisatins A (6), B (7) and C (8), isolated from non-toxic Chinese star anise (/. vernum), caused convulsions and death at 3 mg/kg (p. o.) in mice [15], whereas 2a-hydroxyneoanisatin (4), a positional isomer of the potent neurotoxic anisatin, induced no anisatin/picrotoxin-like convulsions and dramatically decreased the neurotoxicity in mice in
Table 1.
Lethality induced by Illicium sesquiterpenes
Sesquiterpenes
LD50 (mg/kg)
Sesquiterpene
LD50 (mg/kg)
anisatin (1)
1.03*
2-oxo-6-dehydroxyneoanisatin (9)
1.46
neoanisatin (la)
1.62*
(2S*)-hydroxyneomajucin (45)
>40
veranisatin A (6)
<3.00*
2-oxoneomajucin (48)
>40
majucin (43)
>40*
(2/?)-hydroxy-3,4-dehydroxyneomajucin(51)
>40
neomajucin (44)
12.2*
(15}-2-oxo-3,4-dehydroxyneomajucin (52)
>40
pseudoanisatin (12)
>100
(l.ft)-2-oxo-3,4-dehydroxyneomajucin (53)
>40
minwanensin (34)
>50
*Litchfield-wilcoxon method
420
comparison with 1 and la. This is due to an unfavorable interaction of the 2-OH group with the receptor or merely by its high polarity which impairs transport to the target on the basis of comparison of the three dimensional molecular shape and electrostatic properties of active and inactive seco-prezizaane type sesquiterpenes [55]. Other anisatin-type 2-oxo-6-dehydroxyneoanisatin (9) and majucintype neomajucin (44) are also very toxic [7, 11], whereas majucin (43) and its analogues 45, 48, 51, 52 and 53 could not produce any appreciable behavioral changes at dose up to 40 mg/kg [34] as summarized in Table 1. As neomajucin (44) was recognized as a toxic compound, the presence of a spiro (3-lactone moiety in anisatin is not likely to be absolutely responsible for the convulsive toxicity. We have to wait for further investigation in order to prove this kind of problem associated with relationship between structure and toxicity in the Illicium sesquiterpenes. Recently, thirteen seco-prezizaane-type sesquiterpenes were investigated for their structure-activity relationships in GABA receptors to housefly-head and rat-brain membranes. Veranisatin A (6) was found to be the most potent inhibitor in both membranes, followed by anisatin. It is interesting that pseudoanisatin (12) which is not neurotoxic for rats and mice shows a high selectivity for binding to the GABA receptor in housefly membranes. In fact, both anisatin (1) and pseudoanisatin (12) exhibited moderate insecticidal activity against German cockroaches [56]. Neurotrophic Activity
Neurotrophic factors are a subset of biologically active proteins, which are involved in the survival of developing neurons and in the maintenance of mature neurons throughout life [57, 58]. A role of neurotrophic factors in the course of neuronal development is well understood by the example of nerve growth factor (NGF), which enhances neurite outgrowth and maintains cell viability. Such discoveries have raised the hope that NGF may be possible in medicinal treatment of neurodegenerative diseases such as Alzheimer's disease [59]. However, these trophic proteins cannot penetrate into the target brain through the blood-brain barrier due to their high molecular weight, and also bioavailability and stability are problems to be overcome Thus, some endogenous small molecular compounds that are able to mimic the biological effect of the natural neurotrophic factors, or to stimulate their synthesis and secretion, might be promising candidates for pharmaceutical agents of various neurodegenerative diseases. Sex hormones, thyroid hormones, vitamin D and their derivatives are already known to affect survival and differentiation of dissociated mouse embryo brain in cultures or in cultured rat septal neurons. However, application of these hormones to patients with normal hormone function
421
would result in a multitude of undesired effects since they also affect the function of most organs. However, few searches for exogenous NGFlike compounds have been carried out [60, 61]. Thus we have started exploring small molecular compounds from plants-derived natural products having a typical neurotrophic property, which can enhance neurite outgrowth and increase choline acetyltransferase (ChAT) activity in primary cultured fetal rat cortical neurons [62]. Along this line our efforts have resulted in the discovery of novel neurotrophic natural products [63]. HO.
CO,R O
HO 75 R = Bz 76R = H
85
Fig. (17). Illicium sesquiterpenes having neurotrophic activity
As illustrated in the previous chapters, the Illicium plants are shown to be rich in biosynthetically unique seco-prezizaane-type sesquiterpenes and prenylated C6-C3 compounds, some of which were found to exhibit neurotrophic activity [28, 64, 65]. Thus some of seco-prezizaane-type sesquiterpenes which have no neurotoxic action may exhibit an intriguing neurotrophic property. By using the cultures of fetal rat cortical neurons we have evaluated whether Illicium sesquiterpenes isolated by us have neurotrophic properties or not. The neurotrophic sesquiterpenes are shown in Fig. (17). Isodunnianin (32) was found not to be neurotoxic but to have a neurotrophic property such as enhancing neurite outgrowth in primary cultured neurons in the serumcontaining medium at 10 ^M as well as increasing choline acetyltransferase activity at 10 days after seeding [28]. However, under
422 (b)
(a)
M
450 400
313.9
321 1
3» 300 250 200 I.^ll
too 50
Irfl
a 2B2.5
ill conrol hFCF
IIKJ
331.3
330
5
282.5 M
•
j
300 ISO
z a
200 ISO 100 50 D
III oofirol
0.0)
0.1
1.0
10
Fig. (18). Enhancement of neurite outgrowth of rat cortical neurons by compounds 51 and 55 in primary cultured rat cortical neurons; (a) (2S)-hydroxy-3,4-dehydroneomajucin (51); (b) jiadifenin (55); Data are represented as mean + SE (n = 80). Student's test; -kirP <0.01 versus control: Dunnet's test; versus control. (a)
**P < 0.01
(b)
Fig. (19). Neurotrophic effect of 11 -O-debenzoyltashironin (76) in primary cultured rat cortical neurons After the neuronal cells (12000 cells/cm2) cultured for 6 days in the presence or absence of compound 76 were fixed by 4% paraformaldehyde-PBS, the immunohistochemical staining for the microtuble associated protein-2 (MAP-2) was performed. Pictures taken with 200 magnifications, (a): 0.5%EtOH, (b): 0.1 nM, (c): 1 \M, (d): 10 \xM of 76
423
these conditions, the issue on indirect effect of unknown components in the serum remained unsolved. Therefore, the cell cultures have been performed under the culture conditions using 18-day fetal rat cortical neurons in the serum-free Neurobasal Medium (NBM) supplemented with B27 [62]. Anisatin (1) showed no neurotrophic action but toxic effect against the cultured neurons at 10 ^M, and none of anisatin-type sesquiterpenes have exhibited neurotrophic activity. On the other hand, some majucin-type sesquiterpenes, (2.S)-hydroxy-3,4dehydroneomajucin (51), jiadifenin (55) and its carboxylic acid derivative 55a showed potent neurite outgrowth promoting activity in primary cultured rat cortical neurons at the concentration range from 0.01 to 10 \xM [35]. The morphometric analysis, as shown in Fig. (18), proves that 51 and 55 caused the longest axons extended from each cell body, indicating that they can promote neurite outgrowth more than bFGF, basic fibroblast growth factor. In addition, the neurotrophic active potency of 55a was comparative with that of 55. Other majucintype sesquiterpenes showed no neurotrophic property at the concentration range from 1 jxM to 10 ^iM. Although tashironin (75) had neither neurotrophic nor neurotoxic effects in the cultured neurons, 11-debenzoyltashironin (76) was found to promote neurite outgrowth best at the range of concentration from 0.1 jjiM to 10 (xM as shown in Fig. (19) [39]. This big difference is most likely to be attributable to the presence or absence of a hemiacetal group at the C-11 position, and thus an acetal function in a molecule may be responsible for this type of activity. However, all of cycloparivifloranone-type sesquiterpenes 65-72 with acetal or lactol functions have exhibited no neurotrophic efficacy at the same concentration. This means that the structure containing an acetal function is not always in demand for showing neurotrophic activity. Among anislactone-type sesquiterpenes, merrilactone A (85) solely exhibits potent neurite outgrowth promoting activity at the concentration range from 0.01 [xM to 10 jiM [50]. The morphological pictures, compared between neuronal culture in the presence of 0.1 jiM of 85 and control culture containing 0.5% EtOH, are shown in Fig. (20) and also quantitative morphometric analysis of the neurotrophic effect by 85 is summarized in Fig. (21). Estimating from these data, merrilactone A can be regarded as one of the most potent neurotrophic compounds among which we have so far screened in primary cultured rat cortical neurons, and thus has potential as candidates for nonpeptidal neurotrophic agents useful for the treatment of neurodegenerative diseases. Illicium sesquiterpenes, which occur exclusively in the genus Illicium, can be taxonomically regarded as significant markers, and also most of them make up unique highly oxygenated structures. What is
424 (b)
(a)
Fig.
(20).
„
600
g
500
I 5340°
Neurotrophic effect of 85 in a 6-day-old culture of rat cortical neurons, treated with 0.5% EtOH, and (b): culture treated with 85 (0.1 uM)
(a): control culture
531.6 449.8
437.,,
1—M—f
**
441.A 339.8
312.2
tm 100
Control
bFCF (111 u-j. .11.
Fig. (21).
0.01
(I.I
I ii
1(1
I
Morphometric analysis of the neurons affected by merrilactone A (85).
After the neuronal cells (9000 cells cm 2 ) cultured for 6 days in the presence of 0.5 % EtOH, bFGF, and 85 were fixed by 4% paraformaldehyde-PBS, the immunohistochemical staining for MAP-2 was performed. Morphometric analysis was carried out on these neurons according to the criteria [62]. The data are expressed as means±SE ( n = 80); Student's test; **P< 0.01 versus control: Dunnet's test; **P< 0.01 versus control.
425
more interesting for Illicium sesquiterpenes is that some of them have high affinity for some kinds of receptors associated with neuronal functions. This feature may cause neurotoxicity and/or neurotrophic property, presumably depending on their structure types. Relationship between structure and biological activity of Illicium sesquiterpenes has to wait for more examples of neurotrophic compounds and their detailed mechanistic studies. Thus our attention is now directed toward synthesis and biological studies of the natural products having neurotrophic property. It will be our great pleasure if this review stimulates organic chemists and biologist's interests toward seco-prezizaane-type and its related sesquiterpenes. ACKNOWLEDGMENTS The authors gratefully acknowledge research grants from a Grant-in Aid for Scientific Research (No. 12480175) from the Ministry of Education, Sports and Culture, Japan, and the High Tech Research Center Fund from the Promotion and Mutual Aid Corporation for Private School of Japan. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II]
Richard, M. K.; Sauders, F. L. S. Botanical J. Linnean Society, 1995, 117, 333-352. Torre, D. de. C. G.; Harms, H. Genera Siphonogamarum ad Systema Englerianum Conscript, Whilhelm, Leipzig, 1900. Smith, A. C. Sargentia, 1947, 7, 1-224. Lane, J. R; Koch, W. T.; Leeds, N. S.; Gorin, G. J. Am. Chem. Soc, 1952, 74, 3211-3214. Yamada, K.; Takada, S.; Nakamura, S.; Hirata, Y. Tetrahedron, 1968, 24, 199229. Kawano, N.; Matsuo, A. Yakugakuzasshi, 1958, 78, 1220-1223. Kouno, I.; Baba, N.: Hashimoto, M.; Kawano, N.; Takahashi, M.; Kaneto, H.; Yang, C. -S.; Sato, S. Chem. Pharm. Bull., 1989, 37, 2448-2451. Schmidt, T. J.; Schmidt, H. M.; Muller, E.; Peters, W.; Fronczek, F. R.; Truesdale, A.; Fischer, N. H. J. Nat. Prod., 1998, 61, 230-236. Wang, J. -L.; Yang, C. -S.; Yan, R. -N.; Yao, B.; Yang, X. -B. Zhongguo YaoxueZazhi, 1994, 29, 693-696. Huang, J. M.; Yang, C. -S.; Wang, H.; Wu, Q. -M; Wang, J. -H.; Fukuyama, Y. Chem. Pharm. Bull, 1999, 47, 1749-1752. Yang, C. -S.; Hashimoto, M ; Baba, N.; Takahashi, M.; Kaneto, H.; Kawano, N.; Kouno, I. Chem. Pharm. Bull, 1990, 38, 291-292.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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NEW INSIGHTS INTO THE BIOACTIVITY OF CUCURBITACINS JOSE LUIS RIOS, JOSE M. ESCANDELL, M. CARMEN RECIO Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia. Vicent Andres Estelles s/n. 46100 Burjassot, Valencia, Spain ABSTRACT: The cucurbitacins are a group of tetracyclic triterpenoids derived from the cucurbitane skeleton and found primarily in the Cucurbitaceae family. These triterpenoids, present in free or glycosidic form, are generally responsible for the bitter taste of the plants that contain them and are probably the principal cause of the antifeedant effects observed for such plants. Several plants used in traditional medicine to treat both inflammatory diseases as well as various types of tumors are rich in cucurbitacins, a fact which has given rise to several studies concerning their potential use as anti-inflammatory and anticancer agents. Nevertheless, since many cucurbitacins are extremely toxic, relatively few papers have dealt with their pharmacological activity. Recently, however, the relationship between the toxicity of a compound and its chemical pattern of substitution has been established, thus allowing for a more in-depth understanding of this class of triterpenes. In the present review, we provide a compilation of all the studies published in the last ten years on the pharmacological and biological effects of cucurbitacins, focusing principally on their pharmacological properties, especially their anti-inflammatory and anticancer effects.
AN INTRODUCTION TO CUCURBITACINS Occurrence While cucurbitacins are a characteristic group of constituents of the family Cucurbitaceae, they are not exclusive to this family of plants. Indeed, they are present in many other plant families [1], including Begoniaceae, Cercidiphyllaceae, Cruciferae, Datiscaceae, Desfontainiaceae, Elaeocarpaceae, Polemoniaceae, Primulaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Sterculiaceae, and Thymelaeaceae [2-5]. The fact that cucurbitacins are present in various plant families has chemotaxonomic value when differentiating between plant species in cucurbitacin-containing genera [3]. Some particular cucurbitacins are found only in a specific family or genera, as is the case with the nor-
430
cucurbitacins WGi and WG2 from Wilbrandia [6]. The majority, however, are often present in various species and families, as is the case with cucurbitacin B, which has been isolated in Cucurbitaceae, Polemoniaceae, and Rubiaceae [2]. In the Cucurbitaceae family, cucurbitacins have been isolated in the following genera: Acanthosicyos, Benicasa, Brandegea, Bryonia, Cayaponia, Citrullus, Coccinia, Coralocarpus, Cucumis, Cucurbita, Ecballium, Echinocystis, Fevillea, Gerrardantus, Gurania, Hemsleya, Kedrostis, Lagenaria, Luffa, Marah, Melothria, Momordica, Peponium, Sicyos, Telfairia, Trichosanhes, Trochomeria, and Wilbrandia [2,7,8]. Cucurbitacins can be located in various organs of the plants in which they are found. In Cayaponia tayuya, Bryonia alba, and Bryonia dioica, for example, the compounds are concentrated for the most part in the underground organs, such as roots or rhizomes [9-11]. In other species, including Cucurbita andreana [12], Trichosanthes tricuspidata [7], Citrullus colocynthis, Luffa operculata, and Ecballium elaterium [11], the fruits are the best source of cucurbitacins, whereas in others, such as Iberis amara, the compounds are principally found in the seeds [11]. In Kageneckia oblonga [13] and Gratiola officinalis [11], cucurbitacins were isolated from the aerial part of the plants. Biological significance Cucurbitacins have a very strong bitter taste; indeed, they are probably the bitterest compounds known [14]. This characteristic is likely a defense mechanism on the part of the plants to ward off herbivores. In addition, these compounds exert a gibberellin-antagonistic activity, as well as both feeding stimulant and antifeedant effects for insects [15]. Interestingly, while many insect species are deterred or killed by the presence of cucurbitacins in the diet, others are attracted by moderate concentrations of these compounds [3]. Adults and larvae of several species of cucumber beetles, for example, compulsively eat bitter triterpene cucurbitacins [16] and the compounds are known to act as arrestants and phagostimulants for diabroticite chrysomelid beetles, protecting them from predators, parasites, and pathogens [17]. In fact, the systemic resistance against bacterial wilt and feeding by the cucumber beetle vectors of different species induced by rhizobacteria, which generally promote plant growth, is associated with reduced
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concentrations of cucurbitacins, since these comprise a powerful beetle feeding stimulant [18-22]. Some cucurbitacins are also ecdysteroid antagonists [23]. Ecdysteroids are a group of steroidal hormones present in all stages of insect development which regulate many biochemical and physiological processes, including molting and metamorphosis [24]. For instance, cucurbitacin B, a known antagonist of 20-hydroxyecdysone [25,26], is responsible for the antagonistic activity of a methanolic extract of Iberis umbellata (Cruciferae) [26] and of Physocarpus opulifolius (Rosaceae) [4]. Other cucurbitacins with similar properties will be reviewed in the corresponding section of this chapter. Pharmacognostical background The pharmacological activity of medicinal plants, crude extracts, and purified fractions obtained from species with cucurbitacins have been reported in different papers [2]. Citrullus colocybthis, Ecballium elaterium, Bryonia alba, and Momordica charantia have been used as purgatives; Anagallis arvensis, Ecballium elaterium, and Cucumis melo extracts have been employed in treating liver diseases; Cayaponia tayuya, Ecballium elaterium, Bryonia alba, and Wilbrandia ebracteata are used against inflammatory pathologies such as rheumatism; Desfontainia spinosa is a narcotic and a hallucinogen; Momordica charantia has antibacterial activity; Ecballium elaterium is fungicidal; Cucurbita pepo seeds are toxic against taenia, oxyuris nematodes, and cestodes; and Citrullus colocybthis, Momordica charantia, Cucurbita maxima, and different species of Cucumis, Cucurbita, and Luffa all have insecticidal properties [2]. Cucurbitacins seem to be responsible for the major pharmacological and biological effects of the aforementioned plants and their extracts. For example, due to their strong bitter taste, the cucurbitacins act as the purgative principles of different species of Cucurbitaceae by stimulating gastric secretion. They have also been found to decrease the damage in chronic hepatitis and they are also responsible for the antimicrobial, antifungal, and antihelmintic activity of several crude drugs and active extracts. Other ucurbitacins modify the growth, evolution, and metamorphosis of different insects [2,3].
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Some active species have been studied and their pharmacological activity confirmed in different experimental models. In general, the active fractions of such plants were those containing cucurbitacins, as demonstrated by Rios et al. [10] in their work with Cayaponia tayuya roots. This species is used in the Amazon region of South America as an analgesic, anti-inflammatory, and anti-rheumatic agent while the extracts of this species are widely employed in the treatment of skin disorders such as dermatosis and other irritations. The chloroform fraction obtained from the active root methanol extract was found to be more active than the ethyl acetate and butanol fractions. Subsequent phytochemical analysis demonstrated that the chloroform fraction is rich in cucurbitacins, whereas the ethyl acetate and butanol fractions are principally comprised of C-glycosil flavonoids [10]. In a similar vein, species from Wilbrandia have been widely studied as anti-inflammatory, antitumoral, and antifertility agents [6]. Different studies have demonstrated that the pharmacological effects are principally due to the plants' cucurbitacin content, as recently reported by Peters et al. [27-29]. Finally, one intriguing application of these plants is that of combating hypoglycemia. The freeze-dried juice of Cucurbita ficifolia fruits, for example, has been successfully used in the treatment of diabetes type 2 in Mexico. The hypoglycemic effect was demonstrated by estimating blood glucose levels in different experimental models on healthy mice, alloxandiabetic mice, and alloxan-diabetic rats [30]. Chemistry Cucurbitacins are a group of triterpenoids with a 19(10->9P)-a6eo-10alanost-5-ene (cucurbitane) skeleton [15]. They are not steroidal, as the C-10 methyl is located at C-9 rather than at C-10; in addition, the cucurbitane structure includes a gem-dimethyl group at C4 [3]. The potential for various degrees of substitution and insaturation allows for a great number and variety of chemical compounds, but with several common characteristics. For example, all the known cucurbitacins present a double bond between C-5—C-6, and many of them present a double bond at C-l and/or C-23. Often, many of the carbons are substituted by oxygens, thus endowing this family of compounds with a high level of oxidation. A hydroxyl is usually present at C-16(a), C-
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20(P) and C-25, while a carbonyl is typically present at C-ll and C-22. On the other hand, C-2 and C-3 may be indistinctly substituted by a hydroxyl or carbonyl group. Other typical substitutions include a possible double bond at C-l, sometimes a hydroxyl at C-24, and rarely a hydroxyl at C-9. Moreover, the C-25 hydroxyl is sometimes acetylated [2]. The basic skeleton of cucurbitacins is shown in Fig. (1).
23 11 1
^ N i s
27
H 9
!
s j 7 lo
1 \ . 28
29
Fig. (1) Cuourbitane general structure
Although it is uncommon, some cucurbitacins have a fifth ring, e.g. anhydro-22-deoxo-3-ep/-isocucurbitacin D, or are nor-derivatives, e.g. hexanor-cucurbitacin I. In some cases, the A-ring is three-unsatured, which gives rise to aromatized cucurbitacins, Fig. (2) [2,31]. Finally, cucurbitacins are found in either free or glycosidic form. The form is usually linked to the presence of a hydroxyl at C-2P, C-3 and/or C-25 for monosides or at C-26 or C-27 for bidesmosides [2,3]. They are about 50 cucurbitacins described. They are present in plants in the form of glycosides or free aglycones. The most common of these free compounds is cucurbitacins B [3] In general, cucurbitacins are distinguished by use of correlative letters, e.g. cucurbitacin B; however, some of these compounds are described by evoking a chemical modification, e.g. 23-dihydrocucurbitacin B or 3-epicucurbitacin F [2]. In still other cases, the name of the compound comes from the genus or species in which it was originally isolated, e.g. briogenin from Bryonia [9] or cayaponosides from Cayaponia tayuya [32-36].
434
HCT " S ^ ^^
0-
Anhydro-22-deoxo-3-ep(-isocucurbitacin D
Hexanorcucurbitacin I O
6,7-Dehydrofevucordin A Fig. (2). Relevant chemical modification in the general structures of cucurbitacins
Biosynthesis As is the case with other tetracyclic triterpenes, cucurbitacins are formed upon cyclization of the 3S isomer of 2,3-epoxy-2,3-dihydrosqualene. This initial cyclization is followed by a more extensive rearrangement to give, among other compounds, cucurbitacins, as shown in Fig. (3) [3739].
Fig. (3). Formation of 2,3-epoxysqualene from squalene
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Thus, once the appropriate orientation within the corresponding enzyme occurs, steroids, cucurbitacins, or other triterpenes may be formed. In the case of cucurbitacins, the squalene epoxide adopts a chair-boat-chair conformation, and the cyclization leads to a protostane cation, which is the immediate precursor of the cucurbitanes through a series of 1,2-proton and methyl group shifts, Fig. (4) [40].
Protostane
Cucurbitane
Fig. (4). Biosynthesis of cucurbitanes from protostane
Isolation and identification Detection and analytical separation
Thin-layer chromatography (TLC) is a good method for detecting the presence of cucurbitacins in a plant extract. The best results to date have been achieved using high-performance TLC (HPTLC) plates [41] with silica gel and chloroform-methanol (95:10) (Table 1) or toluene-ethyl acetate (25:75) as mobile phases [3,11]. Reversed-phase (RP) HPTLC plates with methanol-water (7:3) have also been used to separate cucurbitacins [3]. The compounds are easily detected with vanillinsulphuric acid or vanillin-phosphoric acid reagents and ultraviolet light (UV)at365nm[llj. Analytical high-performance liquid chromatography (HPLC) produces a good level of separation between cucurbitacins with similar chemical structures [41]. The most effective set-up involves the use of an RP system as the stationary phase with a Cis column, acetonitrile-water mixtures as the mobile phase, and UV detection at 230 nm [11,42]. Thus, using a Cig column and acetonitrile-water (20:80) to (50:50) for 45 min (linear gradient at 2.0 ml/min), Bauer and Wagner [11] analyzed seven
436
extracts from various species and separated and identified 14 different cucurbitacins with excellent resolution (Table 2). Table 1. Analytical separation of cucurbitacins by means of HPTLC (SiO2) and CHCI3-CH3OH (95:10) [11] Cucurbitacin-glucosides Cucurbitacin L-glucoside Cucurbitacin I-glucoside Cucurbitacin B-glucoside Cucurbitacin E-glucoside 23,24-DihydrocucurbitacinE-glucoside
IV
Cucurbitacins in free form
0.14 0.16 0.26 0.29 0.30
Cucurbitacin D Cucurbitacin C Cucurbitacin A Cucurbitacin L Cucurbitacin I Cucurbitacin B 23,24-Dihydrocucurbitacin B Cucurbitacin E 23,24-Dihydrocucurbitacin E
0.63 0.64 0.65 0.67 0.72 0.90 0.90 0.93 0.94
Table 2. Analytical separation of cucurbitacins by means of HPLC (RP-18) and CH3CN-H2OH (gradient) [11] Cucurbitacin-glucosides Cucurbitacin L-glucoside Cucurbitacin I-glucoside Cucurbitacin E-glucoside Cucurbitacin B-glucoside 23,24-Dihydrocucurbitacin E-glucoside
R, (min)
Cucurbitacins in free form
R, (min)
11.1 11.1 19.5 20.2 20.6
Cucurbitacin A Cucurbitacin D Cucurbitacin C Cucurbitacin L Cucurbitacin I Cucurbitacin B 23,24-Dihydrocucurbitacin B Cucurbitacin E 23,24-Dihydrocucurbitacin E
17.3 17.8 18.5 21.8 21.8 28.5 30.0 33.2 34.7
Extraction
The solubility of cucurbitacins is variable, depending on the pattern of substitutions. Free aglycones, for example, are easily soluble in dichloromethane, making this the solvent of choice for the extraction of these compounds, but mixtures with other solvents such as acetone, methanol, or ethanol have also been used. In contrast, for glycosides, which have a higher degree of polarity, an extraction with methanol or ethyl acetate may be required, although many of these compounds are soluble in chloroform or dichloromethane [3]. In all cases, however, it is advisable to eliminate pigments or fats with hexane or light petroleum before commencing with the extraction [11,42]. To this end, in their
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effort to isolate eighteen cucurbitanes and related compounds, Kanchanapoom et a\. [7] successfully employed an extraction with hot methanol and a subsequent liquid-liquid extraction with ether to remove fats. The defatted residue was then submitted to a column of highly porous copolymer of styrene and divinylbenzene and eluted with water, methanol, and acetone, successively. Finally, the fractions underwent column chromatography on silica gel or RP-18. Separation and purification
Purification can be accomplished with different chromatographic protocols, but in most cases, open-column chromatography is used. For example, during the separation process of cucurbitacins from Cucurbita andreana, Jayaprakasam et al. [12] employed a medium pressure liquid chromatography (MPLC) system, using silica gel and mixtures of chloroform-acetone as eluent. A similar protocol was used by Peters et al. [27] during the isolation of the cucurbitacins from Wilbrandia ebracteata, but in this case the elution was performed first with mixtures of petroleum ether / ethyl acetate and then with ethyl acetate / /-propanol. Delporte et al. [13] isolated two cucurbitacins from Kageneckia oblonga using a complex method involving column chromatography on Amberlite, Sephadex LH-20, and MPLC. However, to separate the two major components of Cayaponia tayuya, Recio et al. [43] used a simple method based on gel-filtration through Sephadex LH-20 with dichloromethane as the mobile phase. HPLC also seems to be a good method for separating cucurbitacin mixtures [3]. Since these compounds tend to exhibit at least medium polarity, an RP system is the most appropriate stationary phase (Cis column), with a linear gradient from 20% to 50% of acetonitrile [3] or methanol [42,44] in water being the best mobile phases for separation of both free-aglycones and glycosides. In this case, previous purification of samples by means of a solid-phase extraction (SPE) is advisable. The addition of 0.01% of trifluoroacetic acid (TFA) improves the separation and reduces complications from interfering substances [3]. The presence of different double bonds and carbonyls in the molecule permits adequate monitoring of the separation at 230 nm, but other detection systems have also been used [3].
438 Structural elucidation
Due to the great complexity of this class of molecules, nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are the tools most widely used to identify cucurbitacins. Both one- and two-dimensional NMR techniques have been employed for the structural elucidation of new compounds: 2D NMR, ] H-NMR, 13C-NMR, correlated spectroscopy (COSY), heteronuclear chemical shift correlation (HETCOR), attached proton test (APT), distortionless enhancement by polarization transfer (DEPT), and nuclear Overhauser effect spectroscopy (NOESY) are common techniques for determining the proton and carbon chemical shifts, constants, connectivity, stereochemistry, and chirality of these compounds [1,38,45-47]. Audier and Das [48] published a specific report on the MS fragmentation of cucurbitacins which exemplified how the use of modern MS techniques leads to a better understanding of molecule fragmentation. In a similar vein, Johnson et al. [49] reported the diagnostic fragmentation pathways that occur in cucurbitacins isolated from Fevillea cordifolia after using electron impact (El)-, chemical ionization (CI)-, and fast-atom bombardment (FAB)-MS. Che et al. [15] established the 'H-NMR chemical shifts and coupling constants from a great number of standard cucurbitacins, and Yamada et al. [50] provided the I3C-NMR assignments of the cucurbitacin aglycones. In addition, the 'H-NMR and 13C-NMR data for sixteen cucurbitacin glycosides, a hexanorcucurbitane glucoside, and an octanorcucurbitane were reported by Kanchanapoon et al. [7]. Jacobs et al. [1] used two-dimensional NMR spectroscopy to assign the ' H and 13C chemical shifts of cucurbitacin B, establishing these values as a benchmark for analyzing the spectra of other cucurbitacins isolated from the same source. In fact, the characteristic groups present in all cucurbitacins are easily detectable by means of NMR and typically include the presence of carbonyls at C-ll and C-22, which appear between 212-213 ppm, as well as hydroxyls at C-16(oc) and C-20(P), which appear at approximately 71-73 ppm and 77-79 ppm, respectively. Other characteristic patterns of substitution such as the presence of acetoxyl groups at C-2 and C-25 have also been observed. In this case, the acetylation of the hydroxyl at C-2 modifies both the C-l and C-3 values. Indeed, when a carbonyl is present, this latter change involves a major shift, namely from 213 to 205 ppm. In the case of a second double
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bond, usually at C-23, it is easily observed at about 120 ppm (or at 152 ppm when the bond is at C-24), whereas the constitutive double bond at C-5 or C-6 appears at about 140 ppm or 120 ppm, respectively. PHARMACOLOGICAL AND BIOLOGICAL PROPERTIES Introduction Cucurbitacins are not usually used as medicinal agents because of their toxicity. However, new findings have demonstrated the potential of these natural products for treating different pathologies, including inflammation, cancer, or autoimmune diseases. Moreover, some cucurbitacins affect insect growth or act as ecdysone antagonists by modifying the metamorphosis and evolution of insects. Cucurbitacins as anti-inflammatory agents Some species containing cucurbitacins are used in traditional medicine as anti-inflammatories in different pathologies. In general, they are used in topical applications as they have a certain level of toxicity when applied per os (p.o.). In one paper, Miro [2] compiled the data on this subject that had been published up to 1995. The review cited only the antiinflammatory activity of cucurbitacin B isolated from the juice of Ecballium elaterium, as well as its possible mechanism of action by means of a modification in leukotriene B4 (LTB4) production. In the meantime, other cucurbitacin-containing species have been shown to possess anti-inflammatory activity. The dichloromethane extract from Wilbrandia ebracteata, for instance, has shown antiinflammatory activity on carrageenan-induced paw edema [27] and on carrageenan-induced pleurisy, both in mice [28], as well as on zymosaninduced arthritis in rats [29]. In one study [27], two cucurbitacins were isolated and studied as potential agents for the anti-inflammatory activity on carrageenan-induced paw edema. Cucurbitacins B and E, Fig. (5), significantly reduced the edema after intraperitoneal (i.p.) administration (1 mg/kg), inhibiting the edema by 61% and 58%, respectively, 4 h after application. The authors did not specifically study the mechanism of action, but relying on previously published findings, they hypothesized a
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possible inhibition of the synthesis of cyclooxygenase (COX) products derived from the arachidonic acid pathways [27].
OCOCH 3
Fig. (5). Chemical structure of cucurbitacins B and E (a-elaterin)
In subsequent research, these same authors studied the activity of the same extract and the cucurbitacin B isolated from it on carrageenaninduced pleurisy in mice, demonstrating the activity of both the extract and the isolated compound. Thus, cucurbitacin B administered at 0.1 mg/kg i.p. was found to reduce significantly the prostaglandin E2 (PGE2) levels of the pleural exudates by 53% [28]. Injection of carrageenan into the pleural space of mice is known to induce both an influx of cells, principally polymorphonuclear leukocytes (PMNL), and an increase in vascular permeability, accompanied by a significant increase in PGE2 levels in the exudate. Since the synthesis of PGE2 occurs through the metabolism of arachidonic acid via the COX pathway, the authors suggested that an inhibition of this enzyme, probably the inducible form or COX-2, was the mechanism of action of cucurbitacin B, specifically due to the presence of an acetyl group at C-25 [28]. One weakness of this hypothesis, however, is that it was made on the basis of the observed effects of purified plant extracts rather than on the activity of the isolated compound. The dichloromethane extract from Wilbrandia ebracteata (p.o.) significantly reduced the paw elevation time (1 mg/kg) and cell influx (10 mg/kg) in zymosan-induced arthritis in rats. The same extract inhibited COX-2 activity, as measured by PGE2 production, without affecting that of COX-1. Moreover, nitrite release was clearly and significantly reduced at a dose of 10 mg/kg (p.o.). The analysis of the pharmacological data, together with the HPLC analysis of the extracts, points to an anti-inflammatory effect based on an associated reduction in nitric oxide (NO) release and COX-2 inhibition by the cucurbitacins
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present in the extract. While this effect is most likely caused by the major compounds detected, in this case 23,24-dihydrocucurbitacin B, which was present at a level of 45.8% in the active extract along with cucurbitacin B, which only made up 2.8% of the extract [29], other minor compounds found in the extract, including cucurbitacins R and E and dihydrocucurbitacin E, should also be taken into consideration. Several of the compounds cited above, e.g. cucurbitacins B, D, and E, together with cucurbitacin I, Fig. (6), were isolated from the fruits of Cucurbita andreana and their activity against COX-1 and COX-2 enzymes was studied. All of the compounds studied exerted a moderate, but significant inhibition of the induced form COX-2, but none of them inhibited the constitutive form COX-1. The range of activity at 100 l^g/ml was 32% (cucurbitacin B), 29% (cucurbitacin D), 35% (cucurbitacin E), and 27% (cucurbitacin I), all of which are clearly inferior to the values obtained with specific inhibitors of COX, such as ibuprofen, naproxen, or rofecoxib. Whereas cucurbitacin B was found to inhibit lipid peroxidation by 59% at 100 u.g/ml, cucurbitacin I did so only by 23% at the same concentration while the other two compounds were inactive [12]. This mechanism may well be a complementary pathway of action of cucurbitacins in inflammation; however, other mechanisms such as the inhibition of integrin-mediated cell adhesion [51 ] should also be given further consideration.
Fig. (6). Chemical structure of cucurbitacins D and I
Some of the pharmacological data reported for cucurbitacin B have been corroborated in different studies. In one paper, Yesilada et al. [52] reported on the isolation of cucurbitacin B from the fruit juice of Ecballium elaterium. They further showed how the compound significantly reduced the vascular permeability induced by acetic acid in mice, giving an effective dose-50 (ED50) of 6.1 mg/kg; however, the
442
compound also showed a high toxicity with a lethal dose-50 (LD50) of 10.9 mg/kg. The same research group demonstrated that cucurbitacin B inhibited both the serotonin- and bradykinin-induced edemas in mice, with an ED50 of 3.7 and 3.7 mg/kg, respectively [53]. Moreover, they suggested that the compound did not modify the biosynthesis of interleukin (IL)-la, IL-lp, or tumor necrosis factor (TNF)-a, as the extract tested negative in these assays [54]. In another paper, Rios et al. [10] reported the anti-inflammatory activity of the chloroform extract from Cayaponia tayuya, which exhibited a high potency against carrageenan-induced mouse paw edema, with an ED50 of 122.5 mg/kg (p.o.) and 27.8 (i.p.). From the active extract, Recio et al. [43] isolated two cucurbitacins which were identified as cucurbitacin R and 23,24-dihydrocucurbitacin B, Fig. (7).
OCOCH3
Fig. (7). Chemical structures of cucurbitacin R and 23,24-dihydrocucurbitacin B
Both cucurbitacins were studied as anti-inflammatory agents with both compounds demonstrating a high level of activity in different experimental models of inflammation [43]. Cucurbitacin R showed higher activity in all the experiments, except in that involving carrageenan-induced mouse paw edema, inhibiting the Naja mossambica phospholipase A2 (PLA2)-induced paw edema by 61% (3 mg/kg, i.p.), TPA-induced acute mouse ear edema by 36% (10 mg/kg, p.o.) and 87% (0.1 mg/ear), and serotonin-induced paw mouse edema by 81% (0.5 mg/kg, subcutaneous (s.c.)). In a subchronic model of skin inflammation, cucurbitacin R at 0.1 mg/ear decreased the swelling by 56% and neutrophil infiltration by 69%. In addition, the histological study showed a clear reduction of edema and inflammatory cell infiltration, plus a reduction in epithelium thickness as well as attenuation of other parameters of inflammation such as papilomathosis, acanthosis, hyperkeratosis, and spongiosis [43]. The mechanism of action of
443
cucurbitacin R was not specifically determined, but the authors pointed out that it affects neither 5-lipoxygenase (5-LOX) nor PLA2 activity, nor does it act on the glucocorticoid receptor, as seen by the fact that the antiinflammatory activity was not modified in vivo after administration of the specific antagonist mifepristone [43]. In a later study, however, Park et al. [55] demonstrated that cucurbitacin R decreased the levels of protein and mRNA for inducible NO synthase (iNOS) in murine macrophages by blocking the activation of nuclear factor-KB (NF-KB), which is necessary for transcriptional activation of iNOS. Dihydrocucurbitacin B exhibited a similar pattern of activity to that of cucurbitacin R except in the case of the carrageenan-test. In this assay, the former was clearly more active, but the toxicity against rat leukocytes was higher [43]. Another mechanism in which cucurbitacins may be implicated is the inhibition of the complement pathway. The complement system is one of the most relevant mechanisms for the initiation and amplification of the inflammatory process. There are two possible activation routes, the classical and the alternative pathways, and both can be inhibited by picfelterraenin IA, IB, IV, and VI from Picria fel-terrae, Fig. (8). The last compound, however, has shown the highest potency, with inhibitory concentration-50 (IC50) values of 29 and 21 |^M for the classical and alternative pathways, respectively. The relationship between chemical substitution and anti-complementary activity has not as yet been established, but it has been clearly demonstrated that an increase in the number of sugar moieties decreases the activity [56].
Fig. (8). Chemical structure of picfelterraenin VI
444
Some authors have related the anti-inflammatory properties of cucurbitacins with a possible glucocorticoid-like mechanism. Thus, Panossian et al. [57] demonstrated the adaptogenic activity in preclinical and clinical trials of Bryonia alba, hypothesizing that the pharmacological effects are at least partly due to a mechanism in which the principal constituent of the active extract, namely cucurbitacin Rdiglucoside, modifies the metabolism of eicosanoids and moderately increases the secretion of corticoids. However, Recio et al. [43] demonstrated that there is no direct implication of corticosteroids in the mechanism of action of cucurbitacin R. Similar results were obtained by Witkowski et al. [58], who demonstrated that the effects of cucurbitacins are neither mediated by glucocorticoid receptors nor do they require replication, transcription, or translation. In fact, the presence of two glucoses in the molecule would probably modify the biological response. Ukiya et al. [9] isolated twelve cucurbitacins from Bryonia dioica roots and studied the anti-inflammatory activity of six of them in a 12-0tetradecanoylphorbol 13-acetate (TPA)-induced mouse ear edema. All six of the compounds showed anti-inflammatory activity with an ID50 range from 0.2 to 0.7 mg/ear; however, no relationship was established between chemical structure and anti-inflammatory activity. The most potent compounds were bryoniosides B, E, and G, Fig. (9), which inhibited the edema by 94%, 94%, and 90%, respectively, with ID50 values of 0.2 mg/ear each. Different extracts obtained from species of Wilbrandia exhibited antiinflammatory activity when assayed in different experimental models of inflammation, such as carrageenan-induced rat hind paw edema, carrageenan-induced granuloma in rats, and acetic acid-induced vascular permeability in mice. The authors attributed the anti-inflammatory effect to the two nor-cucurbitacin glucosides isolated previously from the same source, namely cucurbitacins WGi and WG2, Fig. (10) [6]. From the anti-inflammatory, antipyretic, analgesic, and antioxidant extract of Kageneckia oblonga, Delporte et al. [13] isolated two cucurbitacins which were assayed as potential antioxidants and also as inhibitors of enzymes implicated in inflammatory reactions. Isolated compounds, 23,24-dihydrocucurbitacin F and 3(3-((3-D-glucosyloxy)16a,23a-epoxy-cucurbitan-5,24-diene-ll-one, Fig. (11), inhibited the production of superoxide anion as well as elastase release in stimulated human neutrophils. In addition, the compounds inhibited both nitrite and
445
PGE2 production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These inhibitory effects could explain the in vivo mechanism of the active methanol extract.
HO
HO
OH
OH
Fig. (9). Chemical structures of bryoniosides B, G and E
446
OCOCH3
Fig. (10). Chemical structures of nor-cucurbitacins WG] and WG2
HO'1
23,24-Dihydrocucurbitacin F
3P-(p-D-glucosyloxy)-l 6<x,23a-epoxy-cucurbitan-5,24-diene-l 1 -one
Fig. (11). Chemical structures of active cucurbitacins from Kageneckia oblonga
Analgesia Both the extracts containing cucurbitacins and the isolated compounds themselves have been reported as analgesic agents. The dichloromethane extract from Wilbrandia ebracteata roots administered i.p., for example, had an analgesic effect in the acetic acid writhing test in mice, reducing
447
writhing by 26% at 1 mg/kg and by 78% at 10 mg/kg [27]. In the zymosan-induced abdominal constriction in mice, the same extract administered in the same manner inhibited the effect by 46% at 1 mg/kg (p.o.) and by 83% at 10 mg/kg. However, in the host plate test in mice the extract had no effect. These results indicate that the analgesic effect of such extracts is most likely produced by a peripheral mechanism and that cucurbitacins are probably the active principles [29]. In the case of the extract of Kageneckia oblonga, the authors established a relationship between cucurbitacins present in the extract and its antipyretic and analgesic activities, justifying a potential mechanism in which a decrease in PGE2 production through the inhibition of COX activity is implicated [13]. Anticancer and cytotoxic effects of cucurbitacins One of the highest priorities in cucurbitacin research has been the exploration of both the cytotoxic and anticancer effects of these compounds. This is due to the fact that they are generally considered to have a high level of toxicity, presumably also against cancer cells. Almeida et al. [6] thus tested a purified fraction from the rhizome of Wilbrandia sp. and demonstrated not only that the fraction inhibited KB cells with an effective concentration-50 (EC50) of 12 (J-g/ml, but also that it reduced the relative tumor weight of rats bearing Walker 256 carcinosarcoma by 65% [6,59]. These effects were thought to be due to the presence of cucurbitacins WGj and WG2 in the fraction, but the pure compounds were not tested. In another study, cucurbitacins B, D, E, and I from Cucurbita andreana fruits were tested for their inhibitory effects on proliferation of HCT-116 (colon), MCF-7 (breast), NCI-H460 (lung), and SF-268 (central nervous system (CNS)) cancer cell growth. All of the compounds were active at a concentration of 0.4 |aM, with the activity against colon cancer cells ranging from 82% for cucurbitacin B to 65% for cucurbitacin I. Cucurbitacin B was the most active against the four cell lines, inhibiting the proliferation of colon cancer cells by 82% and breast cancer cells by 87%, both at 0.4 uM. It also inhibited lung cancer cells by 96% at 0.1 ^iM and CNS cancer cells by 92% at 0.05 uM. In contrast, cucurbitacin I was the least active cell line [12]. Since these cucurbitacins had been reported as COX-2 inhibitors, and since a high over-expression of COX-2 in different kinds of tumor cell lines has also
448
been observed [60-63], the authors suggested that the compounds' ability to inhibit the COX-2 mechanism was implicated in the mechanism of cancer cell inhibition. Similar compounds, namely cucurbitacins B, D, and R, were isolated from Begonia heracleifolia, and their inhibitory activity on the growth of human nasopharyngal carcinoma (KB), murine embryonic fibroblasts (3T3), human prostate carcinoma (PC3), and murine methylcholanthreneinduced fibrosarcoma (MethA) was demonstrated. The IC50 values ranged from 0.003 to 3.81 u.g/ml, depending on the chemical structure of the compound and the cell line employed in the experiment. In general, cucurbitacin B was the most potent of the assayed compounds; moreover, the glycosylation at C-2 and the reduction of the carbonyl at C-3 to give a hydroxyl were found to curb or eliminate the activity. In contrast, the hydrogenation of the C-23—C-24 double bond did not seem to modify the activity [64]. Cucurbitacin E, isolated from Conobea scoparoides, inhibited cell adhesion by interfering with lymphocyte function associated antigen (LFA-1) whereas intercellular adhesion molecule-1 (ICAM-1) was not affected [51]. A study on the possible mechanism for the cytotoxicity of cucurbitacins revealed that the side chain has a relevant role in several aspects of their pharmacological activity. The molecules with a double bond (C-23-C-24) exhibited increased cytotoxicity while also inhibiting cell adhesion properties as compared to those without the double bond, e.g. cucurbitacin I (IC50 = 0.95 uM) vs its dihydro-derivative, cucurbitacin L (IC50 > 50 uM), Fig. (12); and cucurbitacin D (IC50 = 1.36 (J.M) vs its dihydro-derivative, cucurbitacin R (IC50 > 50 uM). Moreover, the presence of an acetoxyl group at C-25 only modified the potency of the compounds, e.g. cucurbitacin E (IC50 0.18 uM) vs its dihydroderivative, cucurbitacin I (IC50 — 0.95 uM); and cucurbitacin B (IC50 = 0.30 uM) vs its dihydro-derivative, cucurbitacin D (IC50 1.36 uM). Finally, all the compounds tested inhibited actin polymerization in formyl-Met-Leu-Phe (fMLP)-stimulated neutrophils, but did not modify Ca2+ flux or inhibit protein kinase C (PKC) in TPA-activated JY cells. In conclusion, the authors hypothesized that minor changes in the sidechain, but not in the A-ring, markedly affect the potency level for cell adhesion inhibition. This is caused by the disruption of the cytoskeleton, which itself is a consequence of the inhibition of actin polymerization [51].
449
Ito et at. [65], isolated cucurbitacins D and F from Elaeocarpus mastersii and studied their cytotoxicity against a series of human cancer cell lines. Of the two compounds, cucurbitacin D showed higher activity against human lung cancer (Lul), human colon cancer (Col2), human oral epidermoid carcinoma (KB), hormone-dependent human prostate cancer (LNCaP), human telomerase reverse transcriptase-retinal pigment epithelial cells (hTERT-RPEl), and human umbilical vein endothelial cells (HUVEC), with a range of ED50 values from 0.01 to 0.06 u.g/ml. In contrast, the range of cucurbitacin F was from 0.1 to 1.9 ug/ml.
Fig. 12. Chemical structure of cucurbitacin L
Cucurbitacin E inhibited the cell growth of different tumor cell lines, showing remarkable activity in both primary prostate carcinoma explants and immortalized prostate carcinoma cells. It was also found to be a potent inductor of disruption of the actin cytoskeleton; indeed, in a comparative study with similar analogues, the anti-proliferative activity was shown to be directly correlated to the disruption of the F-actin cytoskeleton. Still, the appearance of microtubules remained unaffected [66]. Cucurbitacin E also inhibited a wide range of cancer cell lines with an IC50 range from 13 nM (LOX IMVI melanoma cells) to 295 nM (OVCAR-5 ovarian carcinoma cells). When its anti-proliferative effects were compared with those of a series of cucurbitacin congeners on PC-3 cells (prostate carcinoma), a high correlation between inhibition of cell growth and F-actin disruption was demonstrated. Again, the influence of the side-chain in the chemical structure was clearly supported [66]. Moreover, the same authors [67] demonstrated that cucurbitacin E preferentially inhibits proliferating vs quiescent endothelia. In fact, this compound inhibited the log-phase of ECV and HUVEC endothelial cells at 12 nM and 13 nM, respectively, whereas confluent cells were inhibited at 170 nM and 76 nM, respectively. Cucurbitacin E thus has potential as
450
an anti-angiogenic agent in the treatment of tumor vasculature, especially since proliferating endothelial cells are more sensitive to it than the confluent, low-turnover endothelial cells; however, an in vivo study to evaluate the therapeutic index has yet to be undertaken. Cucurbitacin I suppressed the levels of phosphotyrosine signal transducer and activator of transcription 3 (STAT3) in v-Src-transformed NIH 3T3 cells and human adenocarcinoma A549 cells (IC50 0.5 pM). Moreover, it not only increased mouse survival, but also inhibited growth of human and murine tumors in mice, affecting tumors with high levels of constitutively activated STAT3 while not inhibiting tumors with low levels of activated STAT3 [68]. STAT3 is a key signal transduction protein that, after phosphorylation, plays a dual role of transducing biological information from cell surface receptors to the cytoplasm and translocating to the nucleus where gene expression is regulated. As this protein plays a pivotal role in human tumor malignancy, compounds which suppress its activity may have great potential as anticancer agents [68,69]. Picracin (cucurbitacin Q) and deacetylpicracin (cucurbitacin O) from Picrorhiza scrophulariaeflora, Fig. (13), inhibited phytohemagglutinininduced T-lymphocyte proliferation in a dose-dependent manner with an IC50 of 1 uM. This effect cannot be due to the cytotoxicity of the compounds since in specific experiments the authors demonstrated that neither cucurbitacin exhibits toxic effects for IC50 values up to 50 uM. The mechanism of action seems to be an interference with the cytoskeleton and subsequent abrogation of proliferative signal transduction, which in turn inhibits T-lymphocyte proliferation [70].
0C0CH3
Fig. 13. Chemical structures of picracin (cucurbitacin Q) and deacetylpicracin (cucurbitacin 0)
The methanol extract of Kageneckia oblonga showed a high cytotoxicity against P-388 murine leukemia, A-549 human lung
451
carcinoma, and HT-29 colon carcinoma (IC50 = 2.5 (j.g/ml), but the isolated cucurbitacins showed either weak cytotoxicity (23,24dihydrocucurbitacin F) or none at all (3p-(p-D-glucosyloxy)-16a,23otepoxy-cucurbitan-5,24-diene-ll-one) [13]. The authors compared the cytotoxic effect of 23,24-dihydrocucurbitacin F with that previously reported for cucurbitacin F [71]. Whereas the former had only a weak effect (IC50 = 5 jag/ml), the second exhibited strong cytotoxicity against KB (IC50 = 0.074 ng/ml) and P-388 cell lines (IC50 = 0.04 ng/ml). The authors hypothesized that toxicity increases when a double bond at C23—C-24 is present [13]. However, since the presence of an acetoxyl group at C-25 increases toxicity against non-cancerous cells [43], there is probably more than one structural requirement for increased cytotoxicity. The inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA were examined as a preliminary evaluation of the potential antitumor-promoting activities for eleven cucurbitacins isolated from Bryonia dioica (bryonioside A-G, cabenoside D, bryoamaride, bryodulcosigenin, and bryosigenin). All of these cucurbitacins showed potent inhibitory effects in this test, with the inhibition of induction ranging from 88 to 100% at 1 x 103 mol ratio/TPA, while also preserving the high viability (60-70%) of the Raji cells used in the experiment [9].
OH HO HO
Fig. (14). Chemical structures of cayaponosides B and C2
452
In a similar screening, Konoshima et al. [72] studied the inhibitory effects of twenty-four 29-nor-cucurbitacin glucosides isolated from the roots of Cayaponia tayuya and found that five of them, cayaponosides B, B3, D, D3b, and C2, exhibited significant inhibitory effects on EBV activation induced by the tumor promoter TPA. Moreover, two of the cucurbitacins shown to be active in vitro, cayaponosides B and C2, Fig. (14), inhibited mouse skin tumor promotion in a two-stage in vivo carcinogenesis test.
= R HO-\_^---V--V OH CH 2 OR
CH 2 OR
HO
Fig. (15). Chemical active cucurbitacin-glycosides
Using assays involving EBV-EA activation and two-stage carcinogenesis of skin tumors, the same authors [73] had previously studied the inhibitory effects of nine cucurbitacins isolated from
453
Hemsleya panacis-scandens, eight from Hemsleya carnosiflora, and four from Cowania mexicana. Of the tested cucurbitacins, scandenoside R6, scandenoside R7, carnosifloside III, Fig. (15), 23,24-dihydrocucurbitacin F, 25-acetyl-23,24-dihydrocucurbitacin F, 2-0-P-D-glucopyranosyl23,24-dihydrocucurbitacin F, and cucurbitacin F showed significant activity, inhibiting EBV-EA activation by 85% at 10"3 mol ratio/TPA. Cucurbitacin F and its glucoside both exhibited remarkable anti-tumor promotion effects in a two stage in vivo carcinogenesis test on mouse skin papillomas. Cucurbitacins I, D, and B, along with tetrahydrocucurbitacin I (cucurbitacin R) were found to inhibit the incorporation of radioactive precursors into DNA, RNA, and protein in HeLa S3 cells. The ID50 values of the cucurbitacins, which indicate inhibition of macromolecule biosynthesis, were close to their respective ED50 values, which indicate inhibition of cell proliferation. The authors [58] established a relationship between the capacity of cucurbitacins to inhibit the biosynthesis of DNA, RNA, and protein in HeLa S3 cells and the inhibitory effect on the proliferation of these cells. The inhibitory effects of cucurbitacins on the biosynthesis of cellular macromolecules, as well as the inhibition of cellular growth, originate from a common, as yet unknown target of cucurbitacin activity. However, a correlation has been established between the growth-inhibitory activity of cucurbitacins and dexamethasone, a fact which implies a glucocorticoid mechanism for the former. Notwithstanding, Witkowski et al. [58] assert that the inhibitory effects of cucurbitacins on biosynthesis involve a mechanism resembling the immediate extragenomic effects of glucocorticoids, and are thus not receptor-mediated. Using previously reported data on twenty-four cucurbitacins studied by the National Cancer Institute (NCI), Van Dang et al. [74] established a relationship between chemical structure and cytotoxicity by comparing the data concerning the toxicity against KB cells (nasopharynx human carcinoma) with that concerning toxicity against animals. The most relevant structural features for cytotoxicity are: the presence of an a,(3unsaturated ketone in the side chain (Table 3), a free 16a-0H group in the cucurbitane skeleton, and the presence of an acetoxyl group at C-25 (Table 4). Moreover, the presence of a keto or hydroxyl group at C-2 / C3 and the stereoisomery of the OH group at C-3 were found to modify dramatically the cytotoxic potency (Table 5).
454 Table 3. Influence of C-23 — C-24 substitutions in the cytotoxicity [74] Cucurbitacins
Substitutions
ED5o ng/ml (M)
Cucurbitacin B 23,24-Dihydrocucurbitacin B
A23 C-23,24-dihydro
0.002 (9 xltr 12 ) 2(3.5x10-')
Cucurbitacin Q 23,24-Dihydrocucurbitacin Q
A23 C-23,24-dihydro
30(53x10'') 2900 (5 x 10"6)
Table 4. Influence of C-25 substitutions in cytotoxicity [74] Cucurbitacins
Substitutions
ED.,,, ng/ml (M)
Cucurbitacin B Cucurbitacin D
C-25 (acetoxyl) C-25 (hydroxyl)
0.002 (9 x lO"12) 2 (4 x 10"9)
Cucurbitacin E Cucurbitacin I
C-25 (acetoxyl) C-25 (hydroxyl)
0.00005 (9 x 10"14) 6(11 x 10"')
Table 5. Influence of C-2 and C-3 substitutions in cytotoxicity [74] Cucurbitacins
Substitutions
ED50 ng/ml (M)
Cucurbitacin B Isocucurbitacin B
C-2 (OH) C-3 (O) C-2 (O) C-3 (OHcc)
0.002 (9 x 10"12) 400 (7 x 10'7)
Cucurbitacin D Isocucurbitacin D 3-e/)/-isocucurbitacin D
C-2 (OH) C-3 (O) C-2 (O) C-3 (OHcc) C-2 (O) C-3 (OHP)
2 ( 4 x 10-') 30(58x 10"') 200 (4 x 10'7)
As can be seen above, minor structural modifications not only change the cell cytotoxicity, but also affect the toxicity in animals (Table 6). Thus, while unsaturated C-l cucurbitacins clearly increased both the cytotoxic potency as well as the toxicity in animals (cucurbitacin E vs cucurbitacin B), the saturation of C-23 decreases the toxicity in both cells and animals (cucurbitacin I vs cucurbitacin L).
455 Table 6. Influence of C-l, C-23, and C-25 substitutions on cell and animal toxicities [74] Cucurbitacins Cucurbitacin Cucurbitacin Cucurbitacin Cucurbitacin
E B I L
Substitutions
ED50 (ng/ml)
Toxicity (mg/kg)
A1 A23 C-25 (acetoxyl) C-l,2dihydro A23 C-25 (acetoxyl) A1 A23 C-25 (hydroxyl) A1 C-23,24 dihydro C-25 (hydroxyl)
0.00005 (9 x 10"'4) 0.002 (9 xlO'' 2 ) 6(11 x 10"') 300 (6 x 10"7)
10 2 2 12.5
Finally, blocking C-2, C-3, and C-l6 hydroxyls has been found to reduce the toxicity in all the known cases (Table 7). Comparative data of the cytotoxicity of cucurbitacin Q vs that of its triacetyl-derivative showed a spectacular difference in cytotoxicity, with the former being much more potent than the latter.
Table 7. Influence of C-l, C-2, and C-16 acetylation on cell toxicity [74] Cucurbitacins Cucurbitacin P Cucurbitacin P 1,2,16-triacetate Cucurbitacin O 1,2,16-triacetate
Substitutions
ED 5 0 ng/ml
C-23,24 dihydro C-23,24 dihydro A23
500 45000 20000
On the basis of the data described above, Van Dang et al. [74] used a computer-aided drug design (CADD) to establish a quantitative electronic structure-activity relationship (QESAR) between cytotoxic cucurbitacins and other cytotoxic natural products, including maytansinoids and quassinoids, with the aim of designing new cucurbitacins as future therapeutic agents against cancer. The authors evaluated the pharmacophore of these groups and designed some theoretically active compounds. Of these, the C-25 tygloyloxy derivative seems to be the most effective, Fig. (16), with a theoretical therapeutic index 1727 times higher than that of cucurbitacin E, the most active of the 25-acetyl-derivatives studied.
456
Fig. (16). 25-Tygloyloxy cucurbitacin I (25-tygloyloxy,25-deacetyl cucurbitacin E)
Effects of cucurbitacins as adaptogens and on the immune system As was described above, Panossian et al. [57] demonstrated the adaptogenic activity of Bryonia alba roots in preclinical and clinical trials. The same authors [75] studied the potential mechanism responsible for these adaptogenic effects, focusing on the potential activity of cucurbitacin R-diglucoside, one of the constituents of the active extract. This compound had previously been found to increase the working capacity of mice, and also to increase the survival of mice infected with Staphylococcus aureus as well as that of X-ray irradiated rats. It also reduced stomach ulcers in immobilized rats [75]. In fact, cucurbitacin R-diglucoside protects against stress-induced alterations of eicosanoids in blood plasma and stimulates the adrenal cortex to adapt the organism to stress. Panossian et al. [75] demonstrated that cucurbitacin R-diglucoside increases corticosteroid secretion by stimulating the adrenal cortex, modulating corticosteroid release until optimal levels are obtained, thereby protecting the adrenal cortex from hypotrophy. Moreover, cucurbitacin R-diglucoside modifies the metabolism of eicosanoids, increasing the production of PGE2, which is sub-produced in times of stress. PGE2 has a cytoprotective influence on the gastrointestinal epithelium, which is clearly damaged by stress. On the other hand, cucurbitacin R-diglucoside inhibited the biosynthesis of the pro-inflammatory mediators from LOX such as LTB4 and 5-hydroxy6£,8Z,llZ,14Z-eicosatetraenoic acid (5-HETE), which activate chemotaxis of neutrophils, lysosomal enzyme release, vascular permeability, and superoxide anion generation. Moreover, it inhibited the NADPH-dependent enzymatic and ascorbate-induced non-enzymatic lipid peroxidation. However, cucurbitacin R-diglucoside had no effect in
457
the in vitro assays when these were carried out on isolated leukocytes of immobilized rats, probably because stress significantly suppresses the 5LOX and 12-LOX activity of leukocytes by a mechanism mediated by the increase of corticosterone formation. A previous pretreatment with cucurbitacin R-diglucoside decreased 12-LOX activity, but increased that of 5-LOX. This finding indicates that the systemic effects of cucurbitacin R-diglucoside as an adaptogen occur at a central rather than at a peripheral level [75]. Cucurbitacins B, D, and R were assayed as immunomodulators on mitogen concanavalin A-stimulated IL-2 dependent murine lymphoblasts (IL-2 BL) and mitogen concanavalin A-stimulated murine spleen cells (ConA SC), but the activity was of little interest due to the pattern of activity and its combination with the results of the compounds' influence on growth of permanent cell lines, described above. One interesting finding, however, concerned the activity of cucurbitacin R, the free form of the aforementioned compound, which gave IC50 values of 1.0 and 0.46 (ig/ml against IL-2 BC and ConA SC, respectively [64]. Effects on insects and plant parasites Many secondary metabolites found in plants deter phytophagous invertebrates, sometimes even modifying insect growth and development if included in the diet [8]. Natural products can often act as insecticides via different pathways, as is the case with the analogues of insect juvenile hormones produced by plants. Thus, some derivatives of these analogues are used as commercial insecticides while others act as ecdysteroid antagonists [8]. Ecdysteroids are steroidal hormones responsible for controlling molting and metamorphosis in insects, thereby contributing to their normal development and probably that of other invertebrates as well. Analogues of ecdysteroids, the so-called phytoecdysteroids, occur in some plants, but there is another parallel group of compounds made up of known antagonists of the ecdysteroid receptor [24]. Of this latter group, the cucurbitacins form a widely-cited subset [23]. Cucurbitacin B, for example, is a known antagonist of 20-hydroxyecdysone [25,26], and is responsible for the antagonistic activity of a methanolic extract of Iberis umbellata (Cruciferae) [26] and Physocarpus opulifolius (Rosaceae) [4],
458
which prevents the 20-hydroxyecdysone-induced morphological changes in the Drosophila melanogaster Bn permanent cell line. CH2OH
Carnosoflogenin A
CH2OH
Camosoflogenin C CH 2 OR
Carnosifloside II CH 2 OR
Carnosifloside VI Fig. (17). Chemical structure of cucurbitacins from Hemsleya camosiflora
Four of the seven cucurbitacins assayed in the Drosophila melanogaster Bn bioassay exhibited antagonistic activity [8]. Carnosoflogenin A and C, as well as carnosifloside II and VI, Fig. (17), all isolated from Hemsleya camosiflora, showed weak antagonistic activity at 0.1 mM. The ED50 values obtained to produce a 50% reversal of the reduction in A405 brought about by 5 x 10~8 M 20hydroxyecdysone were 3.4 x 10"4 M and 1.2 x 10"4 M, respectively. These effects were lower than that reported for cucurbitacins B and D,
459
which show activity in the 0.1 uM range. In the last case, the activity was associated with the presence of an a,P-unsaturated C-22 ketone. In the cucurbitacins isolated from Hemsleya carnosiflora, however, the activity was associated with the presence of a trans-A24 double bond. Moreover, dihydrocucurbitacin F and 25-acetoxy-dihydrocucurbitacin F, both isolated from the same source and possessing an oxo-function but lacking a double bond, showed only weak antagonistic activity, with ED50 values in the range of 3 x 10"5 M.
Hexanorcucurbitacin D
OCOCH3
HO1
Cucurbitacin F
Cr'
><"
" ^
Cucurbitacin C
HO"
2p,16a,20p-trihydroxy-cucurbitan-5,24-dien-3,22-dione
3-e/?;'-Isocucurbitacin D
Fig. (I8).Chemical structures of ecdysteroid antagonists
In a complementary study, Dinan et al. [76] screened twenty-six cucurbitacins in the Drosophila melanogaster Bn bioassay and demonstrated the agonist activity of one of them (hexanorcucurbitacin
460
D), as well as the antagonist activity of twelve of them at uM range, along with that of four more at mM range. Of the active compounds, cucurbitacins B, D, and F had EC50 values in the 0.1 uM range, cucurbitacins C, E, and I, 3-epz-isocucurbitacin D, and cucurbitacin Q (picracin) had EC50 values in the 1 uM range, with the rest of the active compounds having EC50 values in the 10 uM range. After analyzing these results against previously reported data, the authors established a relationship between antagonist activity and chemical structure, Fig. (18). The ecdysteroid antagonist activity of cucurbitacins is thus associated with the presence of an a,(3-unsaturated ketone at C-22 and an oxygen containing functional group at C-3. Only 2p,16a,20p-trihydroxycucurbitan-5,24-dien-3,22-dione was active without keto-double bond conjugation, but the authors explain that the antagonism in this case is due to the absence of a C-25 oxygen-containing function, such as occurs in another specific antagonist, ponasterone A (25-deoxy-20hydroxyecdysone), which was found to be more potent than the wellresearched specific antagonist 20-hydroxyecdysone. In some cases, cucurbitacins seem to act as potential phytosteroid supplements. Larvae of the spotted cucumber beetle (Diabrotica undecimpunctata howardi), grew significantly faster when they developed on cucurbitacin-rich roots, surviving as well as larvae on cucurbitacin-poor roots. However, there is no evidence that adults can substitute cucurbitacins for vital phytosteroids. Beetles reared on a cucurbitacin-rich and phytosteroid-poor diet, for instance, laid significantly fewer eggs and died significantly younger than beetles with a full complement of dietary phytosteroids. Even more intriguing is the fact that they also laid fewer eggs than beetles with no access to phytosteroids in their adult diet. The side chain of dietary cucurbitacins probably plays a relevant role in these biological events, as seen by the fact that while cucurbitacins with a hydrogenated side chain play a nutritional role as substitutes or precursors for structural steroids, those with an unsaturated double side chain usually become antagonists of ecdysteroid receptors, negatively affecting the beetle lifecycle [77]. Laccase protein is produced by Botrytis cinerea as an attack mechanism used by the pathogen while invading the host [78]. In previous papers, Viterbo et al. [78-80] reported on the manner in which cucurbitacins selectively inhibit laccase formation in Botrytis cinerea without affecting other enzymes such as polygalacturonidase, cellulase,
461
acid proteinase, carboxypeptidase, and pectin methylesterase [80]. Nevertheless, the authors were unable to demonstrate the interaction mechanism of cucurbitacin during transcription, translation, or posttranslation [78]. Other work with Botrytis cinerea found that cucurbitacin I protected cucumber tissue against fungal infection by actually inducing the fungus to form laccase [81]. In a complementary study, Gonen et al. [82] demonstrated that this cucurbitacin specifically represses the amount of mRNA coding for laccase; however, no clear effects were observed when its deacetylated derivative, cucurbitacin E, was assayed The mechanism by which cucurbitacin I acts is not clear, but it may inhibit transcription by specifically binding to certain regions of DNA to block the transcription process. Cucurbitacin B evokes chemosensory responses at levels as low as 0.1 u,M, probably acting at a different modulatory site than do classical synaptic y-aminobutyric acid (GABA) and glycine receptor-channel complexes. Chyb et al. [83] studied adult western corn rootworm (Diabrotica virgifera virgifera, Coleoptera-Chrysomelidae) beetles and reported the possibility that there are peripheral chemosensory receptor sites that may resemble, both functionally and structurally, synaptic receptor sites in the CNS. Some studies hypothesize about the protection of cucurbitacins against soil borne fungal entomopathogens, both in the adult corn rootworm and in the eggs laid in the soil. However, Martin et al. [84] demonstrated that cucurbitacins do not inhibit fungal growth, as seen by the fact that the sterilized extract containing them showed no activity. These authors thus hypothesize that the activity may actually be due to bacteria associated with the plants. Iberis amara contains both stimulants and deterrents involved in regulating oviposition by Pieris rapae. The most active deterrents were 2-O-p-D-glucosyl cucurbitacin I and 2-0-P-D-glucosyl cucurbitacin E [85]. There also appears to be a quantitative relationship between cucurbitacin C content in Cucumis sativus and spider mite {Tetranychus urticae) resistance [86].
462
Toxicity In general, cucurbitacins and the extracts containing them are considered to be toxic, with the degree of toxicity depending on the plant material, type of extract, and the substitution partner of the compound. For example, exposure to the juice of the anti-inflammatory medicinal plant Ecbalium elaterium, especially in its undiluted form, often leads to a supposedly inflammatory irritation of mucous membranes [87]. These toxic effects seem to correspond to the juice's major active compound, cucurbitacin B. The chloroform extract of Cayaponia tayuya roots was found to have an LD50 of 375 mg/kg (p.o.) [10]. For its part, the dichloromethane extract of Wilbrandia species had an LD50 of 975 mg/kg (p.o.) [6] while that for the dichloromethane extract of Kageneckia oblonga was 940 mg/kg (p.o.) [13]. In the case of isolated compounds, the LD50 values of cucurbitacins range from 5 to 100 mg/kg [6], but there is little information about their toxicity in vivo. One exception, however, is Yesilada's study, in which cucurbitacin B was reported to have an LD50 of 10.9 mg/kg in mice [52]. Some studies with chemical analogues seem to indicate that while side chain substitution is essential for toxicity, substitutions in the A-ring are not. For example, the dihydro-derivatives of cucurbitacin I (cucurbitacin L) and cucurbitacin D (cucurbitacin R) are clearly less toxic than their corresponding dehydro analogues [51]. Moreover, the presence of an acetoxyl group in the side chain (C-25) increases the toxicity of cucurbitacins [43,51]. This data was corroborated by Oh et al. [88], who obtained LD50 values of 0.9 and 11.1 uM for cucurbitacin D and its dihydro-derivative, cucurbitacin R, against B16/F10 melanoma cells. Other pharmacological and biological effects Antifertility Some cucurbitacins have been reported to be antifertility agents. The enriched cucurbitacin extract obtained from Wilbrandia sp. was tested for its potential antifertility effects with different experimental protocols including those involving estrus cycle, implantation, abortifacient, and estrogenic/antiestrogenic activities. The extract suppressed the number of
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occurrences of the estrus stage and uterine implantation, but exhibited no abortifacient or estrogenic/antiestrogenic effects [6]. Tyrosinase and melanin synthesis inhibitor Cucurbitacins D and R inhibited both tyrosinase activity and the melanin synthesis of B16/F10 melanoma cells. Of the two compounds, cucurbitacin D showed higher potency than either cucurbitacin R or the reference drug used in this experiment, with IC50 values of 0.18 uM as an inhibitor of tyrosinase activity and 0.16 )J,M as an inhibitor of melanin synthesis. Cucurbitacin R gave values of 6.7 and 7.5 uM while the reference drug hydroquinone gave values of 11.5 and 7.4 uM against tyrosinase activity and melanin synthesis, respectively [88]. Antihepatotoxic Cucurbitacin B isolated from Ecballium elaterium fruit juice has been shown to have both preventive and curative effects against CCL^-induced hepatotoxicity. Pre-treatment with cucurbitacin B reduced the increased level of serum glutamate-pyruvate transaminase (GPT) in CCU-induced hepatotoxicity in mice by 93%, while also reducing steatosis, necrosis, and inflammation. Post-treatment with the same compound reduced the GPT level by 90%, decreased the degree of steatosis with respect to the control group treated only with CCI4, and abolished both the necrosis and the inflammation [89].
Antimicrobial properties Although some cucurbitacin-containing plants have been reported as being anti-infectious, there is no clear evidence for antimicrobial activity. Using an agar overlay method, Frei et ah [64] screened six cucurbitacins against eight bacteria and one yeast. At 20 jj.g, none of the compounds showed inhibitory activity. Similar results were obtained by Huang et ah [56], who screened four cucurbitacin glycosides against a series of bacteria, fungi, and the herpes simplex virus type 1 (HSV-1). None of
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the compounds exhibited inhibitory activity at 100 ug/ml even though the extract from which the cucurbitacins had been isolated showed both antiviral and antimicrobial effects. FUTURE PERSPECTIVES In the past, cucurbitacins have been viewed as toxic compounds with potential cytotoxic and anti-ecdysone effects. More recently, however, a relevant number of researchers have focused their studies on the potential anti-inflammatory properties of these compounds, especially in light of the good results obtained with extracts containing them. This new knowledge of the activity and toxicity of a wide group of compounds has led to the discovery of several interesting relationships which help avoid negative side effects. Thus, the presence of an acetoxyl group at C-25, the double bond at C-23, or the presence of a carbonyl or hydroxyl at C-3 have been shown to modify both the cytotoxicity and the pharmacological activity of the cucurbitacins. The data reported in this review sheds light on the basic skeleton necessary for a cucurbitacin to be active against different types of cancer cell lines, as well as for one that will be active against different inflammatory diseases. Researchers interested in the former should concentrate their efforts on cucurbitacin B or E in order to obtain new active compounds, whereas in the second case, cucurbitacin R shows the most potential as the basis for a series of anti-inflammatory cucurbitacins. ABBREVIATIONS APT CADD CI CNS COSY COX DEPT EBV-EA EC50 ED50
= Attached proton test = Computer-aided drug design = Chemical ionization = Central nervous system = Correlated spectroscopy = Cyclooxygenase = Distortionless enhancement by polarization transfer = Epstein-Barr virus early antigen = Effective concentration 50 = Effective dose 50
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El FAB fMLP GABA GPT HETCOR 5-HETE HPLC HPTLC HSV-1 IC50 ICAM-1 IL i.p. LD50 LFA-1 LOX LPS LT, LTB4 MPLC MS
= Electron impact = Fast-atom bombardment = formyl-Met-Leu-Phe = y-Aminobutyric acid = Glutamate-pyruvate transaminase = Heteronuclear chemical shift correlation = 5-Hydroxy-6£',8Z,l lZ,14Z-eicosatetraenoic acid = High-performance liquid chromatography = High-performance thin-layer chromatography = Herpes simplex virus type 1 = Inhibitory concentration-50 = Intercellular adhesion molecule-1 = Interleukin = Intraperitoneal = Lethal dose-50 = Lymphocyte function associated antigen-1 = Lipoxygenase = Lipopolysaccharide = Leukotriene, Leukotriene B4 = Medium pressure liquid chromatography = Mass spectrum NF-KB = Nuclear factor-KB NMR = Nuclear magnetic resonance NO = Nitric oxide NOESY = Nuclear Overhauser effect spectroscopy NOS, iNOS = Nitric oxide synthase, inducible nitric oxide synthase PG, PGE2 = Prostaglandin, Prostaglandin E2 PKC = Protein kinase C PLA2 = Phospholipase A2 PMNL = Polymorphonuclear leukocytes p.o. = Per os (orally) QESAR = Quantitative electronic structure-activity relationship RNA, mRNA = Ribonucleic acid, messenger ribonucleic acid RP = Reversed-phase s.c. = Subcutaneous SPE = Solid-phase extraction STAT3 = Signal transducer and activator of transcription 3 TFA = Trifluoroacetic acid
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TLC TNF TPA UV
= Thin-layer chromatography = Tumor necrosis factor = 12-0-tetradecanoylphorbol 13-acetate = Ultraviolet light
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GRISEOFULVIN AND OTHER BIOLOGICALLY ACTIVE, HALOGEN CONTAINING COMPOUNDS FROM FUNGI T. REZANKA, J. SPIZEK
Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnskd 1083, 142 20, Prague 4, Czech Republic ABSTRACT: Fermentation of produced strains, purification, isolation and biosynthesis of griseofulvin are described. Metabolites similar to griseofulvin (precursors, side products of biosynthesis, etc.) are also discussed. Most important fungal metabolites containing halogen atoms are included in the second part of the review. In brief, metabolites are included that were discovered and isolated in 1995 and later. Pharmacological properties are only described when referred to in the original papers. INTRODUCTION Chlorine and bromine forming various compounds are important components of the environment and are among the most common biogenic elements on the Earth. They are thus present in all living organisms, as well as in both inorganic and organic compounds. Compounds containing organic chlorine containing and to a lesser extent bromine are also synthesized by fungi. These often include unusual compounds with fascinating properties including biologically active compounds, antibiotics in the first place. Griseofulvin, a chlorinecontaining antibiotic produced commercially, is one of the most important examples. First Part - Griseofulvin In 1939, the isolation of griseofulvin from mycelia of Penicillium griseofulvum was reported [1]. This compound has the empirical formula Ci7Hi7ClO6 and Grove et al. [2] determined its structure 1 in 1952. Fermentation conditions for griseofulvin production have appeared infrequently in the scientific literature, because this substance is of great economic importance. It is one of the few antifungal compounds that are
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synthesized by fungi themselves and are produced commercially. Table 1 lists numerous organisms that have been shown to produce griseofulvin. The fermentation media and culture conditions for most of the producing organisms appear to be quite similar. Chemical synthesis of griseofulvin is economically not feasible, since a number of intermediate steps are involved in the final product formation. Although a number of synthetic routes to the spiro system have been reported, the methods generally fall into two broad categories, viz., the building up of a spiro ring C onto the A-B ring system and/or the forging together of ring A and C components to develop the oxygen containing ring B. The latter approach is exemplified in the elegant synthesis of griseofulvin developed [3,4]. This synthesis is based on the double Michael addition of vinyl ethynyl ketones to active methylene compounds [5]. The synthesis of griseofulvin by the Merck group represents the second approach to the antifungal antibiotic [6]. Table 1. List of griseofulvin producing microorganisms. Name of microorganism Literature Penicillium griseofulvum [1] Penicilliumjanczewskii [7] Penicillium nigricans [8] Penicillium urticae [8] Penicillium raistrickii [8] Penicillium albidum [9] Penicillium raciborskii [9] Penicillium melinii [9] Penicillium patulum [9] Aspergillus versicolor [10] Carpenteles brefeldianum [11] Khauskia oryzae [12] Nigrospora musae [13] Nigrospora splaerica [13] Streptomyces albolongus [14]
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Fermentation Typically, high yield industrial fermentations in submerged cultures have employed complex media with sugar, inorganic salts and corn steep liquor as the nitrogen source. The optimum chloride concentration for optimal griseofulvin production was found to be 0.001-0.05 % KC1 [15]. Fe2+ shows a specific effect on the formation of mycelianamide, a second compound, which is considered as interfering substance. By a proper strain selection and by keeping FeSO4 content in the media at 0.1% the formation of mycelianamide was eliminated [16]. When the nitrogen concentration was less than 0.04 % or more than 0.4 %, production of griseofulvin was inhibited. The retarding effect of the high nitrogen concentration levels was explained by a specific inhibition of N H / on oxaloacetate formation [17]. Large-scale production of griseofulvin was carried out using 30000 1 of culture medium containing (g/1) corn seed (5-15), corn extract (5-8), lactose (10-14), glucose (7-15), CaCO3 (10-14), KC1 (1-2), KH2PO4 (610), MgSO4 (0.05-0.1), urea (1-2), and hydrogenated sunflower oil (2-10). The medium was inoculated with 5-10 % precultivated inoculum of Penicillium and aerobically fermented for 300-350 hours at pH 5.8-7.0 and 26-30 °C. The maximum production of griseofulvin achieved was 1200 ug/ml without the formation of intermediates [18]. In 450 liter batch fermentation medium [9] containing (in g/1) corn steep liquor (2), lactose (70), CaCO3 (8), K2HPO4 (4) and KC1 (1), the maximum production by the P. patulum mutant was about 1500 ug/ml. A significant improvement in the fermentation technology of griseofulvin production was realized with the introduction of the fed batch process described by Hockenhull [19]. A yield of 6000 ug/ml of griseofulvin was obtained after a 220-h cultivation. When the corn steep liquor was increased to ~5 g/1, the yield of griseofulvin after a 260-hour cultivation increased to 11000 ug/ml [20]. Partial replacement of corn steep liquor with (NtL^SCXt yielded 14000 ug/ml in 3,786 liters fermentor [21]. The precise conditions of the cultivation are described bellow (g/1): corn steep liquor (3.5), (NH4)2SO4 (0.5), KH2PO4 (4), KC1 (1), CaCO3 (4), H2SO4 (0.125), Mobilpar S (0.275, white mineral oil (0.275). Further, the inoculation was performed
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with 10 % of inoculum. The cultivation temperature was 25 C and the rotation impeller speed was about 3.33 Hz. Between 0-5 hours of cultivation, the feeding rate was kept at 2.26 mVmin. In the interval of 5 to 10 hour, the feeding rate was maintained at 4.8 m3/min and this value could be conserved after 10 hours. The additional feeding of a solution containing 50 % of glucose was used to maintain pH in the range 6.8-7.2. The production of griseofulvin was improved by a careful control of the growth conditions of P. urticae. The medium containing (g/1) glucose (60), corn steep liquor (1.5), KH2PO4 (4), KCl (1) and CaCO3 (8) was incubated for 13 days and production was up to 14000 |ig/ml [22]. The use of hydrocarbons as carbon and energy sources for fermentation production of griseofulvin was described in [23] and the yield of 2550 ug/ml after 96 hours was obtained. Further, the production of griseofulvin by a 23-day surface cultivation of P. urticae yielded about 9000 ug/ml [24]. Similarly to the biosynthesis of tetracyclines, even here the bromo analog was produced by simply substituting KBr for KCl in the medium [25]. Deuterated griseofulvin has been obtained by cultivation of P. janczewskii in culture medium with D2O. Methods to improve the production are kept secret and those described in respective patents (see below) are also quite successfully camouflaged. In the fifties and sixties, classical methods based on mutagenesis with UV light or chemical mutagens such as ethyl methane sulfonate (with radioactive sulfur isotopes) and also N-nitrosomethylurea and N-nitrosoN-methylbiuret were mainly used. For example, the sulfur isotope procedure on the 18th day provided a culture that was reported to yield ~146 % more than its parent strain [26]. Kiuchi et al. [27] have reported the transformation of Penicillium urticae with plasmids containing the hygromycin-B resistance. The transformation system used plasmid pDH25MC and its derivatives containing fragments of the Penicillium urticae genome. Tandem repeated integration and random integration of vector DNA were observed. Although Penicillium urticae was able to grow in the presence of 0.2 kg/m3 hygromycin-B, transformants were resistant to more than 5.0 kg/m3 hygromycin-B. The strain of P. patulum was induced by exposure to Na235SO4 (~1 mg/1) and after 13 days the best strain produced 2964 ug/ml of
475
griseofialvin, compared to the production of 1640 ug/ml of the parent strain [26]. Another strain of P. patulum showed a high tolerance to Cl" anions and increased production of griseofulvin with increasing Cl" concentration that reached a maximum at 3 % of Cl" in the cultivation broth [28]. Purification and/or Isolation The mycelium of different strains was extracted three times by homogenization in the presence of CH2CI2. The solvent extracts were concentrated to 10 % of the original volume and cooled to 4 °C to remove some impurities. After decolorization with charcoal, the crystallization was accomplished by evaporation at 50 °C to 6.6 % of the original volume and cooling to 0 °C. The recovery was 95 % pure griseofulvin [29]. Biosynthesis The carbon skeleton of the griseofulvin has been shown by Birch [30,31] to arise from 7 acetates. Radioactive (l-14C)-acetates were incorporated into griseofulvin by P. griseofulvum. The incorporation of 213 C acetate by P. urticae was proved by 13C NMR and so identical carbon sites were reported [32]. Simpson and Holker [33] have similarly shown alternating site incorporation of l-13C-acetates and/or 2-13C acetates into griseofulvin by P. patulum. Incorporation of doubly labeled acetate (13CH3C18C«2H) into griseofulvin by P. griseofulvum revealed that all of its oxygen is derived from the acetate. The location of labeling was determined by pulse sequence in 13C NMR to detect 18O induced isotope shifts [34]. Studies of Sato et al., [35,36] using 2-2H- and 2-3H-acetates incorporation by P. urticae, fully validate the earlier experiments with acetates labeled by isotopes 13C and 14C. Intermediates isolated from P. patulum were reported by Rhodes et al. [37] and a biosynthetic scheme for griseophenones (2, 3) was postulated. Many additional papers in which speculative biosynthetic pathways were proposed were published [11,30,33,38-40]. Final biosynthetic steps including methylation or rather a series of methylations [32,33,40,41] and reductions [31,42] were also investigated. The biosynthetic study of griseofulvin by Penicillium urticae and microbial transformation of (-)- and (+)-dehydrogriseofulvin (4) and their derivatives by Streptomyces cinereocrocatus followed by NMR
476
spectroscopy showed that in the reduction of (-)-dehydrogriseofulvin into (+)-griseofulvin by a partially purified enzyme system of S. cinereocrocatus, the origin of the 6'-a-hydrogen of (+)-griseofulvin was a hydride ion donated by pro-4/?-hydrogen of NADPH [43]. Determination In a recent review, Dasu et al. [44] described determination of griseofulvin by means of different analytical methods. Although published as recently as in 2000, the review unfortunately includes references most typically from the fifties and sixties of the last century. Spectrophotometric methods, {e.g. UV determination [45]), spectrofluorimetric methods and microbiological assay using Microsporum gypseum [46] were developed. A major part of the paper is devoted to chromatographic methods, both to paper and thin-layer chromatography, and to more modern gas chromatography and HPLC. We concluded that just the latter method, i.e. HPLC, is of a real practical significance. For the identification of griseofulvin, standard RP-HPLC with acetonitrile-water as mobile phase with a UV detector or fluorescence detector seems to be the most useful. In addition to the above-mentioned RP-HPLC, other examples of analytical methods, such as determination of griseofulvin by LC-MS, will be described here. The non-volatile metabolites from several different molds were separated by HPLC, and then derivatized and analyzed by GC-MS [47]. New LC-MS and LC-MS-MS methods for the simultaneous determination of mycophenolic acid, griseofulvin, roquefortine C, chaetoglobosin B, verruculogen and penitrem A, and other Penicillium derived mycotoxins in food and feed samples were described. The methodologies involve sample extraction with acetonitrile-water, defatting with hexane and quantification using LC-MS with atmospheric pressure chemical ionization or LC-MS-MS. Detector responses for all mycotoxins were found to be linear over the range 10-1000 ng of mycotoxin/g of extracted food mixture material. The limits of detection for the mycotoxins using MS and MS-MS were 70 and 10 ng/g for griseofulvin, respectively [48]. Biological Activity A review covering most aspects of the mechanism of action of griseofulvin was published in 1974 [49]. Gull and Trinci [50] reported
477
that griseofulvin produced multinucleation. This drug is fungistatic in vitro for various species of dermatophytes, for example Microsporum (see above), Epidermophyton or Trichophyton. The drug has no effect on other fungi including yeast, and actinomycetes or Nocardia. It kills young and actively metabolizing cells and inhibits the growth of older and dormant cells. Griseofulvin was the first available oral agent for the treatment of dermatophytoses and has now been used for more than forty years [51]. Griseofulvin is fungistatic, the exact mechanism by which it inhibits the growth of dermatophytes being still doubtful. However, several mechanisms have been proposed: inhibition of fungal cell mitosis and nuclear acid synthesis and probable interference with the function of microtubules. Griseofulvin has also anti-inflammatory properties and some direct vasodilatory effects when used in high doses. Griseofulvin is poorly absorbed from the gastrointestinal tract but absorption is enhanced by administration with fatty meal, and peak plasma concentration occurs four hours after oral administration. Griseofulvin is detected in the outer layer of the stratum corneum soon after ingestion; it is diffused from the extracellular fluid and sweat. There is no information regarding the mechanism by which the drug is delivered to nails and hair. Deposition in the newly formed cells could be the major factor. It is metabolized by the liver microsomal enzyme system and excreted in the urine. Its half-life is 9 to 21 hours. Griseofulvin has been used in the therapy of dermatophyte onychomycosis; however, treatment periods from 6 to 18 months were necessarily with disappointing results and numerous relapses. Therefore, the newer oral antifungal agents itraconazole, terbinafine and fluconazole have superseded griseofulvin as agents of choice for onychomycosis. Unlike griseofulvin, the new agents have a broad spectrum of action that includes dermatophytes, Candida species and nondermatophyte moulds. Each of the new antifungal agents is more cost-effective than griseofulvin and is associated with high compliance, in part because of the shorter duration of therapy. The main use of griseofulvin currently is to treat tinea capitis, which is the most common dermatophyte infection during childhood [52]. The treatment of tinea capitis requires an oral antifungal agent [53], and griseofulvin is well tolerated particularly in children. However, some
478
clinical studies over the past decade that have investigated the response of tinea capitis to griseofulvin suggest a decrease in sensitivity to this pharmacologic agent [54]. Systemic therapy of scalp ringworm with itraconazole and terbinafine, as well as perhaps fluconazole, seems to be an equivalent or a superior therapeutic approach as compared to the use of griseofulvin. In addition, the data from the use of itraconazole, terbinafine, and fluconazole suggest that they are safe in children [55]and shorten the duration of therapy. Doses of griseofulvin are 15-20 mg/kg/d for 6 to 8 weeks in children with the microsized form. More frequent side effects are minor: headaches, gastrointestinal reactions and cutaneous eruptions. The major drug interactions have been noted with phenobarbital, anticoagulants and oral contraceptives. The absorption of griseofulvin with normal particle size in humans is minimal and unpredictable. However, the absorption can be increased by the costly process of size reduction to microsize or ultramicrosize [56]. A number of griseofulvin derivatives were synthesized, of which some exhibited a significant biological activity. The hitherto data on structurefunction relationships can be summarized as follows: 1. The spatial arrangement of griseofulvin plays a determining role. Only the natural griseofulvin exhibits the fungistatic activity, the 3 remaining isomers are inactive. 2. Chlorine in position 7 of the A ring is not essential for the biological activity. Its elimination or eventually the substitution by fluorine or bromine yields highly active compounds. For a high biological activity, it is required that position 6 of the aromatic ring remains free. Elimination of methoxy groups also does not yield useful compounds. 3. Substitution of oxygen in position 1 with sulfur or methylimino group does not produce compounds with an interesting biological activity as compared to griseofulvin itself. 4. Substitution of the whole ring C with 2,2-dimethyl group results in a significant decrease of the biological activity. 5. A number of modifications of the C ring can be made while preserving a high biological activity. Thus substitution of methoxy group in position with 2' propoxy- or butoxy- group yields compounds 20-50-
479
times more active in vitro than griseofulvin. Substitution of methoxy group with methyl group decreases the activity, elimination of the whole amino group results even in a complete activity loss. Substitution of keto group with CH2 group yields an inactive compound, however, the activity is preserved when carbonyl group is in position 2'. However, two carbonyl groups, in positions 2' and 4', lead to the loss of the activity. Elimination of the 6'-asymmetric center decreases the antifungal activity, whereas a change of the configuration results in a complete activity loss. Similar Metabolites Metabolites similar to griseofulvin, i.e. geodin (2) and erdin (3) were isolated from A. terreus and later also from Penicillium sp. [57-59]. Structurally similar compounds including e.g. dehydrogriseofulvin (4) from P. patulum [60] and P. martinsii (Kamal et ah, 1970) and dihydrogriseofulvin (5) from P. martinsii were also isolated [61]. Further, structurally similar metabolites as geodoxin (6) [62] and gillusdin (7) [63] have been discovered from A. terreus. These fungi also produce the presumed biosynthetic precursors, as mentioned above, griseophenones A, B (8, 9); dihydrogeodin (10) and its structural analogs 11 and 12 [37,64]. The marine annelid, Thelepus setosus produces thelepin (13), a novel brominated spiro compound, which has antifungal activity comparable with griseofulvin [65,66]. OMe
MeO'
2 3 4 7
Ri
R2
R3
R4
OH OH
Cl Cl H Cl
Me Me
CO2Me CO2H
OMe
Me
OH
OMe
OMe
Me
Rs H H H OH
R6
OMe OMe OMe CO2Me
480 OMe
MeO'
Ri
8 9 10 11 12
Me H H H Me
R2 H H Cl H H
R3 OMe OMe Me Me Me
R4
Me Me CO2Me CO2Me Me
Second Part - Other Halogen Containing Compounds from Fungi In the second part of the review, we would like to concentrate primarily on pharmacologically significant compounds containing halogen atom(s) in their molecules and are isolated from fungi. This part aims to review papers published after 1996, since earlier studies had been previously reviewed by Gribble [67,68]. Additional papers published by the same author [69-72], although highly interesting, are not extensive enough, and none of them deals in depth with natural compounds isolated from fungi and containing halogen atom(s) in their molecules. Most organohalogens produced by fungi have an aromatic structure; important groups include the chlorinated anisyl metabolites, drosophihns, and other chlorinated hydroquinone methyl ethers, chlorinated sesquiterpenens, chlorinated anthraquinones and strobulirins [72].
481
Many different reviews have been devoted to the toxicity of mycotoxins [73-76] or their analysis in the natural material [77]. Azaphilones Azaphilones are a large group of pyrano-quinone structures with a high electron acceptor tension determining sensitivity of oxygen in the primary ring yielding y-pyndones, which exhibit chromophore properties. The color of azaphilone pigments depends on their chemical structure. Their name stems from their reaction with ammonia resulting in y-pyridone derivatives. Metabolites in the medium readily react with compounds containing amino groups such as proteins, amino acids, or nucleic acids resulting in water-soluble colored products. Most of them have their absorption maximum within the visible range of the spectrum (400 and 500 nm for yellow and red pigments, respectively). In general, yellow structures are more hydrogenated than orange and red, and amino forms usually have their maximum at higher wavelengths of the absorption spectrum. They were identified in different filamentous fungi indicating that these microorganisms could serve as a useful source of a new group of pigments of natural origin applicable in food industry. Microorganisms tested include the genera Monascus, Penicillium, and Chaetomium, all well-known producers of pigments with the azaphilone basic structure. Some compounds of this type were also identified in Aspergillus ustus, Cochliobolus lunata, Talaromyces sp. and Emericella falconensis. The natural origin of the azaphilone pigments and their easy derivatization, together with increased thermostability in comparison to other natural dyes, open new possibilities of their applications in food industry and cosmetics. Chemical structures of azaphilones isolated from the mycelium and cultivation broth are summarized below. Data concerning their biological activity are contradictory, namely with respect to their antimicrobial, viz. antibacterial and antifungal activities. However, it is generally accepted that they exhibit a significant inhibitory effect on the activity of acyl-CoA:cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT). The azaphilone skeleton is essential for certain biological activities of these metabolites and differences in their activity can be ascribed to differences in their reactivity with amines.
482 Production of azaphilone metabolites (including also non-halogen compounds) by filamentous fungi is summarized in Table 2. Table 2. Production of azaphilone metabolites by filamentous fungi. Metabolite monascin rubropunctatin rubropunctatamine ankaflavin monascorubrin monascorubramine N-glutarylrubropunctatamine N-glutarylmonasconibramine mitorubrin mitorubrinol
mitorubrinic acid monomethyl-(+)-mitorubrin wortmin austdiol chaetoviridin A-D lunatic acid falconensin A-D,H rubrorotiorin luteusin A-E isochromophilone I-VI isochromophilone VH,VIH
Microorganism Monascus purpureus Monascus rubiginosus Monascus rubropunctatus Monascus rubropunctatus
Monascus purpureus Monascus ruber Monascus ruber Penicillium rubrum Talaromyces udagawae Penicillium rubrum Penicillium wortmannii Penicillium vermiculatum Penicillium funiculosum Penicillium vermiculatum Talaromyces tardifaciens Penicillium wortmannii Aspergillus ustus Chaetomium globosum flavoviride Cochhobolus lunata Emericella falconensis Penicillium hirayamae Talaromyces luteus Penicillium vonarxii Penicillium multicolor Penicillium sp.
var.
Isochromophilones I (14, 15), the new gp 120-CD4 binding inhibitors, were isolated from a culture broth of Penicillium multicolor FO-2338 grown on a medium with carbon and nitrogen sources. Their amounts in the medium were ~2 mg/1 and their chemical structures were elucidated
483
by NMR experiments. Both compounds have an azaphilone skeleton substituted by a chlorine atom at C-5 and a side chain, 3,5-dimethyl-l,3heptadien at C-3. Additionally, they contain a y-lactone ring [78]. These metabolites are the first non-peptide compounds, inhibiting the specific binding of the HIVgpl20 protein with the CD4 molecule on the surface of sensitive cells of the organism that is attacked by the virus and thus prevent the HIV virus from entering the cells. Isochromophilones I (14 and 15) inhibited gpl20-CD4 binding with IC50 values of 6.6 and 3.9 uM, respectively, by ELISA method. Isochromophilone (15) exhibited anti-HIV activity at 25 uM, but did not affect cell proliferation in lymphocytes at the same concentration although it inhibited somewhat the cell proliferation at 250 uM. Isochromophilones II (16 and 17) were inactive against bacteria {Bacillus subtilis, Micrococcus luteus, Escherichia coli and Staphylococcus aureus) and fungi (Candida albicans, Aspergillus niger and Piricularia oryzae) at 1.0 mg/ml on paper disc method.
14 (E>A3'-4' 15 (Z>A 3 ' 4 '
New azaphilones named isochromophilones III-VI (18-21) were isolated from the culture broth of Penicillium multicolor FO-3216 as inhibitors of ACAT. Their structures were elucidated by NMR and other spectroscopic analyses. The IC50 values of isochromophilones (17-20) (ACAT) activity in an enzyme assay using rat liver microsomes were calculated to be 110, 50, 50 and 120 uM, respectively [79]. The compound 19 also inhibited the activity of cholesteryl ester transfer protein (CETP) with an IC50 value of 98 uM. The compounds 18, 20 and 21 weakly inhibited the activity of CETP in 300 uM. Antimicrobial and cytotoxic activities of 18, 20 and 21 were tested. They inhibited the growth of Staphylococcus aureus, Bacteroides fragillis, and Pyricularia oryzae at 50 ng/disk. However, they did not
484
inhibit the growth of Bacillus subtilis, Micrococcus luteus, Mycobacterium smegmatis, Escherichia coli, Pseudomonas aeruginosa, Xanthomonas oryzae, Acholeplasma laidlawii, Candida albicans, Saccharomyces sake, Aspergillus niger and Mucor racemosus in the same concentration. The IC50 values of 18, 20 and 21 against the growth of B16 melanoma cells in vitro were 33, 36 and 30 uM, respectively.
R H Ac H
18 19 20
A No No Yes
AcO
21
New isochromophilones VII-VIII (22) and (23) were isolated from the culture broth of Penicillium sp. FO-4164. Both isochromophilones inhibited DGAT activity (assayed in vitro using rat liver microsomes) with IC50 values of 20.0 and 127 uM and ACAT activity with IC 50 values of 24.5 and 47.0 uM, respectively [80]. Antimicrobial activity was tested at a concentration of 10 u.g/paper disk. Both isochromophilones showed antimicrobial activity against Bacillus subtilis, Mycobacterium smegmatis, Micrococcus luteus and Pyricularia oryzae. But no antimicrobial activity was observed against the following microorganisms: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Saccharomyces sake, Mucor racemosus and Aspergillus niger.
485
Isochromophilone IX (24), a novel GABA-containing metabolite, was isolated from a cultured fungus, Penicillium sp. [81]. Cl
AcO'
The azaphilone (25) produced by Penicillium sclerotiorum active in assays for the detection of antagonists of the endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors has been identified. Data for the inhibition of endothelin-1 (ET-1) and endothelin-3 (ET-3) binding in the ET(A) and ET(B) receptor assays, respectively, have been reported for this series. Compound 25 was more selective for the rabbit ET(A) receptor than for the rat ET(B) receptor. The IC50 value for (25) was 9 uM in an assay based on binding of ET-1 to rabbit ET(A) receptors. In an assay based on the binding of ET-3 to the rat ET(B) receptor compound 25 exhibited IC50 of 77 uM. This compound demonstrated an antagonistic behavior in a secondary assay based on blockade of ET-1 stimulated arachidonic acid release from rabbit renal artery smooth muscle cells, when present at concentrations greater than or equal to 30 uM [82].
A novel brominated azaphilone derivative, 5-bromoochrephilone (26) and known derivatives, were isolated from the culture broth of a producing organism Penicillium multicolor, fermented in a medium
486
containing potassium bromide. Nineteen azaphilone-related compounds isolated from the above strain and from other fungi were tested for the inhibition of gpl20-CD4 binding, and the structure-activity relationship was discussed. 5-Bromoochrephilone was found to be the strongest inhibitor (IC50, 2.5 uM). A halogen atom at C-5, a proton at C-8 and a diene structure in C-2 side chain of 6-oxoisochromane ring are necessary for gpl20-CD4 binding [83]. The results in Table 3 indicate that the halogen atom at C-5 and the orientation from C-8 to C-10 in the isochromane ring of azaphilones, in addition to the diene structure in C-2 side chain, are very important for the inhibition of gpl20-CD4 binding. Table 3. Inhibitory activities of azaphilones on gpl20-CD4 binding. Inhibitor isochromophilone I isochromophilone II isochromophilone III isochromophilone IV isochromophilone V tetrahydroisochromophilone I sclerotiorin rubrorotiorin 5-bromoochrephilone rotiorin luteusin A chaetoviridin A chaetoviridin B
IC50 (uM) 6.6 3.9 48 96 14.6 >260 >250 >240 2.5 >240 9.4 >230 140
The effect of thirteen different fungal azaphilones on cholesteryl ester transfer protein activity was tested [84].
487
A new secondary metabolite, 8-O-methylsclerotiorinamine (27), was isolated from a strain of Penicillium multicolor, and its structure was established using NMR spectroscopy and chemical evidence. The metabolite significantly inhibited the binding between the Grb2-SH2 domain and the phosphopeptide derived from the She protein and also blocked the protein-protein interactions of Grb2-Shc in cell-based experiments, with IC50 values of 5.3 and 50 uM, respectively [85].
27 OMe
Four new azaphilones, named helicusins A, B, C and D (28-31), were isolated from Talaromyces helicus. These four new azaphilones showed weak monoamine oxidase-inhibitory effects [86].
28 R
29 R -
Three new azaphilones (32-34), named luteusins C, D and E, were isolated after a 21-h surface cultivation of the ascomycete Talaromyces luteus (anamorph of Penicillium vonarxii) on rice, together with luteusins A and B, previously known as inhibitors of monoaminooxidase [87]. The new compounds had no MAO-inhibitory activity [88]. The potential to inhibit MAO is lost after conversion of hydroxyl group in position C-8 to oxo-group and hydrogenation of the side chain double bonds.
488
Falconensins, i.e. azaphilones esterified by chloroorselinic acid, A (35), B (36) and C (37), were isolated from mycelial extracts of the Venezuelan soil fungus ascomycete Emericellafalconensis [89]. Itabashi et al. [90] later isolated falconensone H (43) together with falconensins A, B, C and D (38) from the same culture. Their chemical structures are shown below.
35 R, - H, R2 = R3 = Cl, R4 = Me 37 R, = Ac, R2 = R3 = Cl, R4 = Me 39 R, = R3 = H, R2 = Cl, R4 = Me 4OR,=R3 = R4 = H , R 2 - C I 42 R, = R 4 - H , R 2 - R 3 = C 1 F>3
Further falconensins, i.e. falconensins E (39), K (40), L (41), M (42) and N (44), were isolated also from Emericella falconensis. Three new azaphilone derivatives designated falconensins E, F and G were isolated from the mycelium of Emericella falconensis, along with falconensins AD and H. The structures of new falconensins were established by spectroscopic investigation and chemical correlations. The absolute stereochemistry of falconensins A-G was also established [91].
489
38 R, = Ac, R2 - Rj = Cl, R, = Me 41R|=R 3 =R4=H,R 2 -C1 44R 1 -R 4 -H,R 2 =R 4 -C1
Six new hydrogenated azaphilones designated falconensins I-N were isolated as minor components from mycelia of Emericella falconensis and/or E. fruticulosa along with nine azaphilone derivatives, falconensins A-H. The structures of falconensins I-N were determined by spectroscopic investigation and chemical correlation [92]. Chaetoviridines A (45), B (46), C (47) and D (48), also belonging to the azaphilone group, can be isolated from the fungus Chaetomium globosum var. flavoviride grown on wheat. Spectral data show that chlorine-containing azaphilones are involved with a conjugated y-lactone connected angularly with the azaphilone unit. Chemical structure of chaetoviridin A was determined by NMR, and so the absolute configuration has been established [93]. The red pigment chaetoviridin A contains an unsaturated y-lactone ring, whereas the yellow chaetoviridins B, C and D contain a saturated y-lactone ring as shown. Chaetoviridin A was shown to inhibit monoaminooxidase and growth of P. oryzae at 2.5 ug/ml.
490
O
46R=H 47 R=OH
OH
48
OH
The fungal metabolite, sclerotiorin (49) was first isolated independently by two groups from Penicillium sclerotiorum [94,95] and later from P. multicolor [95]. The 7-epimer (50) is also a known metabolite of P. hirayamae [96]. Sclerotiorin was isolated for the first time from lichen mycobiont Pyrenula japonica [97]. The related rubrorotiorin (51) was also found in P. hirayamae [98].
49 50 7-epimcr
Chlorofusin (52) was one of the major components produced by fermentation of Fusarium sp. 22026. The DELFIA-modified ELISA was used to guide to the purification of inhibitors of the p53/MDM2 interaction from these fermentation extracts. Chlorofusin was the most abundant inhibitory compound. Titration of purified chlorofusin in the
491 DELFIA-modified ELISA gave an IC50 of 4.6 uM. In simultaneous crossscreen testing, chlorofusin was inactive at a concentration of 4 uM in the TNFa:TNFa receptor protein-protein interaction, which was configured in the same format as the primary assay. The compound showed no cytotoxic effects against Hep G2 cells at a concentration of 4 uM. At a test concentration of 7.3 uM, chlorofusin did not exhibit any antimicrobial activity against the following test strains: Escherichia coli, Staphylococcus aureus, Serratia marcescens, Bacillus subtilis, Klebsiella pneumoniae, Proteus vulgaris, Candida albicans, Cryptococcus neoformans and Aspergillus niger [99].
Cl
OH
J ^
CONH 2
52 H 2 NOC
Indoles Indole-alkaloid isoprenoid was isolated from extracts of Penicillium crustosum grown on rice. This compound, designated thomitrem (53), contains a 18(19)-double bond and lacks the characteristic penitrem 17(18)-ether linkage [100]. Penitrems are a group of tremorgenic mycotoxins produced by a variety of Penicillium and Aspergillus species, amongst which Penicillium crustosum is generally regarded as the most important producer of this group of mycotoxins. They have been reported to intoxicate animals. Generally, penitrem A is considered to be the most significant of the series of P. crustosum mycotoxins, which includes
492
penitrems B, C, D, E and F, and other related metabolites such as PC-M4, PC-M5, PC-M51 and PC-M6.
,OH
Cl 53
A fungus, Penicillium crustosum, is known to produce the tremorgenic mycotoxins penitrems A, C, F (54-56), when grown in surface culture [101-103]. All of these tremorgens have a common core structure composed of an indole moiety, biosynthetically derived from tryptophan, and a diterpenoid unit from four mevalonate-derived isoprenes. Penitrems are also produced by other Penicillium species and by Aspergillus sulphureus. This group of metabolites is capable of eliciting tremors in vertebrates, and some specific members have also shown insecticidal activity. For example, compound 54 shows convulsive and insecticidal activities against Bombyx mori, Spodoptera frugiperda, and Heliothis zea, and its use as an insecticide was patented in 1990. Natural penitrem analog 6-bromopenitrem E [104] also exhibits insecticidal activity.
,OH
54 R, = OH, 23a,24a-epoxide 55 R, - H 56 R, = H, 23a,24a-epoxide
Twenty-five Penicillium species were isolated and mycotoxins produced by several of these species, including penitrems A-F, were detected. The levels of penitrem A in these samples were in the range 357500 ug/kg [105]. HPLC and diode array detection were used to confirm
493
the chemical structure of the mycotoxins, e.g. penitrem A and ochratoxin A in extracts from three mycotoxigenic fungi (Penicillium crustosum, Penicillium glabrumlspinulosum, and Penicillium discolor) that dominated on Castanea sativa nuts sold in Canadian grocery stores [106]. Another paper includes data for detection and dereplication of >400 fungal metabolites using MS/ESI+ methods [107]. Sporidesmins, the series of sulfur-containing physostigmine-like metabolites, were isolated from Sporidesmium bakeri A (57), B (58) and C (59), a fungus that causes facial eczema and liver damage in farm animals, e.g. New Zealand sheeps [108-110]. Later studies identified sporidesmins D (60) [111], E (61) [112], F (62) [111], G (63) [113,114], H (64) [115]) and J 65) [115] from P. chartarum. Protonated [M+H]+ and deprotonated [M-H]" ions were observed in positive and negative ion ESI modes, respectively [116]. In a further paper, complexation of sporidesmin A, with metals, was used for its analysis [117].
MeO
O
OMe 57 R = OH, n= 2 58 R = H, n= 2 61 R = OH, n= 3 63R = OH, n=4
MeO OMe
59
494
,,,iv\\SMe
MeO OMe 60
MeO
MeO OMe
OMe
Me
64
62
MeO 65
Saccharothrix aerocolonigenes produces an indolocarbazole antitumor agent rebeccamycm (66) under submerged fermentation conditions. Adding D,L-6-fluorotryptophan to culture of S. aerocolonigenes induces the formation of two novel indolocarbazoles, fluoromdolocarbazoles A (67) and B (68). Feeding A^-5-fluorotryptophan to culture of S. aerocolonigenes induces the production of a novel indolocarbazole, fluoroindolocarbazole C (69). These fluoroindolocarbazoles have been isolated from culture broth and purified to homogeneity by vacuum liquid chromatography and column chromatography. All three fluoroindolocarbazoles are more potent than rebeccamycin against P388 leukemia via ip route in murine model [118].
495
66 67 68 69
Ri
R2
R3 R4
Cl H H H
H F F H
H
H H F
Me Me
H H
Antraquinones Like the fungal and lichen xanthones, anthraquinones, which are also produced both by lichens and fungi, are derived from extended polyketides by cyclization. Several chlorinated compounds were described. The structures of novel topoisomerase I inhibitors, topopyrones A and B (70 and 71), were elucidated by spectral analysis of the chemical derivatives. It was suggested that topopyrone B is converted from topopyrone A [119]. Topopyrones A and B selectively inhibited recombinant yeast growth dependent on the expression of human topoisomerase I with IC50 values of 1.22 and 0.15 ng/ml, respectively. The activity and selectivity of 71 were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds, however, they did not inhibit human DNA topoisomerase II. Both topopyrones were cytotoxic to all tumor cell lines when tested in vitro. Topopyrone B has potent inhibitory activity against herpesvirus, especially varicella tester virus (VZV). It inhibited VZV growth with EC50 value of 0.038 ug/ml, which is 24 fold stronger than that of acyclovir (0.9 ug/ml). Both topopyrones were inhibitory against Gram-positive bacteria.
496
OH
OH
O
O
"OH
Aspergillus ustus produces several novel pentacyclic metabolites, the austocystins, two of which, i.e. A (72) and C (73), contain chlorine [120]. OMe
O
72 Rj = Me, R 2 = H
The fungus Cercospora beticola, which is a highly destructive disease of sugar beets worldwide, has been shown by several groups to produce a series of highly intricate metabolites, beticolins 1 (74) (= cebetin A), 2 (75), 3 (76), 4 (77), 6 (78), 8 (79) and cebetin B (not shown), the latter of which is bis-M% complex of cebetin A [121-126]. Following some initial confusion regarding the complexity of structures and the fact, that beticolin 2 and cebetin A are in equilibrium, the situation now appears to be in order [125,126].
497
ci HO,
HO,
OH
O
OH 76R = Me;a-COOMe 77 R - CH2OH; a-COOMe 78 R = Me; P-COOMe 79 R - CH2OH; 0-COOMe
The cheese mold Penicillium nalgiovensis produces nalgiolaxin (80), which appears to be the first natural chlorinated anthraquinone isolated [127,128], although fragilin (81) was the first fully characterized [129]. A summary of the known chlorinated anthraquinones (80-88) is given below.
R,0
498 No. Ri
R2
80 81 82 83 84 85 86 87 88
Me Me H H Me Me
Me
OH OH OH OH OH OH OH OH
R3
Rt
R5
MeCHOH H H H H H
H H H
Cl - H OH - H OH H
OH OH OH OH
OMe OMe OMe OH OH
Several chlorinated metabolites closely related to anthraquinones are also known. For example, the aspen tree fungus Phialophora alba which protects the tree against attack by the decaycausing fungus Phellinus tremulae produces anthrone, in addition to other derivatives [130]. Anthrone was found in cultures of Aspergillus fumigatus [131], and the corresponding bromo compound was formed in the presence of bromide [131]. The novel bis-anthrones, flavoobscurin A (89), B (90) and B2 (the latter two are rotational isomers) were produced by Anaptychia obscurata [132]. OH Cl
Cl
Cl
Cl
OH
Macrocycles Several relatively large cyclic peptides have been found to contain halogen. Islanditoxin (91) which was isolated in 1955 from a culture of
499
Penicillium islandicum is a chlorine-containing peptide whose structure was determined later. This organism also produced cyclochlorotine (92), which is an infectant of yellowed rice [133]. The fungus Metarhizium anisopliae produces the chlorohydrin cyclic peptide (93) [134]. Cyclochlorotine, a hepatotoxic mycotoxin, was also isolated from Penicillium islandicum.
Monorden (94) and the novel resorcylic acid lactones pochonins A (95), B (96), C (97), D (98) and E (99) were isolated from cultures of the clavicipitaceous hyphomycete Pochonia chlamydosporia var. catenulata strain P 0297. Fermentation of P 0297 in bromide-containing culture media led to a shift in secondary metabolite production and yielded monocillins (compounds without bromine in the molecule) as major metabolites besides monorden (94) as well as the novel compounds pochonin F and a monocillin II glycoside as minor metabolites, Fig. (1). Most of these compounds showed moderate activities in a cellular replication assay against herpes simplex virus and against the parasitic protozoan Eimeria tenella. In contrast to the structurally related zearalenone derivatives, none of the metabolites of strain P 0297 was
500
found to be active in a fluorescence polarization assay for the determination of modulatory activities on the human estrogenic receptor ERbeta[135].
95R = H 96R = OH
OH
98R-H 99R-OH
—B— pHvittK —*- MonDtden (mgfll
1.0-
-O-
Mycdialdiywd(h<
5
r 2t
0 IOf)
l.VJ
200
FcmBitiIiontime(h]
Fig. (1). Time course of production of monorden (94) in Q6/2 medium (150 ml shake flasks) by strain P 0297.
501
Phomopsin A (100), the main mycotoxin isolated from cultures of Phomopsis leptostromiformis and the cause of lupinosis disease, is the hexapeptide containing unusual amino acid [136]. The ^-configuration of the indicated amino acids was established by comparison of the Ntrifluoroacetyl n-butylester derivatives of the acid hydrolysis products of phomopsin A with samples prepared from authentic amino acids, using capillary gas chromatography on a chiral stationary phase. The E configuration of the two 2,3-didehydro amino acids is based on the products obtained by catalytic hydrogenation and sodium borohydride reduction of phomopsin A followed by acid hydrolysis (for 2,3didehydroisoleucine) or by analysis of the coupled 13C NMR spectrum of phomopsin A (for 2,3-didehydroaspartic acid). Evidence was presented, which shows that the glycine formed during the acid hydrolysis of phomopsin A is derived from the 3,4-didehydrovaline moiety. The sequence of the amino acids was established by heteronuclear C H selective population inversion experiments and by fast atom bombardment mass spectrometry of phomopsin A and its derivatives. An X-ray crystallographic study of phomopsin A confirmed the amino acid sequence and showed that the hexapeptide is modified by an ether bridge in place of the 5-hydroxy group of the iV-methyl-3-(3-chloro-4,5dihydroxyphenyl)serine and the hydroxy group of the 3hydroxyisoleucine units. In addition, the X-ray study specified the absolute configuration of phomopsin A as 22E, 25E, 3R, 45, 75, 105, 115, 195. The consumption of lupins or post-harvest lupin roughage infected with the fungus Phomopsis leptostromiformis has been identified as the cause of lupinosis, mycotoxicosis of sheep, cattle, and horses. The condition, characterized by severe liver damage, is of considerable economic importance in Australia, and field cases have also been reported in South and New Zealand [137].
502
The structures of decatromicins A and B (101 and 102) that strongly inhibit the growth of methicillin-resistant staphylococci MRSA were elucidated. Decatromicins A and B inhibited the growth of Gram-positive bacteria including multi-drug resistant strains such as Staphylococcus aureus and methicilin resistant S. aureus. The antimicrobial activity of decatromicin B was stronger than that of decatromicin A. This observation suggests that the antibacterial activity against Gram-positive bacteria of decatromicins might increase with increase in the number of chlorine atoms attached to the pyrrole ring. However, these antibiotics did not inhibit growth of Gram-negative bacteria and yeast at 100 ug/ml. The acute toxicities (LD50) of decatromicins A and B in mice {ip) were estimated to be more than 100 mg/kg [138].
COOH
Depsides and Depsidones Similarly, to lichens, depsidones were also detected in fungi. The fungus Aspergillus unguis contains haiderin (103), rubinin (104), shirin (105) and nasrin (106) [139]. The 2-chlorounguinol and emeguisins A-C (107-109) were found in Emericella unguis [140,141]. A marine isolate of the fungus Emericella unguis gave a new antibacterial depside, guisinol. The structure determination was based on mass spectrometry and NMR spectroscopical studies. The fungus, Chaetomium mollicellum produces several new depsidones three of which are chlorinated, mollicellins D (110), E (111) and F (112). The lactones canesolide (113) and buellolide (114), which may arise from depsidones by catabolism, were also found in Buellia canescens. Structurally, guisinol (115) resembles the depsidones emeguisin A-C previously isolated from another strain of the same species. Five other isolates of E. unguis also produced the same
503
qualitative profile of secondary metabolites including guisinol and dechloronidulin. Other species of Emericella did not produce any of these compounds that are thus of chemotaxonomic significance [142].
103 Me
H OH Cl
H
R7 OH
Me
104 Me
H OH Cl
H
OMe
Me
105 Me 106 Me
Cl OH Cl Cl OH Cl
Cl Cl
OH OH
Me Bu
107
H OH Me
Cl
OH
Me
108
H
OMeMe
Cl
OH
Me
109
Cl OH Me
Cl
OMe
Me
110 Me
Cl OH CH2O H H
OH
111 Me
Cl OH CHO
R2 R3
R»
i-Bu
Me
Me
OMe
OH
504
ci
.OH OMe
Cl
MeO
115
The cherry rot fungus, Monilinia fructicola, which was earlier shown to produce chloromonilinic acids A and B, also contains 4-chloropinselin (116) [143] and its biosynthetic product chloromonilicin (117) [144-146]. In the paper [143], the structural elucidation of a new antifungal metabolite, chloromonilicin (117), isolated as a growth self-inhibitor of phytopathogenic fungus Monilinia fructicola, was reported. It contained a novel, seven-membered lactone ring presumably formed by oxidative cleavage of a benzene nucleus in a xanthone system. Subsequently was isolated a new chlorine containing metabolite, 4-chloropinselin (116), a probable precursor of (117). In addition, bromomonilicin (118) and bromopinselin (119) were prepared for activity tests and biosynthetic studies. Biosynthetic incorporation of (119) into (118) was accomplished using M. fructicola culture. COOMe
OH
116R1 = H,R2 = n9R,=H,R2 =
117R=C1 118R=Br
505
Table 4. Antimicrobial spectrum of bromomonilicin (118), by agar dilution method on glucose nutrient agar. MIC (ug/ml)
Test organism Staphylococcus aureus Escherichia coli Shigella flexneri Pseudomonas aeruginosa Candida albicans Trichophyton asteroides T. interdigitale T. rubrum
>50 >50 >50 >50 25 6.2 6.2 6.2
A new chlorinated depsidone (maldoxone 120) and a new spirocyclohexadienone (maldoxin 121) have been isolated from the culture medium of an as yet unidentified Xylaria species. Their role in the grisan-depsidone biosynthetic pathway was discussed [147]. OH COOMe
Cl
OMe OMe 120
MeOOC
Terpenes Over 30 sesquiterpene aryl esters have been isolated from Armillaria spp.; of these, eight are esterified with chlorinated orsellinate. The honey mushroom, A. mellea, produces 70 mg of the chlorinated compound armillaridin (122) per kg dry mycelium [148], while liquid cultures of A. ostoyae contain the chlorinated compounds, melledonal C (123) (50 mg/1) and melleolide D (35 mg/1) (124) [149]. Clitocybe elegans is the only other genus for which chlorinated sesquiterpene aryl esters have been reported [150], including melledonal D (125), which has not yet been detected in Armillaria spp. The pathogenic basidiomycete, Armillaria causes root disease in both coniferous and deciduous trees. Armillaria
506
spp. were the most frequently isolated fungi associated with root in living spruce and balsam fir trees in Ontario, Canada [151]. It is not known yet what makes some of the Armillaria spp. such virulent parasites, but secondary metabolites are thought to be the major cause. In a test for fungi or phytotoxicity of fourteen Armillaria metabolites, four of them chlorinated, the toxicity decreased with increasing hydrophobicity, e.g. methylating the hydroxy group of the orsellinate and/or adding a chlorine atom [152]. Sonnenbichler et al. [149] found that increased amounts of melledonal C (123) and other nonchlorinated sesquiterpene aryl esters were produced in cultures of A. ostoye growing in the presence of an antagonistic fungus or host plant cells [149,152]. The biosynthesis of the chlorinated metabolite was enhanced up to 5-fold upon antagonization [149], indicating that the physiological purpose of the sesquiterpene aryl esters is their antibiotic activity.
R4
122 123 124 125
Ri
R2
CHO CHO CHO CH2OH
H OH OH OH
R3 H H OH H
R4
R5
H OH OH OH
Me Me Me Me
Two novel sesterterpenes, neomangicols A (126) and B (127), were isolated from the mycelial extract of a marine fungus belonging to the genus Fusarium. The carbon skeleton of the neomangicols is undescribed and constitutes a new class of C25 rearranged sesterterpenes. Neomangicol A was most active against MCF-7 (human breast carcinoma) and CACO-2 (human colon carcinoma) cell lines, displaying IC50 values of 4.9 and 5.7 uM, respectively. Neomangicol B was less active, having a mean IC50 value of 27 uM across the entire cell panel (versus 10 uM for neomangicol A), while neomangicol B displayed antibacterial activity similar to that of the known antibiotic, gentamycin, against the Gram-positive bacterium Bacillus subtilis [153].
507
HO
HO,
126 R = Cl 127R = Br
The fungal metabolite ascochlorin (128), which is produced by Ascochyta viciae [154-156], was simultaneously isolated as "LL-Z1272y from Fusarium sp. [157] and as "ilicicolin D" from Cylindrocladium ilicicola, a fungus of dead beech leaves [158,159]. Other metabolites from these fungi include: LL-Z1272a (129), LL-Z1272X, (130) [157,158], and cylindrochlorin (131) [160]. The fungus Nectria coccinea also produces several of these metabolites, including the new chloronectrin (132) [161]. The hydroxy analog (133) was produced by Ascochyta viciae [162]. The tobacco pathogen Colletotrichum nicotianae produces colletochlorin B (134), C (135), and D (136) [163-165]. The hypolipidemic active metabolites, ascofuranone (137) and ascofuranol (138), have been isolated from Ascochyta viciae [166]. The fungus Acremonium luzulae also produces ascochlorin (138) [167], while Strobilurus tenacellus and Mycena spp. contain the new strobilurin B (139) [168].
508
CHO
128R = H 130 R = OAc OH
129 CHO
CHO
CHO 132 R = Ac 133 R = H
509
OH
136 CHO OH
CHO
138 X = H, OH COOMe
MeO OMe
510 The strobilurins were first isolated from Strobilurus tenacellus [169]. The chlorinated strobilurin B (140) has been isolated from three genera and seven species, e.g. Mycena alkalina, M. avenacea, M. crocata, M. vitilis, Xerula longipes, X. melanotricha, and S. tenacellus. Chlorinated oudemansin B (141) is produced by X. melanotricha [170]. The chlorinated and nonchlorinated strobilurins and the closely related oudemansins are new respiration inhibitors, binding to cytochrome b. Thirteen strobilurins and three oudemansins have been isolated so far. Their high antifungal activity against phytopathogenic fungi and insects and their low toxicity toward mammals and bacteria make them attractive lead compounds for the synthesis of agricultural fungicides [171]. Most fungi that produce strobilurins and oudemansins grow on wood. Oudenmansiella mucida also produces a nonchlorinated strobilurin on sterilized beech wood. Therefore, it appears that the strobilurins play a role in securing nutrient resources for the producers from competing fungi [171]. The aromatic ring and the benzylic carbon atom in the strobilurins and oudemansins are derived from the shikimate pathway, whereas the side chain is built up from acetate units [171]. MeCX
V
Y "V^ ^^ "V' JDMe
MeC
141
Aromatic Compounds Two strains of the basidiomycete, Bjerkandera adusta produces in static liquid culture, phenyl, veratryl, anisyl, and chloroanisyl metabolites as well as a series of compounds not previously known to be produced by Bjerkandera species. A new metabolite, for which the name bjerkanderol B (142) was given, has been proposed. Experiments with static liquid cultures supplied with 13C6- and 13C9-Z-phenylalanine showed that all
511
identified aromatic compounds (with the exception of phenol) could be derived from L-phenylalanine. For the aryl propane diols, the 13C label appeared only in the phenyl ring and the benzylic carbon, suggesting a stereoselective resynthesis from a C7 and a C2-unit, likely aromatic aldehyde and decarboxylated pyruvate, respectively. For both strains, cultures supplied with Na37Cl showed incorporation of 37C1 in all identified chlorometabolites. The compounds have been reported to exhibit important physiological functions in this white rot fungus. Possible mechanisms for their formation through the newly discovered compounds have been discussed [172]. OH
OH
MeO'' 142
A new D-glucose-6-phosphate phosphohydrolase (G6Pase) inhibitor (143), CJ-21,164 was isolated from the fermentation broth of the fungus Chloridium sp. The structure was elucidated to be a novel tetramer of the salicylic acid derivatives by spectroscopic analyses. The compound inhibited G6Pase in rat liver microsomes with an IC50 of 1.6 uM. Glucose output from hepatocytes isolated from rat liver was inhibited when it was present in the incubation medium, consistent with the role of this compound as a G6Pase inhibitor. It dose-dependently inhibited G6Pase, with an IC50 value of 1.6 uM. At a concentration of 133 uM, this compound inhibited the rate of glucose output stimulated with 25 nM glucagon by 81 %. Hepatocytes incubated with this compound did not show significant cytotoxicity at these concentrations, suggesting that the reduction in glucose output might not be a consequence of cytotoxicity [173].
512
!OOH
OH
OH
OMe
143
In the search for new, naturally occurring, anti-angiogenic compounds, it was found that a culture broth of an unidentified fungal strain B90911 exerted inhibitory activity on capillary-like tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Active compounds were isolated by bioassay-guided separation and their structures were identified to be two new asterric acid derivatives, i.e. 3-chloroasterric acid (144) and 3,5-dichloroasterric acid (145), by spectroscopic analyses. These compounds significantly inhibited (10 ug/ml) the VEGF-induced tube formation of HUVEC, suggesting that asterric derivatives could be useful for further study as anti-angiogenic agents [174]. COOMe
OMeMeOOC 144 R, = H, R2 = Cl 145 R! = R2 = Cl
Simple naphthalene metabolites (146 and 147) were produced by Verticillium lamellicola [175] and (148) found in the fungus Scolecobasidiella avellanea.
513 COOR,
O
COOR,
Cl
146 R! = H, R2 = Me 147 R, = Me, R2 = H
148
The structures of two novel fungal antibiotics, isolated from a Pterula species that interfere with the NADH: ubiquinone oxidoreductase and inhibit the respiration of eucaryotes, were determined by spectroscopic techniques. The compounds, pterulinic acid (149) and pterulone (150), contain a 1-benzoxepin ring system and are chlorinated [176]. In the serial dilution assay, both compounds showed antibacterial activities at concentrations up to 100 ug/ml {Acinetobacter calcoaceticus, Escherichia coli, Salmonella typhimurium, Bacillus brevis, Bacillus subtilis, Micrococcus luteus, etc.). They exhibited weak cytotoxicity activities toward L1210 and BHK cells but showed moderate activity toward HL60 and HeLa cells. In contrast, 150 was not cytotoxic against L1210, HL60, BHK, and HeLa cells at concentrations up to 100 jig/ml and has phytotoxic activities. The germination of Lepidium sativum and Setaria italica was inhibited at concentrations 10-50 ug/ml. Both compounds neither showed nematocidal activity against Meloidogyne incognita and Caenorhabditis elegans, nor hemolytical effects at concentration up to 100 ug/ml. HOOC
149 150
Antibiotic aspirochlorine was originally isolated from Aspergillus tamarii [177], later from A. flavus [178] and A. oryzae [179], and shown
514
to have the novel structure (151) by X-ray crystallography and total synthesis [180]. Armillaridin (152) is a novel phenolic sesquiterpene containing a cyclobutane ring that is produced by Armillaria mellea [181]. Later work with this organism revealed the related metabolites: melleolide D (153) [182], melledonals B (154), C (155) [183] and armillaricin(156)[184].
151
OMe
OMe
In one of the few studies of marine fungi, the novel chloriolins A-C (e.g. the structure of chloriolin B, 157) have been reported from a cultured, unidentified fungus associated with the sponge Jaspis johnstoni [185].
515
1 157
Coumarins and Isocoumarins One of the most ubiquitous of all fungal metabolites is the isocoumarin derivative ochratoxin A (158), which was first isolated from Aspergillus ochraceus [186,187] and later from Penicillium viridicatum [188], P. cyclopium, P. commune, P. variabile, P. purpurescens [189], Aspergillus melleus and A. sulphureus [190]. The related ochratoxin C (159) is produced by Aspergillus ochraceus [191], and 4-hydroxyochratoxin A (160) was isolated from cultures of Penicillium viridicatum [192]. When ochratoxin A was administered in the diet, hepatocellular tumors, renalcell tumors, hepatomas and hyperplastic hepatic nodules were observed in male mice. Incidence of and mortality from urothelial urinary tract tumors have been correlated with the geographical distribution of Balkan endemic nephropathyin. Ochratoxin A has been detected in moldy cereals including wheat, maize, rye, barley and oats, peanuts, coffee beans, bread, flour, rice, peas and beans from 0.03 to 27.5 ppm. Residues of ochratoxin A have been detected in samples of meat from animals slaughtered immediately after consuming contaminated feed and were detected at levels of 10 to 920 ug/kg in sausage, ham and bacon samples. The quantification of ochratoxin A, at levels within the range 0.25-10 ng/ml from wine by HPLC-fluorescence detection, was described [193]. RP-HPLC - fluorescence method for the detection of ochratoxin A in wine with a detection limit of 0.05 ng/ml was also published [194]. A stable isotope dilution assay by LC-MS/MS was developed for quantification of the ochratoxin A by using [Ds]-ochratoxin A as internal standard with a low detection and quantification limits of 0.5 and 1.4 ug/kg, respectively [195]. The LC-MS/MS method (ESI and APCI) was also applied to the analysis of contaminated coffee samples by ochratoxins A and B with absolute minimum detection limit around 10-20 pg per injection. Fragment ions from the [M+H]+ and [M+Na]+ ions of
516
both ochratoxins were monitored in the multiple reaction monitoring mode [196].
159 R, -Et, R 2 - H 160 R, - H, R2 - OH
The metabolite profiles of three different isolates of Penicillium nalgiovense from cheese were analyzed. The novel isocoumarin metabolite, dichlorodiaportin (161) was isolated from the cultures of P. nalgiovense along with further metabolites. The reason isocoumarins occur naturally and the possible toxicity or health benefits of these compounds in cheese and other relevant food products should be investigated [197]. MeO OH
The structures of four new biologically active nematocidal halogenated dihydroisocoumarins (162-165) isolated from submerged cultures of the wood-inhabiting ascomycete Lachnum papyraceum have been elucidated by spectroscopic methods. The compounds are structurally related to lachnumon and mycorrhizin A, which are also produced by the fungus [198]. The brominated metabolites isolated from L. papyraceum in this investigation are further examples of how bromine can be introduced into normally chlorinated fungal metabolites when bromide salts are added to the culture medium. Besides obtaining derivatives that are useful for QSAR studies and for assessing the importance of the halogen atom for the biological activity of the metabolites, the shifts in the secondary metabolism of the fungus induced by the addition of bromide to the
517
culture medium may also be helpful during studies of the biosynthesis of mycorrhizins. R,O
O
Ri
162 163 164 165
R2 H OH OH Me H
H H H
R3
Br Cl Cl Cl
R4
(R)-Me (S)-Me (R)-Me (R)-Me
Chlorflavonin (166) was discovered in the culture broth of Aspergillus candidus [199-201]. A biosynthetic study of this fungal metabolite indicates that it is a true metabolite and is synthesized de novo by this microbe [202]. It was also detected in Aspergillus candidus and A. campestris [203]. The fungus Monilinia fructicola produces chloromonilinic acids A (167) and B (168) derived from chloromonilicin [204], to be discussed later. OH
166
The strain of Actinamadura spiralis isolated from a soil sample produced new antibiotics, pyralomicins (169-175). Pyralomicins inhibited the growth of Micrococcus luteus and Escherichia coli at the concentration of 0.2-25 ug/ml by agar dilution method. Pyralomicin la (169) and pyralomicin 2a (173) did not show any acute toxicity in mice at 100mg/kgip[205].
518 OH
OR,
169 170 171 172
Ri
R2
R3
R4
H H H Cl
Cl Me Cl Cl
Me Cl Me Me
Me Me H H
OH
OR4 OH
173 174 175
Ri
R2
R3
R4
H H H
Cl Me Cl
Me Cl Me
Me Me H
A new antifungal agent, CJ-19,784 (176) was isolated from the fermentation broth of a fungus, Acanthostigmella sp. This compound inhibits the growth of pathogenic fungi, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus with IC50 values of 0.11, 20 and 0.54 ug/ml, respectively. Compound 176 and 3'-Cl analog did not exhibit significant activity against HeLa cells. It is well known that the production of halogenated microbial metabolites depends on the presence of halogen atoms in the fermentation medium. For example, chlortetracyclin and chloromonilicin are produced well under the fermentation in chlorine-containing media, and the chlorine atom is easily exchanged for a bromine atom by replacing the media with bromine-containing media. It is very interesting that
519
compound 176 but not 3'-Cl analog was produced as a major product under the fermentation in chlorine-containing medium (0.2 % sodium chloride). This suggests that the fungus Acanthostigmella sp. would have unknown biosynthetic mechanisms enabling prior use of the bromine atom [206]. OH
.OMe
OMe
A novel inhibitor of STAT6 activation, named as TMC-264 (177), was discovered from the fermentation broth of Phoma sp. TC 1674. TMC-264 suppressed expression of IL-4 driven luciferase and germline C epsilon mRNA with IC50 values of 0.3 uM and 0.4 uM, respectively. It exhibited a potent inhibitory activity against tyrosine phosphorylation of STAT6 with an IC50 value of 1.6 uM, whereas weakly inhibited tyrosine phosphorylation of STAT5 with an IC50 value of 16 uM, but did not inhibit the phosphorylation of STAT1 up to 40 uM. TMC-264 blocked formation of the complexes between phosphorylated STAT6 and STAT6 oligonucleotides in a dose-dependent manner, while it did not affect the formation of phosphorylated STAT1/STAT1 oligonucleotides complexes. These results suggested that TMC-264 selectively inhibited IL-4 signaling by interfering both with phosphorylation of STAT6 and binding of the phosphorylated STAT6 to the recognition sequence [207]. The structure was elucidated on the basis of NMR analyses of normal abundance and biosynthetically 13C enriched TMC-264 (177) [208].
520
OMe
MeO
177 O
In the process of screening microbial extracts from endophytic Cytospora sp., three novel compounds cytosporins A, B and C (178) were found as specific inhibitors of angiotensin II binding to receptors of AT2. The IC50 of component A in the biochemical assay was found to be approximately 25-30 uM in ATi and 1.5-3 uM in AT2. These results were obtained by blocking one of the two angiotensin II binding sites in rat adrenal glands. Further analysis suggested reversible inhibition by this compound, and thus it appears to be a competitive inhibitor [209].
178
Miscellaneous Compounds In addition to the numerous fungal and lichen metabolites discussed in the preceding sections, there are several others that do not easily fit into the well-defined structural categories. Several other chlorinated aliphatic metabolites are produced; a volatile organohalogen, l-chloro-5-heptadecyne (179) was detected in an edible wild milk cap Lactarius spp. [210]. Lepiochlorin (180), an antibacterial lactol, was isolated from liquid cultures of a Lepiota sp., a fungus cultivated by gardening ants [211]. Another aliphatic halogenated compound with an interesting tnchloromethyl group was isolated from the mycelium of the fungus Resinicium pinicola in 120 mg/kg yield [212].
521
The compound, pinicoloform (181), showed antibiotic and cytotoxic activities. -(CH 3 ) 10 — 179 OH
181
Kaitocephalin (182) was isolated from Eupenicillium shearii and protected chick primary telencephalic and rat hippocampal neurons from kainate toxicity at 500 uM with EC50 values 0.68 uM and 2.4 uM, respectively, without showing any cytotoxic effect. Although a wellknown AMPA/KA antagonist CNQX with a quinoxalinedione skeleton effectively protected chick primary telencephalic neurons from kainate toxicity with EC50 value 0.53 uM, it exhibited cytotoxicity against chick primary telencephallic and rat hippocampal neurons at the concentrations of 20 uM and 2 uM, respectively. Kaitocephalin also protected chick primary telencephalic and rat hippocampal neurons from AMPA/cyclothiazide (500 uM/50 uM) toxicity with EC50 values 0.6 and 0.4 uM, respectively. Kaitocephalin is the first AMPA/KA antagonist from nature, consisting of a quite different skeleton from other known AMPA/KA antagonists [213]. H
COOH
COOH
NH,
The salt-water culture of Aspergillus ochraceus separated from the Indo-Pacific sponge Jaspis coriacea has yielded two new chlorine containing polyketides, chlorocarolide A (183) and B (184). These compounds have an overall structural analogy to penicilic acid whose biosynthesis has been intensely studied. The structures and stereochemical features of the chlorocarolides were reported [214].
522
O'
183 7R* 8R* 184 7S* 8S*
Three new chlorine-containing compounds (185-187) together with penicillic acid were obtained from a marine-derived fungus Aspergillus ostianus isolated from a marine sponge at Pohnpei. Compound 185 inhibited the growth of R. atlantica at 5 ug/disc (inhibition zone 12.7 mm), while 186 and 187 were active at 25 ug/disc (10.1 and 10.5 mm, respectively). The growth of E. coli and S. aureus was also inhibited by these compounds. Compounds (185-187) did not inhibit the growth of S. cerevisiae and M. hiemalis even at 100 ug/disc. Compound 185 was the most potent among the three new components. Thus, the position of Cl affects the activity of these compounds [215].
R,
185R, = C1,R2 = OH 186 Kx = OH, R2 = Cl
187
Two new compounds named aranochlors A (188) and B (189) were detected as minor components from the fermented broth of the fungal strain Pseudoarachniotus roseus [216]. Fermentations were carried out in shake flasks as well as in laboratory fermenters. For the isolation of 188 and 189 six batches of each 100 liters were processed. Both compounds were present both in the culture filtrate and the mycelium. Both aranochlors exhibited antibacterial and antifungal activities. The
523 minimum inhibitory concentrations (MIC) of 188 and 189 required to inhibit a variety of bacterial and fungal strains are listed in Table 5. Table 5. Inhibitory activities of aranochlors against microorganisms. Test Organism Staphylococcus aureus Bacillus subtilis Micrococcus luteus Escherichia coli Pseudomonas aeruginosa Candida albicans Saccharomyces cerevisiae Aspergillus niger
different
MIC (ug/ml) Aranochlor A Aranochlor B 3.12 1.56 3.12 3.12 0.39 0.39 25 50 >200 >200 >200 >200 1.56 6.25 >200 >200
189
The red pigments, auxarconjugatins A and B (190,191) were isolated from mycelia of Auxarthron conjugatum, an ascomycetous fungus belonging to the Onygenaceae, in which the causative fungi of severe mycoses are also found [217].
524
OMe 190 R=H 191R = Me
Selective growth inhibition of IL-6 dependent cells was detected in fermentation extracts of a fungal strain, which was characterized as Sporothrix species. An active metabolite, 192 termed chlovalicin was isolated. Chlovalicin dose-dependently inhibited the growth of IL-6 dependent MH60 cells (IC50, 7.5 uM) in the presence of 0.2 U/ml IL-6 and, to a lesser extent, the growth of B16 melanoma cells (IC50, 38 uM). This compound did not show any antimicrobial activity at a concentration of 1 mg/ml [218].
192
The structure determination of lachnumon (193), brominated derivatives of lachnumon (194) and mycorrhizin (195) and brominated derivatives of mycorrhizin A (196) was described. The compounds, which exhibit similar antimicrobial and nematocidal activities as their chlorinated analogues, were isolated from extracts of cultures of the ascomycete Lachnum papyraceum to which CaBr2 had been added [219].
525
MeO' O
Br
193 R = H
195 R = H
194R = C1
196R = C1
A novel fungal metabolite, Sch 202596 was discovered from the fermentation of a fungal culture Aspergillus sp. [220]. The fungus, Aspergillus sp. was isolated from the tailing piles of an abandoned uranium mine in Tuolemene County, California. Compound 197 was revealed to be a new spirocoumaranone by spectroscopy, related to the griseofulvin family of compounds. In the in vitro galanin receptor GALRl assay compound (197) exhibited inhibitory activity with an IC50 value at 1.7 uM. Cl
MeOOC
MeOOC
197
HIOH
"OH
The structure and absolute configuration of microline, a new metabolite isolated from culture filtrates of Gilmaniella humicola, has been shown to be (198) by spectral data, chemical transformations and Xray analysis [221].
526
H
The structures of mikrolin and mycorrhizinol, two new metabolites isolated from culture filtrates of Gilmaniella humicola, have been shown to be (199) and (200), respectively, by spectral and chemical studies [222].
OH
OH
O 199
200
The structure and configuration of gilmaniellin, a metabolite of Gilmaniella humicola, has been shown to be (201) by X-ray analysis [223].
527
OH
O
201
The novel substances designated as ICM0301 A-H were isolated from the culture broth of Aspergillus sp. F1491 [224, 225]. ICM0301s inhibited the growth of human umbilical vein endothelial cells induced by basic fibroblast growth factor with IC50 values of 2.2-9.3 ug/ml. ICM0301C (202) and ICM0301D (203) showed significant anti-angiogenic activity at a concentration lower than 101 ug/ml in the angiogenesis model using rat aorta cultured in fibrin gel. ICM0301s showed very low cytotoxicity against various tumor cells. The taxonomy of the producing organism, and the fermentation, isolation and biological activities of ICM0301s were described. The structures were elucidated by spectroscopic analyses. ICM0301C and D have a substituted decalin skeleton containing one chlorine atom.
528
OH
H 202 R = Me 203 R = H
Vertihemipterin A, the ascochlorin glycoside, and its aglycone, 4',5'dihydro-4'-hydroxyascochlorin, and a new analog, 8'-hydroxyascochlorin, were isolated from the fermentation broth of the pathogenic fungus Verticillium hemipterigenum [226]. Structures of these compounds were elucidated by spectroscopic methods. Cytotoxic activities of these ascochlorin analogs were evaluated. All compounds were tested for their cytotoxic activities against three cancer cell-lines, KB, BC-1 and NCIHI 87, as well as Vero cells. The compounds exhibited significant cytotoxicities against all cell lines, e.g. for compounds 204-206, see Table 6.
529
Table 6. Cytotoxic activities of compounds 204-206. Cytotoxicity (IC50, ug/ml) Compound KB BC-1 1 NCI-H178C Verod 19 8.4 7.9 6.9 204 >20 38 >20 >20 205 2.2 3.4 206 2.7 1.4 1 oral human epidermoid carcinoma; human breast cancer; a
;
human small cell lung cancer; d African green monkey kidney fibroblast OH
OHC
204 R = - O 205 R = OH
OHC
OH
206
Trichodermamides A and B (207), two modified dipeptides, have been isolated from cultures of the marine-derived fungus Trichoderma virens [226]. Trichodermamide B displayed significant in vitro cytotoxicity against HCT-116 human colon carcinoma with an IC50 of 0.32 ug/ml. This metabolite also exhibited moderate antimicrobial activities against amphoterocin-resistant C. albicans, methacillin-resistant S. aureus, and vancomycin-resistant E. faecium with MIC values of ca. 15 ug/ml against
530
all three strains. Trichodermamide A was completely inactive in all of these bioassays, suggesting that the chlorine atom is an essential part of the pharmacophore. Chlorination is known to play an essential role in the activity of numerous, structurally diverse natural products including the antibiotics vancomycin and chloramphenicol and the antitumor compounds cryptophycin and rebeccamycin [227]. In the case of trichodermamide B (207), the chlorohydrin moiety at C4 and C5 might be a precursor of an epoxide, which could be the biologically active molecular form of this molecule. .OMe
Cl
Or
T^
OMe
207
The spiroxins 208-210 were purified from the culture extract of a marine-derived fungus, isolated from a soft orange coral collected from the waters near Vancouver Island, Canada [228]. Their unique bisnaphthospiroketal structures were established by spectroscopic methods. In addition to cytotoxicity, these compounds showed antibiotic activity and were active in a mouse xenograft model against human ovarian carcinoma. The mechanism of action of these compounds was shown to be due, in part, to their effect on DNA. Spiroxin A (208) showed some activity against Gram-positive bacteria but only marginal activity against Gram-negative bacteria. Compound 208 showed antitumor activity in nude mice against ovarian carcinoma (59 % inhibition after 21 days) at 1 mg/kg/dose given IP on day 1, 5 and 9 post staging. In a cytotoxicity assay, 208 exhibited a mean LC50 value of 0.09 ug/ml against a panel of 25 diverse cell lines. In evaluating its probable mechanism of action, it was observed that in the presence of e.g. 2-mercaptoethanol, 208 caused a concentration-dependent nicking of pBR322 DNA, suggesting that the compound partly exerts its cytotoxicity effect through a single-stranded DNA cleavage. Cytotoxicity of quinones has been attributed to DNA
531
modification, alkylation of essential protein thiol groups, oxidation of essential protein thiol groups by superoxide radicals or a combination of these mechanisms. The oxidation state of the spiroketal carbon, a masked ketone, could allow the spiroxins to behave chemically as quinone epoxides, possibly causing DNA cleavage under reducing conditions via an oxidative stress mechanism involving the formation of thiol conjugates. Thus, a variety of mechanisms may play a role in spiroxinmediated cytotoxicity.
o
OH
208 R, = H, R 2 + R3 = O 209 R, = Cl, R 2 + R 3 = O 210 R, = Cl, R 2 = OH, R3 = H
Gymnastatins A-E have been isolated from a strain of Gymnascella dankaliensis originally separated from the sponge Halichondria japonica [229]. Cytotoxic activities of compounds 211-215 were examined in the P388 lymphocytic leukemia test system in a cell culture [230]. The results showed that three of the compounds (211-213) exhibited potent cytotoxic activity and two (214 and 215) exhibited weak cytotoxic activity (ED50 0.018, 0.108, 0.106, 10.5 and 10.8 ug/ml, respectively). Gymnastatin A of these compounds showed strongest cytotoxicity. This evidence suggested that conjugated ketones were important for the enhancement of cytotoxicity in gymnastatin analogs, and hence the cytotoxic activity of compound 213 resulted from a conjugated ketone, which might be derived from compound 213 in the test system. Pericosines A (216) and B have been isolated from a strain of Periconia byssoides, originally separated from the sea hare Aplysia
532
kurodai, and their structures have been established based on spectral analyses. Pericosine A exhibited significant cytotoxicity (ED50 0.12 ug/ml) in the P388 lymphocytic leukemia test system in cell culture.
Cl
Cl
RHN 211aR 1 =OH,R 2 = 211bR!=H,R 2 = O
R=
OMe
i
\
RHN
OMe
212
213
214b R, = H, R 2 = O H
215a R, = OH, R 2 = H 215b R, = H, R 2 = O H
533
The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (217), is described [232]. The new compound was produced by a cultured marine fungus only when a unicellular marine bacterium strain, CNJ-328, was cocultured in the fungal fermentation. The fungus, isolated from the surface of the brown alga, Rosenvingea sp., collected in the Bahamas Islands, was identified as an undescribed member of the genus Pestalotia. Pestalone (217) exhibits moderate in vitro cytotoxicity in the National Cancer Institute's 60 human tumor cell line screen (mean GI50 = 6.0 uM). More importantly, pestalone showed potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MIC 37 ng/ml) and vancomycin-resistant Enterococcus faecium (MIC 78 ng/ml). The potency of this agent toward drug-resistant pathogens suggests that pestalone should be evaluated in more advanced, whole animal models of infectious disease.
OH HO,
OMe
COOMe 216 217
Structures of three novel compounds designated monordens C to E (218-220), isolated from the fermentation broth of amidepsine-producing fungus Humicola sp. FO-2942, were elucidated by spectroscopic evidence [233].
534
o.
220
FR225659 and four related (221-225) compounds are novel gluconeogenesis inhibitors that consist of a novel acyl-group and three abnormal amino acids [234]. Spectroscopic analysis concluded that FR225659 is an N-acyl tripeptide consisting of a novel acyl, a 3-chloro-4hydroxyarginine, a 3-hydroxy-3-methylproline, and a dehydrovaline. They were isolated from the culture broth of Helicomyces sp. No. 19353 and were purified by chromatography. These compounds inhibited the glucagon-stimulated gluconeogenesis of rat primary hepatocytes and had hypoglycemic effects in two different in vivo models [235].
535
N
Ri R2 R3 OH Me 221 OH OH Et 222 OH OH H Me 223 224 OMe OH Et 225 OMe OH Me Two new biologically active cyclopentenones, VM 4798-la (226) and VM 4798-lb (227) were obtained [236] as a 3:1 inseparable mixture from fermentations of Dasyscyphus sp. A47-98. The mixture of the two isomers showed cytotoxic and weak antibacterial and antifungal properties (e.g. Micrococcus luteus, Mycobacterium phlei, Candida parapsilosis, Rhodotorula glutinis, Aspergillus ochraceus and Zygorhynchus moelleri). In the serial dilution assay, 226 and 227 inhibited the growth of fungi and bacteria at 10-100 ug/disk. 226 and 227 caused a 50 % lysis of HeLa S3-, HL 60- and L1210-cells at a concentration of 10 ug/ml. The cytotoxic activity on Jurkat cells is quite significant with a 50 % lysis at 1.4 uM. The 3:1 mixture of 226 and 227 completely inhibited the incorporation of the appropriate radioactive precursors into DNA, RNA and proteins in Jurkat cells at a concentration of 1.9 uM. This is suggested to be a consequence of the breakdown of the mitochondrias membrane potential.
536
COOMe HO 227
CONCLUSION Fermentation of produced strains, purification, isolation and biosynthesis of griseofulvin were discussed in this review. In addition, compounds similar to griseofulvin were described. Griseofulvin is among the oldest antibiotics and a few that have been successfully used in the treatment of fungal diseases of the skin, nails, and hair. It inhibits e.g. Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum audouini, M. canis, M. gypseum, and Epidemophyton floccosum and decreases growth of Aspergillus spp. and Phialophora spp. Usually, topical application is not sufficient and has to be accompanied by peroral application. In addition to its therapeutic uses, griseofulvin and its derivatives are interesting with respect to their biosynthesis, which has some specific features including introduction of the halogen atom in a reaction catalyzed probably by a halogen peroxidase, similarly to other chlorine containing antibiotics such as chlorotetracycline and chloramphenicol. It would be worth investigating, whether targeted genetic modification of production strains would lead to new derivatives of griseofulvin and related compounds and, possibly also to hybrid antibiotics with better biological activity or physico-chemical properties. In addition to antimicrobial activity, many compounds referred here exhibit other interesting biological activities, such as nematocidal, cytotoxic, antitumor and antiangiotensic etc. that might be used in the therapeutic practice. ACKNOWLEDGEMENTS This work was supported by the Grant Agency of the Czech Republic (grant no. 204/01/1004) and by the Institutional Research Concept no. AV 0Z 5020 903. The authors wish to express their thanks to Mrs G.
537
Brou5kova for administrative help. Excellent technical assistance of Mr. M. Rezanka (student of Faculty of Science of the Charles University, Prague) and Mr. P. Rezanka (student of Institute of Chemical Technology and Faculty of Science of the Charles University, Prague) is gratefully acknowledged. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18]
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Bioactive Alkaloids of Fungal Origin Hideo Hayashi Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan ABSTRACT: In order to obtain fungal isolates, which produce bioactive compounds, random screening was carried out using okara (an insoluble residue of the whole soybean homogenate) as a cultural medium. We observed three kinds of activities against silkworms: insecticidal activity, convulsive activity and paralytic activity. A soil isolate, Penicillium simplicissimum ATCC 90288, produced novel insecticidal indole alkaloids that we designated as okaramines. Okaramines were found to be produced by not only this strain but also other strains belonging to P. simplicissimum. The isolate, Aspergillus aculeatus KF-428 also produced two okaramine congeners: okaramines H and I. These data strongly supported the fact that okaramines are widely produced by fungi. Till now, eighteen okaramine congeners have been isolated; their biogenesis and structure-activity relationships are described in this review. We also describe the results of synthetic studies for the okaramines J and N. The isolate, Penicillium expansum MY-57 produced five insecticidal compounds: the new communesin congeners, communesins D, E, and F, and the known communesins A and B. Convulsive compounds, verruculogen and penitrems, were produced by the isolates P. simplicissimum MF-24 and P. simplicissimum ATCC 90288, respectively, indicating that our convenient bioassay system with silkworms could be used to search for convulsive compounds. Novel convulsive compounds, brasiliamides A, B, C, D, and E, were found in the cultural media of Penicillium brasilianum JV-379. Finally, chance observation led to the isolation of new paralytic compounds, asperparalines A, B, and C, from Aspergillus japonicus ATCC 204480. Asperparalines have unique structures consisting of a bicyclo[2,2,2]diazaoctane core and a spirosuccinimide moiety.
INTRODUCTION Combinatorial chemistry has become a powerful methodology for the construction of new compounds. Natural products, however, also have enormous potential as a source of new compounds. In particular, numerous useful microbial products have been isolated as antibiotics, herbicides, fungicides and enzyme-inhibitors. Moreover, microorganisms have provided various compounds with diverse bioactivities, such as immunomodulatory, antitumor and antihelmintical activities [1].
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Many efforts have been made to identify strains producing insecticidal components. In the 1960s, piericidins [2,3] and aspochracin [4,5] were isolated from Streptomyces sp. 16-22 and from Aspergillus ochraceus, respectively. In the 1970s, milbemycins were isolated as insecticides and acaricides from Streptomyces hygroscopicus subsp. aureolacrimosus [6-8]. Avermectins were isolated from Streptomyces avermitilis and developed as antiparasitic agents [9]. In the 1980s, our group also isolated a new insecticidal compound, A^-norphysostigmine, from Streptomyces sp. [10]. Spinosyns A, B, C and D (formerly known as A83543A-D), which were isolated from Sacchropolyspora spinosa in 1991, were found to possess potent mosquito larvicidal activity [11]. Spinosyns have also been used for crop defense [12]. However, we should emphasize that, among the various microbial products, only a few, such as the above-described avermectins and spinosyns, have practical applications. In the 1980s, our group began to screen microbes for insecticidal compounds that could be used in practice, or become lead compounds for the generation of new carbon skeletons. As a result, various strains were found to exhibit insecticidal, convulsive and paralytic activities against silkworms. This chapter deals with the procedures used for isolation of bioactive strains and their active principles. An overview of their chemical structures, activities, synthesis, and structurally related compounds is also given. INSECTICIDAL COMPOUNDS Various media were used for culturing fungi, and the fungal secondary metabolites are known to depend on the cultural conditions, such as the medium constituents and temperature. In our study, okara, which is an insoluble residue of whole soybean homogenate and a waste material in tofu (soybean curd) production, was used as a culture medium. This is the first time that this material has been used for culturing fungi. Fungal strains isolated from soil samples in the usual manner were cultured with okara media for about 10 days. The okara and mycelia were soaked in acetone, and aliquots of both acetone extracts were added to an artificial silkworm diet. Third instar silkworm larvae were introduced into a Petri dish containing the artificial diet, and the mortality rate was determined 24 h after initiating the administration. Using this screening method several hundred isolates were checked for their activity, and two strains, Penicillium simplicissimum Thom ATCC 90288 (originally AK-40) and Penicillium expansum Link MY-57, exhibited the insecticidal activity.
551
Okaramines Discovery of Okaramines A (1) andB (2)
The material from an acetone extract of okara fermented with P. simplicissimum ATCC 90288 was partitioned between ethyl acetate and water. The ethyl acetate layer thus obtained exhibited insecticidal activity. The ethyl acetate fraction was repeatedly chromatographed on silica gel with a hexane-acetone mixture, and then on alumina with a hexane-ethyl acetate mixture. Crystallization of the 50% ethyl acetate eluate and of 60-80% ethyl acetate eluates gave two active compounds, which were named okaramines A (1) and B (2), respectively [13-16]. The molecular formula of okaramine A (1) was determined to be C32H32N4O3 by HR-EIMS together with 13C- and !H-NMR spectra, implying nineteen degrees of unsaturation. The 'H-NMR spectrum is shown in Fig. (1). The 13C-NMR spectrum indicated that 1 had two amido carbonyl carbons and twenty olefmic carbons, suggesting that 1 was a heptacyclic compound. The UV absorption maximum at 374 nm indicated the presence of an indole chromophore with an expanded conjugation. Precise analysis of ' H - ' H COSY and HMBC spectra led to the planar structure of 1. Acetylokaramine A (3), giving a good crystalline structure for X-ray analysis, and the structure thus established is shown in Fig. (2). Okaramine A (1) was shown to be composed of two moieties, i.e. 3a-hydroxy-A^-(reverse-prenyl)-l,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid and 6,6-dimethyl-7/7-3,6dihydroazocino[5,4-6]indole-2-carboxylic acid. These two moieties formed a diketopiperazine ring, resulting in the formation of 1.
10.0
UllKlJJL 8.0
7.0
6.0
JL 5.0
4.0
Fig. (1). 300 MHz 'H-NMR spectrum of okaramine A (1) in acetone-rf6
3.0
2.0
1.0
552
1, okaramine A R = H 3, acetylokaramine A R = Ac
Fig. (2). ORTEP drawing of acetylokaramine A (3)
Okaramine B (2), C33H34N4O5, seemed to be an analog of 1. The H-NMR spectrum shown in Fig. (3) and 13C-NMR data strongly suggested that the azocinoindole moiety in 1 existed unchanged in 2. The essential difference between the l H-NMR spectra of 1 and 2 consisted of the absence of a vinyl group and a methine proton, and the appearance of an ethylidene group. Okaramine B (2) also contained an additional !
_JU ,
8.0
,
1
.
1
1
7.0
1
.
.
1
1
6.0
.
1
1
.
1
,
5.0
r—1
1
1
1
1
1
4.0
Fig. (3). 270 MHz 'H-NMR spectrum of okaramine B (2) in DMSO-rf6
1
1
3.0
_prxn
r-
2.0
1.0
553
hydroxyl group and a methoxyl group. To determine the connectivity of each functional group, a long-range 2D !H-13C COSY experiment was carried out for 2, and the results indicated that C-ll was bound to C-8a, and that an azetidine ring was newly formed. In addition, it was shown that two hydroxyl groups were located at C-2 and C-3a, and a methoxyl group at C-3. The relative stereochemistry was confirmed by the nuclear Overhauser effect difference spectra of 2, indicating the a-orientation for 2-OH, 3a-0H and H-ll and the p-orientation for 3-OCH3. The absolute configurations at C-2, C-3a and C-8a of 2 were deduced to be the same as those in 1 by comparing the CD spectrum of 2 with that of 1. Okaramine B (2) has two moieties, a hexahydropyrroloindole and a dihydroazocinoindole, and it is of particular interest that the pyrroloindole part is condensed with a newly formed azetidine ring.
"OH
2, okaramine B
4, okaramine C
Discovery of Okaramine C (4)
The unique structures of okaramines A (1) and B (2) led us to investigate whether okaramines or similar compounds were produced by other strains. First, we examined four strains belonging to P. simplicissimum, i.e. IFO 5762, AHU 8065, AHU 8402 and MF-24, and found that strain AHU 8402 showed the highest activity [17]. From an okara medium fermented with P. simplicissimum AHU 8402, three insecticidal compounds were isolated. Two of them were identified as 1 and 2, and the third one seemed to be a new, related compound, which was thus named okaramine C (4). Okaramine C (4), C32H36N4O3, proved to be a tetrahydro-derivative of 1. The ]H-NMR and 1H- H COSY spectra indicated that 4 had an additional reverse-prenyl group, an exchangeable proton and a -CH2-CH< group instead of -CH=CH- and -CH=C<, suggesting that the C-l'=C-2' double bond was saturated and
554
that the N-3'-C-4' bond was reductively cleaved [17]. This finding that P. simplicissimum AHU 8402 produces okaramines A (1), B (2) and C (4) suggests the probability for various strains to produce okaramines. Discovery of Okaramines D (5), E (6), F (7), and G (8)
Minor congeners of the okaramines were investigated from the culture extracts of P. simplicissimum ATCC 90288 in order to elucidate the structure-activity relationship and also to clarify the biosynthesis of okaramines. The congeners were purified by detection using Ehrlich reagent followed by staining with vanillin-sulfuric acid to identify okaramine-related compounds. Four new okaramine congeners were isolated and termed okaramines D (5), E (6), F (7), and G (8) [18, 19]. Okaramine D (5), C33H34N4O6, contained one additional oxygen atom when compared with okaramine B (2). The ^-NMR spectrum of 5 showed the presence of >CH-CH2OH, strongly suggesting that a secondary methyl group at C-12 in 2 had been replaced in 5 by a hydroxymethyl group. All spectral data and acetylation of 5 led to the conclusion that 5 was 12-hydroxyokaramine B [18]. Okaramine E (6), C32H32N4O4, showed IR and UV spectra quite similar to those of 5, indicating, that 6 had the same functionalities and conjugated systems as 5. In the H-NMR spectrum of 6, signals assigned to a hydroxymethyl group at C-12 were observed, suggesting that the substitution on the azetidine ring in 6 was the same as that in 5. The NMR data of 6 also showed the existence of a dihydroazocinoindole moiety and a 1,2-disubstituted benzene ring. Compared with 5, the signals of two hydroxyl groups at C-2 and C-3a, a methine group at C-3a, and a methoxyl group at C-3 had disappeared, and signals attributable to a hydroxyl group and a -CH2-CFK linkage were newly observed, suggesting that 6 had a hydroxyl group at C-2 or C-3 a. This assumption helped complete the molecular formula for 6. The location of the hydroxyl group was concluded to be at C-3a from a comparison of the chemical shifts of C-3b and C-8a between 5 and 6. This conclusion and the configuration of the hydroxyl group were supported by the similarity in the chemical shifts of C-2, C-3 and C-3a between 6 and 1 [18]. The molecular formula of okaramine F (7), C32H30N4O4, was determined by HR-EIMS, and was identical to the molecular formula of okaramine E (6), but with two fewer hydrogen atoms. In the 'H-NMR spectrum of 7, almost all the signals observed in 6 were found, with the exception that the signals assigned to H-2 and H2-3 in 6 were replaced by one singlet signal, indicating that the bond between C-2 and C-3 had become unsaturated. Another possibility that a newly introduced double
555
bond was located between C-3 and C-3a was ruled out by a comparison between the UV spectra of 6 and 7. Okaramines generally show UV absorption at around 380 nm, which is characteristic of the azocinoindole ring, but 7 showed an absorption maximum at 402 nm, strongly suggesting that the double bond was between C-2 and C-3 [18]. The molecular formula of okaramine G (8) was C32H34N4O3, indicating that 8 has two more hydrogen atoms than okaramine A (1) and two less than okaramine C (4). From an inspection of the 'H- and 13 C-NMR spectra of 8, 8 was found to possess a 3a-hydroxy-A^-(reverseprenyl)-l,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid moiety, a 2,3-disubstituted indole and a diketopiperazine ring. The spectra indicated an additional reverse-prenyl and another exchangeable proton in 8, both of them resulting from the reductive cleavage between N-3' and C-4'. This structure was confirmed by HMBC experiments. From the NOESY experiments, the conformation of 8 was deduced to be quite different from that of 1—namely, 2-(reverse-prenyl)indole moiety in 8 turned around the C-l'-C-llb' axis and a reverse-prenyl group at C-6a' was closer to a carbonyl at C-12' [19].
«OH
OCH-,
5, okaramine D
6, okaramine E
"'OH
7, okaramine F
"OH
8, okaramine G
556 Discovery of Okaramines H (9) and I (10)
In further screening microbes for insecticides, an isolate Aspergillus aculeatus Iizuka KF-428 was obtained from a soil sample. This strain also exhibited the insecticidal activity when grown on okara, and three active principles were isolated. Two of them were identified as okaramines A (1) and B (2). The third one also seemed to be an okaramine-related compound and was named okaramine H (9) [20]. Okaramine H (9) had a molecular formula of C32H32N4O3, which was identical to that of okaramine A (1). The 1H- and C-NMR spectra of 9 were very similar to those of 1, suggesting the existence of azocinoindole and pyrroloindole moieties in 9. Signals assignable to a prenyl group in 9 were observed instead of the signals assigned to a reverse-prenyl group in 1. In long-range *H and 13C shift-correlated 2D-NMR experiments, the signal of C-7a was correlated with H2-IO, H-6, and H-4, revealing that the prenyl group was located at C-7. The orientations of hydrogen atoms at C-2 and C-8a and of a hydroxyl group at C-3a in 9 were considered to be the same as those of 1, because of the similarity between chemical shifts and coupling constants of H-2 and H-3 with those of 1. Okaramine H (9) might be formed through an aza-Claizen type rearrangement in which a reverse-prenyl group at N-8 of 1 is transferred to C-7 via the formation of a six-membered ring [20]. An inactive okaramine-related compound was isolated from the okara culture of A. aculeatus KF-428. This compound, okaramine I (10), had a molecular formula of C27H24N4O3. The ! H- and 13C-NMR spectra of 10 were the same as those of depentenylokaramine A, which was formed by hydrogenolysis of 1 with Pd/C [15, 20].
"OH
"OH
9, okaramine H
10, okaramine I
557 Discovery of Okaramines J (11), K (12), L (13), M (14), N (15), O (16), P (17), Q (18), andR (19)
Okaramines have attracted considerable attention due to their molecular complexity and intriguing biogenesis. We thoroughly searched the fermented material of P. simplicissimum ATCC 90288 for new okaramine congeners, with the result that nine members of the okaramine family were isolated. Okaramine J (11) had a molecular formula of C32H36N4O3, which is identical to the molecular formula of okaramine C (4). The ' H-NMR spectrum of 11 is shown in Fig. (4). The critical differences were the absence of one of two reverse-prenyl groups that were observed in 4 and the appearance of a new prenyl group and one exchangeable proton coupled to the methine proton at C-8a. The HMBC spectrum of 11 indicated that the prenyl group was bound to C-7 in the pyrroloindole ring [21]. Okaramine K (12), C32H34N4O3, had a molecular formula identical to that of okaramine G (8) [21]. The essential difference between the 'H-NMR spectra of 8 and 12 was the absence of a reverse-prenyl group
JL 10.0
pplll -
9.0
8.0
7.0
6.0
5.0
4.0
Fig. (4). 270 MHz 'H-NMR spectrum of okaramine J (11) in acetone-rf6
3.0
2.0
1.0
558
and the appearance of a prenyl group and one exchangeable proton. The HMBC spectrum of 12 indicated that the prenyl group was bound to C-7 in the pyrroloindole ring. Okaramine K (12) had 'H- and 13C-NMR spectra each consisting of signal accompanied by a 1/9-fold weaker signal of the same multiplicity. This suggested that 12 occurred as a 9:1 mixture of isomers. Furthermore, two isomers were obtained in pure form by HPLC, although each pure isomer was found to rapidly convert into a mixture of the original composition. To assign the stereoisomer, we carried out NOESY experiments on 12. The correlation observed between H-N3' and H-ll' from the major isomer supports the idea that the major isomer has the (Z) configuration at the C-l'=C-2' bond. On the other hand, the minor isomer of 12 was shown to have the {E) configuration at the same bond. It has been reported that aplysinopsin-type indole alkaloids, which are structurally similar to echinulin, underwent photoisomerization in a solution under either UV irradiation or ordinary daylight [22]. Therefore, 12 underwent facile photoisomerization in a solution under UV irradiation to become appreciably enriched by the (E) configurational isomer {ZIE = 6/4). Interestingly, the {ZIE) ratio of the stereoisomers of 12 reverted to a mixture of the original composition in one or two days at room temperature under condition of laboratory daylight. These facts can be interpreted as indicating that (Z)-12 is more thermodynamically stable. However, 8 did not exist as a mixture of {ZIE) configuration because of the steric repulsion between H-ll' of the indole nucleus and I3-CH3, I4-CH3.
iOH
•"'OH
O
11, okaramine J
12, okaramine K
Okaramine L (13) had a molecular formula of C32H36N4O3, which is identical to the formulae of okaramines C (4) and J (11). In the 'H-NMR spectrum of 13, the signal at H-N8 that was observed in 4 had disappeared, and a new signal assigned to the benzene ring of the
559
pyrroloindole moiety was observed. Careful comparison of the 'H-NMR spectrum of 13 with that of 11 revealed that methylene protons at C-10 of the prenyl moiety were shifted upfield, suggesting that this moiety was located at N-8. This consideration was confirmed by HMBC experiments, in which correlation was observed between H2-IO and both C-7a and C-8a, and between H-8a and C-10 [21]. Okaramine M (14) had a molecular formula of C29H30N4O3. The presence of a reverse-prenyl group was established by the NMR spectra. The presence of an acetyl group was also indicated in the NMR spectra. The placement of the reverse-prenyl group at C-3a in the indoline moiety was confirmed by HMBC correlations between C-3a and each of I3-CH3, I4-CH3, and H-ll. The placement of the acetyl group at N-8 was indicated by the fact that signals of H-8a and H-7 were recognized at a lower-field position than those of the corresponding protons of the other okaramines. Furthermore, ! H and 13C long-range correlation between H-8a and an acetyl carbon was observed [21]. Okaramine N (15) had a molecular formula of C32H34N4O3, indicating that 15 had two more hydrogen atoms than okaramine A (1). The precise analysis of NMR experiments suggested that the C-l'=C-2' double bond o -"OH
13, okaramine L
14, okaramine M
MOH
••'lOH
15, okaramine N
16, okaramine O
560
was saturated, and this assumption was supported by the fact that 15 lacked the UV absorption at 374 run present in 1. The results of the NOESY experiment indicated that the relative configurations at C-2, C-2', C-3a, and C-8a of 15 were all of c/s-type [23]. Okaramine O (16) had a molecular formula of C32H34N4O4, which was identical to the formula of 15, but with one more oxygen atom. The NMR spectra of 16 indicated the presence of an oxymethine group and a hydroxyl group, and the location of the hydroxyl group was also determined to be at C-l'. The relative configurations at C-2, C-2', C-3a, and C-8a of 16 were determined to be the same as those of 15 on the basis of the NOESY experiment. The hydroxyl group at C-l' was determined to have an a-orientation on the basis of the NOESY experiment and the lR-lH coupling constants [23]. Okaramine P (17), C32H34N4O4, had the same molecular formula as 16. The ^ - N M R spectrum of 17 is characterized by the disappearance of the reverse-prenyl signals in 16 and the appearance of a prenyl group. The HMBC spectrum indicated that the prenyl group was connected to C-7. The relative stereochemistry of 17 was the same as that of 16 at all chiral
"OH
"OH
18, okaramine Q
17, okaramine P
19, okaramine R
561
centers, as judged by the NOESY and the 13C-NMR chemical shifts [23]. Okaramine Q (18) had a molecular formula of C32H32N4O4. The UV spectrum (A,max 234, 288, 376 nm) indicated the presence of an indole chromophore with an expanded conjugation. The ^ - N M R spectrum of 18 resembled that of okaramine B (2), except for the absence of the methoxyl proton signal in 2 and the presence of signals due to isolated methylene protons. A precise comparison between the NMR spectra of 18 and 2 led to the conclusion that 18 is a demethoxyl derivative of 2 [23]. Okaramine R (19) appeared to possess the molecular formula of C32H32N4O4 by HREIMS, suggesting the presence of an additional oxygen atom as compared with okaramine A (1). The ^-NMR spectrum of 19 was identical to that of 1, with the exception that 19 lacked a methine signal at C-8a, and showed a new amide proton signal. In the 13 C-NMR spectrum of 19, the signal at C-8a that was obviously observed in 1 also disappeared, and a new signal assigned to an amide carbonyl
24
562
carbon was observed. The 'H-NMR signals for 3a-0H and H2-3 were correlated with this carbonyl carbon signal in the HMBC experiments. These data indicated that the carbonyl must be at C-8a, forming an oxyindole moiety [23]. Possible okaramine precursors, i.e. cyclo (Trp-Trp) (20), cyclo (2-(reverse-prenyl)-Trp-Trp) (21), cyclo (A^reverse-prenyl)-frp-Trp) (22), cyclo (2-(reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (23), and cyclo (Arl-(reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (24), were isolated during the course of the investigation of okaramine congeners [21]. Three of these compounds, 21, 22, and 24, are new compounds. cyclo(2-(Reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (23) was synthesized by Schkeryantz and coworkers as a precursor of gypsetin [24], but it was isolated for the first time from natural sources in our study. One year after our findings, Kozlovsky and coworkers reported the isolation of fellutanines A, B, C, and D from Penicilliumfellutanum [25], which were found identical to the compounds 20, 22, and 23, respectively. Absolute Configuration of Okaramines
In order to clarify the absolute configuration of okaramines, we determined the absolute stereochemistry of the above-mentioned derivatives of cyclo (Trp-Trp) (20). Acid hydrolysis of 20 gave L-tryptophan, which was identified by comparison with standard D,L-tryptophan samples by chiral HPLC analysis. Thus, the absolute configuration at C-2 was proved to be S [21]. The absolute stereochemistries of 21, 22, 23, and 24 were defined by a CD comparison with cyclo (L-Trp-L-Trp) (20). Furthermore, acid hydrolysis of 23 afforded L-tryptophan through the loss of a reverse-prenyl side chain [26, 27]. Hydrolysis of 21, 22, and 24 also gave L-tryptophan. Accordingly, these results elucidated the absolute stereochemistries of 21, 22, 23, and 24 as those depicted [21]. The stereochemistries of okaramine C (4), okaramine J (11), okaramine K (12), okaramine L (13), and okaramine M (14), including the absolute configurations, were then established [21]. The absolute configuration at C-2' of 4, 11, 12, and 13 was determined to be S by chiral HPLC analysis of the acid hydrolysate of each of these compounds. NOESY and NOE difference experiments were carried out to define the stereochemistry of 11. Because NOEs were observed between H-2 and H-2', between H-2 and H-8a, and between H-8a and 3a-OH, the absolute stereochemistry of 11 was determined. Based on NOESY and NOE difference data, the absolute stereochemistry of 11 was found to be identical to those of 4 and 13. The relative configurations at C-2, C-8a,
563
and C-3a in 12 were also determined based on the NOESY and NOE difference experiments. It was assumed that the absolute stereochemistry of 12 was the same as those of 4, 11, and 13, because all these compounds are produced by the same strain. The chiral HPLC analysis of the acid hydrolysate of okaramine M (14) revealed the presence of L-tryptophan. The absolute configuration at C-2' was determined to be S. The V( H ,H) coupling observed between H-2 and H-2' of 14 is consistent with the cis relationship between these protons. Thus, C-2 of 14 had the S configuration. In the NOE difference spectra of 14, significant NOEs were observed between H-2 and H-2', and between H-2 and H-8a. In addition, NOE enhancement was observed for 13-CH3, 14-CH3, and H-ll upon irradiation of H-8a. Therefore, the absolute stereochemistry of 14 was determined. The absolute stereochemistries of okaramines N (15), O (16), P (17), Q (18), and R (19) were considered to be the same as those of other okaramines because of biogenetic consideration. Insecticidal Activity of Okaramines
Insecticidal activities of okaramines and their derivatives are summarized in Table 1 using the LD50 values. Acetylokaramine A (3) showed the same activity as okaramine A (1). Okaramine C (4), whose azocine ring is cleaved, was also as active as 1, suggesting that the azocine ring moiety does not play an essential role in exhibiting the activity. On the other hand, okaramine G (8), whose azocine ring is also cleaved, showed much less activity than 1. This reduction in activity seems to have been caused by the conformational change in 8. Okaramines H (9) and I (10) exhibited no activity, indicating the importance of a reverse-prenyl group at N-8. Okaramines J (11), K (12), and L (13) exhibited no activity. This Table 1. Insecticidal Activity of Okaramines against Silkworms. Compound
1LD50 (ng/g diet)
okaramine A (1) acetylokaramine A (3) okaramine B (2) okaramine C (4) okaramine D (5) okaramine E (6) okaramine F (7) okaramine G (8) okaramine H (9) okaramine I (10) okaramine J (11)
8 8 0.2 8 20 >100 >100 40 >100 >100 >100
okaramine K. (12)
>100
Compound
LD50 (Hg/g diet)
okaramine L (13) okaramine M (14) okaramine N (15) okaramine O (16) okaramine P (17) okaramine Q (18) okaramine R (19) 4',5'-dihydroxyokaramine B (25) r,2',4',5'-tetrahydroxyokarmaine B (26) derivative of okaramine B (27) derivative of okaramine B (28)
>100 >100 >100 >100 >100 8 >100 6 80 >100 >100
564
fact also strongly suggested that the reverse-prenyl group at N-8 was very important and could not be substituted by a prenyl group. Okaramines N (15) and O (16) showed no activity, indicating that the resulting conformational change of the azocine ring moiety must be one reason for the reduction in activity. The LD50 values of okaramines B (2) and D (5) were 0.2 and 20 fxg/g diet, respectively, indicating that the hydroxylation at C-12 had drastically reduced their activity. Okaramines E (6) and F (7) exhibited no activity at a dose of 100 ug/g diet, suggesting that the functional groups in the pyrroloindole moiety play an important role in the insecticidal activity. To determine the effects of the azetidine and azocine ring moieties on the activity, chemical modification of 2 was carried out. Hydrogenation of 2 over 10% Pd/C in acetic acid provided 4',5'-dihydrookaramine B (25), l',2',4',5'-tetrahydrookaramine B (26), and two azetidine opened-ring compounds (27 and 28) [28]. Dihydrookaramine B (25) and tetrahydrookaramine B (26) had LD50 values of 6 (ag/g diet and 80 |ag/g diet, respectively, indicating that the reduction of activity was due to conformational change of the azocine ring moiety. Because two azetidine opened-ring derivatives (27 and 28) showed no activity, the azetidine ring was suggested to be the essential component responsible for the activity. The silkworm is a useful insect rather than a pest, and thus it was necessary to determine whether okaramines would also exhibit activity against harmful insects. Okaramines A (1) and B (2) were tested against various harmful insects. As a result, the most active of the 18 okaramines, compound 2 exhibited the same activity against the second instar larvae of the beet armyworm (Spodoptera exigua) as against silkworms, and thus this compound was considered to have potential use in practical applications. Biosynthetic Pathway
Biogenetic consideration of the structures of okaramines and related compounds hitherto isolated leads to the plausible biosynthetic scheme for okaramines outlined in Fig. (5). The basic framework of okaramines is derived from two molecules of L-tryptophan and two isoprene units. The sequence of events in the biosynthesis of okaramines is of crucial importance to the following discussion. According to our proposal, cyclo (L-Trp-L-Trp) (20) derived from L-tryptophan is biosynthetically considered to be an efficient precursor of okaramines. The formation of cyclo (2-(reverse-prenyl)- L-Trp-L-Trp) (21), cyclop1-(reverse-prenyl)L-Trp-L-Trp) (22), and cyclo (A^1-(reverse-prenyl)-L-Trp-2'(reverse-prenyl)-L-Trp) (24) is thought to arise via prenylation of 20.
565
Okaramine C (4) is derived from 24 by intramolecular cyclization and further oxidation at C-3a (Fig. (5) part 1). Intramolecular cyclization of 4 forms a tetrahydroazocine ring, leading to okaramine N (15). Oxidation at C-l' of 15 gives okaramine O (16), which yields okaramine A (1) through dehydration between C-l' and C-2'. On the other hand, aza-Claisen rearrangement of a reverse-prenyl group in 4, 16, and 1 leads to okaramines J (11), P (17), and H (9), respectively (Fig. (5) part 2). Intramolecular cyclization of 1 forms an azetidine ring, resulting in the formation of a biosynthetically significant postulated intermediate (29). Oxidation of this intermediate leads to okaramines Q (18) and E (6). Subsequent modification of 18 leads to okaramine B (2) and okaramine
=x, 0=
I cyclization
OCS.N^
19
H
A^.
4
Fig. (5). Proposed biosynthetic pathway for okaramines (part 1)
566
D (5) successively. Okaramine D (5) could be formed from the intermediate via 6 (Fig. 5 part 3). Removal of a reverse-prenyl group from 1 leads to okaramine I (10).
rearrangement
HN'
Hi'hrWoH CU.N;
jhydroxylation
I desaturation
HN
Hl'hpH'IOH
VN
H
"
rearrange -ment 17
16
I dehydration [d
Idehydration
HN
Hl-rrrl'lOH ^ J rearrange -ment
HI'hrH'IOH O^N^J , cleavage N"-"O
Fig. (5). Proposed biosynthetic pathway to okaramines (part 2)
I
rearrangement l]
567
HI'hpj-WOH
T^
elimination
1
Postulated intermediate i 29
v hydroxylation
Fig. (5). Proposed biosynthetic pathway to okaramines (part 3)
Synthetic Study of Okaramines N (15) andJ (11)
In 2003, Corey and coworkers described a remarkably simple synthesis of okaramine N (15) that took advantage of the new and
568
powerful Pd-promoted construction of the tetrahydroazocinoindole subsystem (Fig. (6)) [29]. (5)-A^-Boc-tryptophan methyl ester (30) was converted to the known indoline (31). Introduction of a reverse-prenyl group and oxidation furnished 32. The removal of the Boc-protecting group from 32 and saponification gave an amino acid. Schotten-Baumann acylation of the amino acid with FmocCl afforded
a) NaBH4CN, AcOH b) i) CuCl, ;-Pr2NEt, 2-acetoxy-2-methyl-3-butyne ii) DDQ iii) H2, Pd/C, quinoline c) i) SOC12 ii) LiOH iii) FmocCl d) 3-methyl-2-butenal,NaBH4 e) i-Pr2NEt, bis(2-oxo 3-oxazolidyl)phosphinic chloride f) Pd(OAc)2 g) Et2NH h) A'-methyltriazolidinedione Fig. (6). Synthesis of okaramine N (15) by Corey [29]
569
reverse-prenylated indole (33). 7V-Alkyl tryptophan methyl ester (34) was acylated with 33 to afford a tetracyclic intermediate (35). Treatment of 35 with Pd(OAc)2 provided tetrahydroazocinoindole (36). Exposure of 36 to excess diethylamine in THF resulted in Fmoc cleavage and cyclization to furnish diketopiperazine (37). The bisindole (37) underwent highly
11 Anth = 9-anthracenyl a) i) AnthSO2Cl, Et3N ii) tert-butyl isourea b) i) NBS, Et3N ii) 3,3-dimethyldioxirane iii) NaBH c) 1,1 -dimethylpropargyl bromide, CuCl, ;-Pr2NEt d) H2, Pd/Al2O3 e) TFA f) TMSOTf, 2,6-lutidine g) PyBop, Et3N h) Al/Hg i) i) KOH/MeOH ii) HBTU, <-Pr2NEt Fig. (7). Synthesis of okaramine J (11) by Ganesan [30]
570
selective reaction with the commercially available "ene" reaction reagent TV-methyltriazolinedione to form the ene product at C-3 of the iV-unsubstituted indole subunit. Subsequent photooxidation followed by reduction of the resulting product afforded the hydroxylated octacycle (38). Finally, thermolysis of 38 furnished okaramine N (15). Total synthesis of okaramine J (11) was achieved by Ganesan and coworkers in 2003 [30]. A key reaction in their synthesis was the acid-catalyzed, room-temperature, aza-Claisen rearrangement of an A^-reverse-prenylated hexahydro[2,3-Z>]pyrroloindole to a C-prenylated derivative (Fig. (7)). Hexahydro[2,3-£]pyrroloindole (40) was obtained by oxidative Witkop cyclization of L-tryptophan tert-buty\ ester. The alkylation of 40 afforded alkyne (41). The resulting alkyne (41) was hydrogenated to afford alkene (42). Treatment of 42 with TFA produced rearranged 43, indicating that this transformation was a charge-accelerated, aza-Claisen rearrangement. Removal of the tert-butyl ester provided acid (44). The indole C-2 reverse-prenylated derivative (45) was made in four steps from L-tryptophan according to the procedure described for the total synthesis of gypsetin [24]. Coupling 44 and 45 afforded pentacycle (46). Reductive removal of the anthracenylsulfonamide protecting group afforded 47. The methyl ester was hydrolyzed to the free amino acid, which underwent cyclization under peptide-coupling conditions to give okaramine J (11). Okaramine-Related Compounds
Okaramine A (1) is a novel heptacyclic compound containing a hexahydropyrroloindole moiety and a dihydroazocinoindole moiety. The azocinoindole moiety has been reported to constitute only two compounds: a metabolite (48) of Aspergillus ustus [31] and cycloechinulin (49) produced by A. ochraceus [32] (Fig. (8)). One of the structural characteristics of the okaramine family is the presence of a reverse-prenylated hexahydro[2,3-&]pyrroloindole moiety. Some related
H 3 CO H
48 Fig. (8). Compounds with azocinoindole moiety
49, cycloechinulin
571
compounds are shown in Fig. (9). Brevianamide E (50) was isolated from the culture medium of Penicillium brevicompactum by Birch and Wright in 1970 [33]. Amauromine (51) was isolated as a vasodilator from the culture broth of Amauroascus sp. No. 6237 by Takase and coworkers in 1984 [34, 35]. A family of new compounds, including ardeemin (52), iV5-acetylardeemin (53), and 15b-hydroxy-iV5-acetylardeemin (54), were isolated from the fermentation broth and mycelia of a strain of Aspergillus fischeri var. brasiliensis by Karwowski and coworkers in 1993 [36. 37]. A^5-Acetylardeemin (53) potentiates the cytotoxicity of the anticancer agent vinblastine in multi-drug resistant human tumor cells [36]. In 1994, Shinohara and coworkers isolated gypsetin (55) as an inhibitor of acyl-CoA:cholesterol acyltransferase from the cultured broth of Nannizzia gypsea var. incurvata IFO 9229 [38, 39]. In 2000, Kozlovsky and coworkers reported the isolation of fellutanine D (56) from the cultures of Penicillium fellutanum; and since then it has been reported cytotoxic [25].
50, brevianamide E
51, amauromine
o
o
52, ardeemin R = H 53, A^-acetylardeemin R = Ac
H
54, 15b-hydroxy-7V5acetylardeemin
OH
55, gypsetin
56, fellutanine D
Fig. (9). Compounds with hexahydro[2,3-6]pyrroloindole moiety
572
Each of the tryptophan metabolites shown in Fig. (10) has prenyl groups, reverse-prenyl groups, and a diketopiperazine ring. Echinulin (57), which contains a tryptophan moiety, was isolated by Birch and Farrar in 1963 [40]. Neoechinulin (58), which contains a dehydrotryptophan moiety, was also isolated as a pigment from the same molds that produced 57 by Barbeta and coworkers in 1969 [41]. Neoechinulins A (59), B (60), and C (61) were isolated as ivory crystals, yellow crystals, and yellow crystals, respectively, from sugar beet molasses cultures of Aspergillus amstelodami by Dossena and coworkers in 1974 [42]. Neoechinulins D (62) and E (63) were also isolated from the neoechinulins A-, B-, and C-producing strains by Marchelli and coworkers in 1977 [43]. Cryptoechinulin A, which is identical to compound 61, was isolated in small amounts from cultures of A. amstelodami along with a large quantity of 57 by Cardillo and coworkers in 1974 [44], and cryptoechinulin G (64) was isolated from the same strain by Gatti in 1978 [45]. In 1976, Nagase and coworkers isolated isoechinulins A (65), B (66), and C (67) from the course of their search
59, neoechinulin A
60, neoechinulin B
61, cryptoechinulin A neoechinulin C
62, neoechinulin D
Fig. (10). Structures of echinulin family (part 1)
573
for indole metabolites in the mycelia ofAspergillus rubber [46]. In 1999, Fujimoto and coworkers reported the isolation of tardioxopiperazines A (68) and B (69) as immunomodulatory constituents from an Ascomycete Microascus tardifaciens [47]. It is noteworthy that all compounds shown in Fig. 10 have a reverse-prenyl group at C-2 in the indole ring.
63, neoechinulin E
65, isoechinulin A
67, isoechinulin C
64, cryptoechinuline G
66, isoechinulin B
68, tardioxopiperazine A
69, tardioxopiperazine B Fig. (10). Structures of echinulin family (part 2)
574
Communesins Identification of Communesins A (70) and B (71) and Discovery of Communesins D (72), E(73), and F (74)
The acetone extract of okara fermented with Penicillium expansum Link MK-57 was found to exhibit the insecticidal activity against silkworms. The acetone extract of okara fermented with this strain was purified by solvent extractions, column chromatography, HPLC, and crystallization to yield five active compounds—i.e., two known compounds, communesins A (70) and B (71), and three new ones, communesins D (72), E (73), and F (74) [48]. The structures of the known compounds, 70 and 71, were assigned by comparing their physicochemical properties and spectral data with those reported in the literature [49]. o. ,o
70, communesin A
71, communesin B
Communesin D (72) was obtained as colorless needles and gave a protonated molecular ion [M+H]+ at m/z 523.2687 by HRFABMS, consistent with the molecular formula of C32H34N4O3. The UV spectrum showed an absorption maximum at 266 nm, suggesting that 72 had the same chromophore as 71. The 1H- and 13C-NMR data are similar to those for 71, indicative of the presence of a 1,2-disubstituted benzene and a 1,2,3-trisubstituted benzene ring moieties. The NMR data also strongly suggested that 72 had the same carbon skeleton—including the seven-ring system—as 71. Communesin D (72) was also found to have a (2£,4£)-2,4-hexadienoyl moiety by the ^ - N M R signals. The methyl signal at N-15 in 71 was not observed in 72, and a new signal assignable to an aldehyde proton was observed at 5H 8.91 (1H, d, J= 0.5). This fact, together with the difference in molecular formula between 71 and 72, suggested that the methyl group in 71 was substituted by a formyl group in 72. Key HMBC correlations between H-l' and C-6, and between H-5 and each of C-6, C-4, and C-8a, clearly established the location of this formyl group as N-15 and allowed the planar structure of 72 to be fully assigned [48]. Communesin E (73) had a molecular formula of C27H30N4O2, as
575
determined by HRFABMS and NMR data, suggesting that 73 was a demethyl compound of 70. The 'H-NMR spectra of 73 and 70 were nearly superimposable, but 73 lacked the signal for an iV-methyl observed in 70, indicating that 73 was an N-\5 demethyl derivative of 70. In addition, the presence of an acetyl group at N-16 and a 2-methyl-l,2-epoxypropyl moiety at C-ll was also suggested by the 'H-NMR data. Consequently, 73 was elucidated to be the A^5-demethylcomrnunesin A [48]. Communesin F (74) was found to have the molecular formula of C28H31N4O from the HRFABMS and NMR data. The ^ - N M R (Fig. (11)) and 13C-NMR spectra of 74 differed from those of 70 only by the absence of the epoxyl group and the appearance of a double bond, consistent with the difference in molecular formula between 74 and 70. The whole structure of 74, including the heptacyclic skeleton, an acetyl group at N-16 and a methyl group at N-15, was determined by a precise analysis of the ^ H COSY, HSQC, HMBC, and NOESY spectra of 74 [48]. The relative stereochemistry of 72, 73, and 74 was deduced to be the same as that of 70 and 71 at all chiral centers on the basis of the close similarity of the spectral parameters, especially the 13C-NMR chemical shifts, with the corresponding values for 70 and 71.
H
H
H r I
CHO
72, communesin D
73, communesin E
74, communesin F
^O
576
.ill 7.0
6.0
I i JIAL 5.0
4.0
3.0
PpTTT
2.0
Fig. (11). 400 MHz 'H-NMR spectrum of communesin F (74) in CDC13
The insecticidal activity of communesins A (70), B (71), D (72), E (73), and F (74) against third instar larvae of silkworms was examined by an oral administration. The LD50 values for 71 and 74 were 5 and 80 [ig/g of diet, respectively. Communesins A (70), D (72), and E (73) exhibited lower insecticidal activity than did 71 and 74, with the LD50 values for 70, 72, and 73 being 130, 130, and 200 ug/g of diet, respectively.
Biosynthetic Pathway of Communesins
May and coworkers proposed the plausible biosynthetic root to communesin A (70) shown in Fig. (12) (part 1) [50]. A quinone methide (75) derived from tryptamine and the related natural product, aurantioclavine (76), undergo a Diels-Alder reaction to form a polycyclic intermediate (77). This highly twisted lactam (77) should be easily cleaved by the residual primary amine to produce spiro lactam (78). Reduction of 78 and aminal closure afford a common intermediate (79) of communesins. Epoxidation and acylation of 79 afford 70. Expansion
577 H,N HOOC
Tryptamine
•N
R 76 R = H (aurantioclavine) Quinone methide R = Me imine
NH epoxidation al d N H ] . I acylation
79 Fig. (12). Plausible biosynthetic pathway to communesins (part 1) [50]
of this pathway suggests the pathway to communesins B (71), D (72), E (73), and F (74) (Fig. (12) part 2.). Acylation of 79 affords 74. On the other hand, epoxidation and acylation afford 71. Elimination of a methyl group at N-15 of 70 generates 73, while oxidation of a methyl group at N-15 in 71 generates 72. Communesin-Related Compounds
Communesins A (70) and B (71) were originally isolated from the mycelia of a strain of Penicillium sp. adhering to the marine alga, Enteromorpha intestinalis, and reported to exhibit cytotoxic activity in the P-388 lymphocytic leukemia test system in cell cultures [49]. The ED50 values for 70 and 71 are reported to be 3.5 and 0.45 u-g/ml, respectively, in the test system.
578
9t,. b NH
^
I
H
NH
^
epoxidation
H
^
acylation
79 acylation
Jdemethylation
74
Fig. (12). Plausible biosynthetic pathway to communesins (part 2)
Communesin-related compounds are shown in Fig. (13). Jadulco and coworkers quite recently isolated communesins B (71), C (80), and D (72) from the fungus Penicillium sp. derived from the Mediterranean sponge Axinella verrucosa [51]. These three communesins have been shown to exhibit moderate antiproliferative activity in several bioassays performed on different leukemia cell lines. Nomofungin (81), which had a pyran oxygen instead of an NH in 71, was isolated from the fermentation broth of an unidentified endophytic fungus obtained from the bark of Ficus microcarpa [52]. Later synthetic studies of nomofungin revealed that this compound was identical to 71 [53, 54]. Perophoramidine (82) was isolated from the Philippine ascidian Perophora namei [55]. Perophoramidine is a hexacyclic substructure of
579
80, communesin C
81, nomofungin
82, perophoramidine Fig. (13). Communesin-related compounds
the core heptacyclic ring system of communesins; it exhibits cytotoxicity toward the HCT116 colon carcinoma cell line and induces apoptosis via PARP cleavage. CONVULSIVE COMPOUNDS Verruculogen Identification of Verruculogen (83)
During the course of searching for okaramine-related compounds OH :
o OHM
H3CO
83, verruculogen
580
produced by strains belonging to P. simplicissimum, we observed interesting convulsive activity against silkworms in a strain of P. simplicissimum MF-24. The purification guided by the convulsive effect on silkworms led to the isolation of an active principle. The active principle, C27H33N3O7, was identified as verruculogen (83) [56]. Verruculogen (83) was originally isolated from the culture of P. verruculosum as an agent responsible for the tremor producer activity in mice or 1-day old cockerels [57]. Verruculogen (83) caused convulsive activity in the silkworms at a dose of 0.1 ug/g diet. Verruculogen-Related Compounds
H3CO
84, acetoxyverruculogen
85, fumitremorgin B o H3C0
H3CO
86, fumitremorgin A OH
=
o
OH 1
87, fumitremorgin C o
H-,CO
88, 12,13-dihydroxyfumitremorgin C Fig. (14). Verruculogen-related compounds
91, demethoxyfumitremorgin C
581
Verruculogen-related compounds are summarized in Fig. (14). In 1982, Uramoto and coworkers reported the isolation and structural elucidation of acetoxyverruculogen (84) from P. verruculosum as a tremorgenic metabolite [58]. In 1974, Yamazaki and coworkers reported the planar structure of fumitremorgin B (85) [59], which had been isolated as one of two toxins (fumitremorgins A and B) from Aspergillus fumigatus, growing on rice and miso (soybean paste) [60]. The structures of fuitremorgins A (86) and B (85) were determined in 1980 [61, 62]. These two compounds cause severe tremors and convulsion in experimental animals. Fumitremorgin C (87), the simplest member of the fumitremorgin family, was isolated from A. fumigatus by Cole and coworkers in 1977 [63]. Hermkens' group reported the total synthesis of 87 in 1988 [64], and Hino's group also reported the synthesis of 87 in 1989 [65]. Abraham and Argmann described the isolation of 12,13-dihydroxyfumitremorgin C (88) from A. fumigatus DSM 790 [66]. In 1995, Cui and coworkers reported the isolation of trypanostatins A (89) and B (90) from a marine fungal strain of A. fumigatus BM939 [67]. Trypano statins completely inhibit the cell-cycle progression of tsFT210 cells in the G2/M phase [68]. Cui and coworkers also isolated demethoxyfumitremorgin C (91), 83, 85, 87, and 88, showing the co-occurrence of these compounds, and 89 and 90 in the secondary metabolite of the strain BM939 [69, 70]. These findings suggested the OR 7
O
H 3 CO
89, tryprostatin A R = OCH3 90, tryprostatin B R = H
94, cyclotryprostatin C Fig. (15). Tryprostatin-related compounds
92, cyclotryprostatin A R = H 93, cyclotryprostatin B R = CH3 o o
95, cyclotryprostatin D
582
possible intermediacy of 91 in the biogenesis of the verruculogen and fumitremorgins. In 1997, Cui and coworkers also isolated cyclotryprostatins A (92), B (93), C (94), and D (95) as new inhibitors of the mammalian cell cycle from the same strain, A. fumigatus BM939 [71]. The structures of the tryprostatin family are shown in Fig. (15). Penitrem A and 6-Bromopenitrem E Identification of Penitrem A (96) and Discovery of 6 Bromopenitrem E (98)
As mentioned earlier, P. simplicissimum ATCC 90288 produced insecticidal okaramines. Moreover, this strain induced the same effect on the silkworms as verruculogen (83). The acetone extract obtained from the mycelia and media of this strain was concentrated and the aqueous residue was extracted with dichloromethane. The dichloromethane extract was partitioned between hexane and methanol, containing 10% water. The activity was found only in the lower layer. The active ethyl acetate extract obtained from the lower layer was chromatographed on silica gel with a hexane-ethyl acetate mixture. The 40-60% ethyl acetate eluates were rechromatographed on silica gel with a hexane-chloroform mixture. HPLC of the active 90-100% chloroform eluates on a Capcell pack Cig column, using 65.7% aqueous methanol with a flow rate of 1.0 ml/min, yielded two active compounds, AC 1 and AC 2. The convulsive principle AC 1, C37H44CINO6, was determined to be penitrem A (96) by means of the spectral data (MS, UV, IR, ! H-, and C-NMR) [72], which were indistinguishable from those reported previously for 96 [73]. The convulsive principle AC 2, C37H44BrNO6, showed spectroscopic characteristics quite similar to those of 96. The only difference between the two principles was that AC 2 had a bromine atom in the place of the chlorine atom of 96. The !H-NMR spectrum of AC 2 is shown in Fig. (16). In the 13C-NMR spectrum of 96, signals assignable to C-6 and C-7
OH Cl
H
96, penitrem A
H
98, 6-bromopenitrem E
583
UlUUu ppm •*—r-<—•
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
Fig. (16). 500 MHz 'H-NMR spectrum of 6-bromopenitrem E (98) in acetone-
were observed at 124.5 and 111.8 ppm, respectively. Penitrem E (97) [73], which had no chlorine atom at C-6, showed C-6 and C-7 signals at 120.3 and 111.6 ppm, respectively. AC 2 showed signals corresponding to those at 114.7 and 115.0 ppm. If a bromine atom was at C-7, signals of a quaternary carbon and a methine carbon should have been observed in a higher field than 111.6 ppm and in a lower field than 120.3 ppm, respectively. Therefore, it was deduced that a bromine atom was located at C-6 of AC 2, indicating that AC 2 is 6-bromopenitrem E (98), a new congener of the penitrems [74]. Penitrem A (96) and 6-bromopenitrem E (98) showed convulsive activity against silkworms at a dose of 0.3 ug/g diet. Penitrems and a Related Compound
In 1983, de Jesus and coworkers isolated tremorogenic mycotoxins named penitrems A (96), B (99), C (100), D (101), E (97), and F (102) from Penicillium cructosum [73, 75]. Penitrems and related compounds are shown in Fig. (17). Penitrems are indole alkaloids combined with
584
H H ~ R
H
H
99, penitrem B R = H 102, penitrem F R = C1
97, penitrem E
OH
c
H H " H
100, penitrem C R = C1 101, penitrem D R = H
103, pennigritrem
Fig. (17). Structures of penitrems and pennigritrem
terpenoid moieties. Penitrems A (96), C (100), and F (102) are chlorinated at a benzene ring, de Jesus and coworkers also investigated the biosynthesis of 96 both with 13C- and 2H-labeled precursors and showed that 96 is derived from tryptophan, geranylgeranylpyrophosphate, and two isopentenylpyrophosphate units [76]. In 1992, Penn and coworkers isolated an analog of 96, pennigritrem (103), from Penicillium nigricans [77]. Pennigritrem (103) is a fungal indole-diterpenoid, but 103 has an oxetane ring, like taxol [78] and cephalomannine [79], unique among the members of penitrem family. Brasiliamides Discovery of Brasiliamides A (104), B (105), C (106), D (107), andE (108)
The convulsive effect induced by penitrem A (96), 6-bromopenitrem E (98), or verruculogen (83) in silkworms suggested that this bioassay using silkworms may be valuable as an initial screening in the search for compounds that act on the nervous system. Further random screening using this bioassay resulted in the finding that the isolate Penicilium
585
brasilianum Batista JV-379 exhibited remarkable convulsive activity [80]. The strain P. brasilianum JV-379 was cultured with okara. After cultivation for two weeks, the cultured okara was soaked in methanol. The methanol extract was concentrated and the aqueous residue was extracted with hexane and ethyl acetate successively. The active ethyl acetate extract was chromatographed on silica gel with a solvent system (hexane-ethyl acetate-methanol). The 100% ethyl acetate and 5% methanol eluates were combined and chromatographed further to afford brasiliamides A (104) and C (106). The 70% ethyl acetate eluate was purified by column chromatography to afford brasiliamide B (105). The 10% methanol eluate was rechromatographed on silica gel and further flash-chromatographed on Chromatorex ODS to yield brasiliamides D (107) and E (108).
ppm i
i
i
'
10.0
'
i
i
i
i
9.0
8.0
7.0
6.0
5.0
4.0
Fig. (18). 270 MHz 'H-NMR spectrum of brasiliamide A (104) in CDC13
3.0
2.0
1.0
586
The molecular formula of brasiliamide A (104) was determined to be C24H26N2O6 from the HR-EIMS and NMR data, indicative of thirteen degrees of unsaturation. The IR spectrum revealed the presence of aromatic rings, a ketone carbonyl and amide groups. The 'H-NMR (Fig. (18)), C-NMR, and 2D NMR spectra indicated the presence of a phenyl group, a 3-methoxy-4,5-methylenedioxyphenyl group, two acetoamides, and a trisubstituted double bond. The presence of three isolated methylenes was also suggested from the NMR data. An olefinic proton and an amide proton were mutually coupled, indicative of the linkage between a trisubstituted double bond and NH in the acetamide group. The ketone carbonyl should form -CH2-CO-CH2- comprising two isolated methylenes. The remaining methylene protons were correlated to three non-oxygenated carbons in the 3-methoxy-4,5-methylenedioxyphenyl group, suggesting that 104 had a 3-methoxy4,5-methylenedioxybenzyl moiety. The methylene protons were also correlated to olefinic carbons, indicative of the linkage between the 3-methoxy-4,5-methylenedioxybenzyl group and a quaternary carbon in the >C=CH-NH-COCH3 moiety. The signals for an isolated methylene showed cross peaks with carbons in the phenyl group together with a ketone carbonyl, indicating the presence of a <|)-CH2-CO-CH2-. The geometry of the double bond (C-8=C-1') was confirmed as Z-configuration by the NOE correlation between an olefinic proton and aromatic protons (H-2 and H-6). Consequently, the structure of 104 was elucidated to be A^1,A^2-diacetyl-A/2-(2-oxo-3-phenylpropyl)-3-(3methoxy-4,5-methylenedioxyphenyl)-l,2-(Z)-propenediamine [80].
„ 104, brasiliamide A
Ao
r
3"
OCH3
105, brasiliamide B
Brasiliamide B (105) had a molecular formula of C24H26N2O5, indicative of thirteen degrees of unsaturation as in the case of brasiliamide A (104). In the 'H-NMR spectrum of 105, the signals were complicated, with almost all being doubled or broadened in CDCI3 at 20°C. This phenomenon was observed in various deuterated solvents, e.g., acetone-c/6, CeD6, CD3OD, and DMSO-^6- These observations suggested that conformational isomers of 105 were present in the solutions.
587
lAJ I
IAJAJUV^A'L^ ppm
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
Fig. (19). 270 MHz 'H-NMR spectrum of brasiliamide B (105) in acetone-rf6 at -60 °C
Lowering the temperature to -60°C in CDCI3 sharpened all signals, and some signals derived from minor conformers were observed as shown in Fig. (19). The alteration of an acetyl methyl region was particularly remarkable, and four pairs of acetyl methyl signals were observed in the spectrum. The ratio of these signals was approximately 76:15:7:2, suggesting that 105 existed in equilibrium with four conformational isomers in solution. We therefore tried to elucidate the structure of 105 with a major conformer in CDCI3. The !H-NMR, 13C-NMR, and 'H-'H COSY spectra revealed the presence of the same partial structures as in 104, a phenyl group, a 3-methoxy-4,5-methylenedioxyphenyl group, a trisubstituted double bond, and two acetoamides. The spectra also indicated the presence of a -CH2-CH-CH2- moiety. These partial structures were connected from the COLOC spectrum, and the whole structure of 105 was determined to be l,4-diacetyl-2-benzyl-5-(3-methoxy-4,5-methylenedioxybenzyl)-
588
Fig. (20). ORTEP drawing of dihydrobrasiliamide B (109)
be l,4-diacetyl-2-benzyl-5-(3-methoxy-4,5-methylenedioxybenzyl)1,2,3,4-tetrahydropyra-zine. This structure was completely supported by the following X-ray crystallographic data. Hydrogenation of 105 with 5% Pd/C afforded reductive products. The major product was purified by reversed-phase ODS column chromatography to give a dihydroderivative, C24H28N2O5. Crystallization of dihydrobrasiliamide B (109) from methanol yielded rhombic prisms that were suitable for an X-ray crystallographic analysis. The ORTEP drawing of 109 shown in Fig. (20) established the complete structure of 109 as frvms-l,4-diacetyl2-benzyl-5 -(3 -methoxy-4,5 -methylenedioxybenzyl)piperazine [80]. Brasiliamide C (106), C74H26N2O5, had the same molecular formula as brasiliamide B (105). The TH-NMR, l3C-NMR, and ' H - ' H COSY spectra indicated the presence of a 3-methoxy-4,5-methylenedioxyphenyl group, a phenyl group, two acetamide groups, a trisubstituted double bond, an isolated methylene and a -CH2-CH-CH2- linkage. The HMBC spectrum indicated that the phenyl group was bound to the double bond and that the 3-methoxy-4,5-methylenedioxyphenyl group was located at C-l". The geometry of the double bond in the benzylidene moiety was determined to be an is-configuration, based on the NOE correlation between the acetyl methyl (8-CH3) and the olefinic proton (H-T) [81]. The molecular formula of brasiliamide D (107) was determined to be C24H28N2O5 from the HR-EIMS and NMR data, indicative of twelve degrees of unsaturation. Precise analysis of the NMR data revealed that 107 had the partial structures of a benzyl group, a 3-methoxy-4,5methylenedioxybenzyl group, two acetoamides, and a disubstituted piperazine ring. These partial structures were connected from the HMBC spectrum. The planar structure was determined to be the same as that of dihydrobrasiliamide B (109), but the spectral data for 107 were completely different from those of 109. The piperazine ring conformation of 107 was not a chair-form but rather a twist boat-form conformation based on the coupling constants between the methine and methylene protons (J2,3a = 6.4 Hz, J2,ib = 11.3 Hz and J5)6a = 6.4 Hz, J5;6b =11.3 Hz) in the piperazine ring, while that of 109 was a typical chair-form conformation. These observations suggested that both methine protons
589
(H-2 and H-5) were pseudo-axial in configuration and that the relative configuration of these protons was 2,5-cis [81]. The molecular formula of brasiliamide E (108) was determined to be C22H26N2O4. The13C-NMR spectrum closely resembled to that of 107, except for the absence of one acetyl group, strongly suggesting that 108 was a deacetyl derivative of 107. The position of an acetyl group was inferred from the HMBC spectrum to be at N-l [81].
0CH3
0CH3
109, dihydroxybrasiliamide B (2,5-trans)
106, brasiliamide C
0CH3
107, brasiliamide D (2,5-cis)
108, brasiliamide E (2,5-cis)
Conformational Analysis of Brasiliamides
Xxxx x \xx E l form Ratio
42%
Z l form V.
Z2 form J 35%
E2 form 23%
Fig. (21). Rotaitional properties of (ra«.s-l,4-diacetyl-2,5-dimethylpiperazine (112)
590 brasiliamide B (105) (at -60 °C in CHC13)
V
V N
F
"Y Ti
LNAR A15%o
A" 76%
R
X
7%
dihydroxybrasiliamide B (109) (at room temperature in CHCI3)
Ph'
35%
14%
18%
brasiliamide C (106) (at -15 °C in CHCI3)
v Ph—
v
N
°r
^
2%
64%
R
1%
33o/o
brasiliamide D (107) (at -15 °C in CHCI3)
V 51%
Y 10%
V
32%
Fig. (22). Rotaitional properties of brasiliamides (R = 3-methoxy-4J5-methylenedioxyben2yl)
Brasiliamides B (105), C (106), D (107), and E (108) showed conformational change due to the restricted rotation of amide bonds in a solution, and four or two sets of conformers appeared in the NMR spectra. To clarify their conformational properties, the conformational change
591
was first examined using a model compound, trans-1,4diacetyl-2,5-dimethylpiperazine (110). Three sets of signals due to each rotamer were completely assigned, and the direction of the acetamide group on each rotamer was determined from differential NOE measurements. In these experiments, the chemical shifts of the ring protons were markedly changed due to the direction of the carbonyl group in acetamides. When the carbonyl group in the acetamide (N-l) is directed to the C-2 side, the neighboring methine proton (H-2) is deshielded. On the other hand, when the carbonyl group is directed to the C-6 side, the equatorial proton of methylene (H2-6) is deshielded. The ratio of three rotamers, El, Zl (Z2) and E2, was evaluated to be approximately 42:35:23, respectively, on the basis of integral values of the ring methyls (Fig. 21). In the case of 105, 106, 107, 108, and 109, the same method was used to analyze conformational changes. The results are shown in Fig. (22). Bioactivity of Brasiliamides
The convulsive activity of brasiliamides against the third instar larvae of silkworm was also examined. The activity of brasiliamides A (104) and B (105) was evaluated according to the ED50 values of 300 and 50 p.g/g diet, respectively, upon oral administration. The functional moiety for the activity was expected to be a 3-methoxy4,5-methylenedioxybenzyl moiety, since natural compounds having a 3-methoxy4,5methylenedioxybenzyl group, such as myristicin [82], have been reported as insecticides. Dihydrobrasiliamide B (109), brasiliamide C (106) and brasiliamide D (107) showed weaker activity than brasiliamide B (105), indicating that a double bond in the piperazine ring was more significant than a 3-methoxy-4,5-methylenedioxybenzyl group. Brasiliamide E (108), which is equivalent to deacetoxybrasiliamide D, showed no activity, suggesting that the acetyl group is essential to exhibit the activity. Biosynthesis of Brasiliamides
Brasiliamides are comprised of two phenylpropane moieties and acetates. The plausible biosynthetic pathway of brasiliamides starting with the formation of diketopiperazine ring is outlined in Fig. (23). Phenylalanine and 3-methoxy-4,5-methylenedioxyphenylalanine are combined into a diketopiperazine (111). Reduction of ketones, dehydration reaction, and rearrangement of the double bond lead to intermediates 112 and 113. Acetylation of the two compounds 112 and 113 leads to brasiliamide C (106) and brasiliamide B (105), respectively. The two compounds 112
592
and 113 give an intermediate (114) after reduction of each double bond. Acetylation of 114 leads to brasiliamide E (108) and brasiliamide D (107) successively. On the other hand, oxidative cleavage of the tetrahydropiperazine ring in 105 affords 104.
Y PrV
106
o
104
PIT
107 Fig. (23). Proposed biosynthetic pathway to brasiliamides (R = 3-methoxy-4,5-methyIenedioxyphenyl) The configuration shown is relative one.
593 Brasiliamide-Related Compounds
Piperazine-containing compounds are shown in Fig. (24). In 1969, Caesar and coworkers reported the isolation of nigragillin (115) from Aspergillus phoenicis [83]. Piperazinomycin (116) was isolated as an antifungal antibiotic from the cultured broth of Streptoverticillium olivoreticuli subsp. neoenacticus by Tamai and coworkers in 1982 [84, 85]. Piperazinomycin (116) has a unique cyclic structure in which two benzene rings and one piperazine ring are linked together by three
116, piperazinomycin o
118, nigerazine B
117, nigerazine A
OH
119, dragmacidon
120, dragmacidon A
H N
(I 11
121, dragmacidon B Fig. (24) Compounds with piperazine ring
122,2,5-bis(6'-bromo-3'-indolyl)piperazine
594
separate atoms (one oxygen atom and two carbon atoms). Iwamoto and coworkers isolated nigerazines A (117) and B (118) as new metabolites positive to Dragendorff's reagent from Aspergillus niger 1-639 [86, 87]. Nigerazines reduce the root elongation of lettuce seedlings. A number of cytotoxic bis-indole alkaloids have been discovered in marine organisms. Dragmacidon (119) was isolated from deep water Caribbean sponge, Dragmacidon sp., by Kohmoto and coworkers in 1988 [88]. Dragmacidons A (120) and B (121) were isolated from the Pacific Ocean sponge Hexadella sp., collected off the coast of British Columbia by Morris and coworkers in 1990 [89]. Dragmacidon A (120) shows cytotoxic activity, while 121 shows no activity. In 1991 Fahy and coworkers isolated 2,5-bis(6'-bromo-3'-indolyl)piperazine (122) from the encrusting grey tunicate Didemnum candidum collected in the southern Gulf of California [90]. PARALYTIC COMPOUNDS Asperparalines Discovery of Asperparalines A (123), B (124), and C (125)
The hint, which led us to this finding, was the accidental observation of paralytic syndrome in silkworms that had digested an extract of a certain isolate. We had been screening numerous soil isolates for their insecticidal activity against silkworms upon oral administration in the usual manner. It was of great interest that the strain Aspergillus japonicus Saito JV-23 resulted in paralysis in silkworms. The isolate A. japonicus JV-23 was also cultured on okara as in other experiments mentioned earlier. The fermented okara together with mycelia was soaked in methanol. The methanol extract was extracted with dichloromethane after removal of methanol. The dichloromethane extract was purified by solvent partition, column chromatography, and crystallization, to finally yield three active compounds, asperparalines A (123), B (124), and C (125) [91, 92]. Asperparaline A (123) was shown to have the molecular formula of C20H29N3O3 by HR-EIMS together with *H-NMR and 13C-NMR spectra, indicative of eight degrees of unsaturation. Resonances at 171.9, 175.3, and 181.6 ppm in the C-NMR spectrum of 123 indicated the presence of three carbonyls, one of which had to be an amide carbonyl, while two other carbons in the five-membered ring were imido carbonyls based on the absorption bands at 1773 and 1698 cm'1 in the IR spectrum, revealing 123 to be pentacyclic. The 'H-NMR spectrum shown in Fig. (25) confirmed the presence of five methyl groups (two jV-methyls, two
595
JUL_ 3.0
2.5
iI
JuJ 2.0
1.5
JL JL. 1.0
Fig. (25). 270 MHz 'H-NMR spectrum of asperparaline A (123) in acetone-rf6
tertiary methyls, and a secondary methyl), three isolated methylenes, a -CH 2 -CH< linkage, and a -CH2-CH2-CHCH3- linkage. For the connectivity of partial structures, HMBC experiments were carried out. Consequently, the planar structure of 123 was determined. Furthermore, the conformation of the structure of 123 was obtained by the application of X-ray crystallographic analysis. The ORTEP drawing of 123 is shown in Fig. (26) [91, 92]. The spectral features of asperparaline B (124), C19H27N3O3, were quite similar to those of asperparaline A (123). In the H-NMR spectrum of 124, one of two iV-methyl groups in 123 disappeared, and a signal assignable to NH was newly observed, suggesting that 124 lacked either 22-CH3 or 23-CH3. In the HMBC spectrum of 124, an TV-methyl signal at
1
\v^.-
o.
o
123, asperparaline A Fig. (26). ORTEP drawing of asperparaline A (123)
596
8H 3.15 was correlated with signals of C-7 and C-14, confirming that 124 was iV20-deinethylasperparaline A [92]. Another analog, asperparaline C (125), had the molecular formula of C19H27N3O3, which was the same as that of asperparaline B (124). In the 'H-NMR spectrum of 125, a doublet methyl signal assigned to 3-CH3 in asperparaline A (123) disappeared, and a -CH2-CH2-CH2- linkage was observed, indicating that 125 was C3-demethylasperparaline A. This assumption was supported by the HMBC and NOESY experiments [92]. The structures of asperparalines A (123), B (124), and C (125) were determined to be spiro compounds made up of an A^methyl succinimide and a cyclopent[f]indolizine having an Af-methyl amide bridge. Another structural characteristic of asperparalines is a bicyclo[2,2,2]diazaoctane core, and various compounds, such as those of the paraherquamide family mentioned in the following section, contain the same core in their structures. However, all these compounds have an indole moiety in their structures- so, it is of great interest that asperparalines have no indole part in their structures.
o /\ 124, asperparaline B
o
125, asperparaline C
Bioactivity of Asperparalines
The biological activities of asperparalines against several insects were also examined. Asperparaline A (123) induced paralysis in silkworms at a dose of 10 |ig/g diet within 1 h of oral administration, and the paralysis lasted for 7 to 10 h. When injected with a microsyringe, 123 induced paralysis at a dose of 3 (j,g/g body weight within 20 min, and the paralysis lasted for 4 to 5 h. Asperparalines B (124) and C (125) exhibited almost the same effect on silkworms. Asperparaline A (123) showed remarkable insecticidal activity against third instar larvae of Nilaparvata lugens, third instar larvae of Nephotettix cincticeps, and the adults of Musca domestica. Biosynthetic Study
The paraherquamide family and other related compounds cited in the
597
following section have a bicyclo[2,2,2]diazaoctane core. Biosynthetic studies on these compounds support the notion that this structural motif is formed by a biosynthetic intramolecular [4+2] cycloaddition of the isoprene-derived olefin across a preformed azadiene moiety derived from an oxidized piperazine-dione as shown in Fig. (27). In 2003, Williams and coworkers performed feeding experiments on A. japonicus JV-23 to determine the primary amino acid building blocks that comprise asperparaline A (123) [93]. Incorporation of acetate, L-methionine,
PH
x
V
Fig. (27). Formation of bicyclo[2,2,2]diazaoctane core
H
O
OPP
III!
OH + NH 2
L-Isoleucine
i
r*
L-Tryptophan
• .v
oxidation
127
126
H
dimethylallyl pyrophosphate
H
methylation
* "H
128 Fig. (28). Possible biosynthetic pathway to asperparaline A (123) [93]
123
598
L-isoleucine, and L-tryptophan was observed, suggesting that 123 likely shares a common biosynthetic pathway with the paraherquamides as shown in Fig. (28). Prenylation of the cyc/o-L-tryptophanL-P-methylproline and intramolecular [4+2] cyclization (via 126) would provide the putative bicyclo[2,2,2] core (127). Williams and coworkers have already demonstrated that 127 serves as a biosynthetic precursor to paraherquamide A (146) shown in Fig. (33). Oxidation of 127 leads to the catechol derivative (128). Oxidative cleavage of four carbon atoms from the oxygenated aromatic ring in 128 could furnish the spirosuccinimide ring of 123. Synthetic Study
The first synthesis of the model compound of asperparaline A (123) was reported in 1999 by Williams and coworkers [94], as shown in Fig. (29). They developed a novel synthetic approach to a 3-spirosuccinimide system from a 2,3-disubstituted pyrrole. The synthesis was begun with the commercially available 3,3,5,5-tetramethylcyclohexanone (129), which was transformed into oxime (130). The pyrrole (131) was obtained from 130 through a Trofimov reaction. The pyrrole (131) was then iV-methylated with iodomethane to afford JV-methylpyrrole (132). The iV-methylpyrrole (132) was oxidized through a photooxygenation reaction using Rose Bengal as a photosensitizer under UV light irradiation to afford hydroxypyrrolidone (133). Treatment of 133 with NOH c)
132
133
134
a) H2NOH-HC1 b) C2H2 c) Mel d) O2, hv, Rose Bengal e) NaH, A Fig. (29). Williams' model study on the spirosuccinimide ring system of asperparaline A (123) [94]
599 CN
={
a)
b)
c)
CN
136
137 O O
138
N
139
140
a) CH2(CN)2, piperidine, PhCOOH b) NaCN, AcOH c) HBr d) AcCl e) MeNH2 Fig. (30). Tanimori's approach to the spirosuccinimide ring system of asperparaline A (123) [95]
sodium hydride in DMSO at 180°C furnished the desired spirosuccinimide (134). In 2000, Tanimori and coworkers also reported a method for synthesizing the spirosuccinimide moiety of asperparaline A (123) [95], as shown in Fig. (30). 2,2-Dimethylcyclopentanone (135) was treated with malononitrile in the presence of piperidine and benzoic acid to
C
NH a)
-NH
'COOH
COOH
b)
•NH2CI COOMe
c)
d)
•N
COOMe
141 Co 2 (CO) 6 e) COOMe
COOMe
142
a) ref. [97] b) SOC12, MeOH c) propargyl bromide, Lil d) Co2(CO)8, Ar e) 48% aq. MeNH2, MeNH-HCl Fig. (31). Tanimori's approach to asperparaline C (125) [96]
600
afford unsaturated dinitrile (136). Michael addition of the cyanide anion to 136 proceeded smoothly to provide trinitrile (137). Acid-catalyzed hydrolysis of 137 was accompanied by decarboxylation to give acid (138). The acid (138) was converted into anhydride, a crude product reacted with methylamine to afford the desired spirocyclic N-methylsuccinimide (140). In 2001, Tanimori and coworkers described a Pauson-Khand cyclization reaction to construct the tetracyclic indolidine core of asperparaline C (125) [96], as shown in Fig. (31). The starting enyne (141) was synthesized from L-proline by the standard procedure [97]. The [2+2+1] cycloaddition of 141 with Co(CO)s gave tricyclic indolidinone (142) as a single diastereomer. Condensation of 142 with methylamine resulted in conjugate addition of the amine to the enone moiety followed by ring closure to provide bridged tetracyclic lactam (143). Although some model compounds have been synthesized, total synthesis of asperparalines has not been reported. Asperparaline-Related Compounds The unique bicyclo [2,2,2] diazaoctane ring system constitutes one of the structural characteristics of brevianamides (Fig. (32)). Brevianamide A (144) was originally isolated from cultures of Penicillium brevicompactum by Birch and Wright in 1969 [98]. Brevianamide A (144) was also isolated from cultures of Penicillium vindication by Wilson and coworkers in 1973 [99]. Bird and coworkers observed that 144 is formed only after conidiation has begun in solid cultures of P. brevicompactum [100]. Birch and Russell isolated brevianamide B (145)
o /\ H 144, (+)-brevianamide A
145, (+)-brevianamide B
o H
ent-145, (-)-brevianamide B Fig. (32). Structures of brevianamides
601
from the culture of P. brevicompactum and reported that 145 is a stereoisomer of 144 [101]. In 1987 and 1990, Paterson and coworkers reported that 144 is a potent antifeedant against pests [102, 103]. The structure and absolute configuration of 144 was determined by Coetzer in 1974 through X-ray crystallography on a semisynthetic derivative, 5-bromobrevianamide A [104]. Williams and co workers achieved the first total synthesis of 145 in 1988 [105] and also reported that P. brevicompactum constructs 144 and 145 in optically pure form and that the natural 145 and semi-synthetic one (ent-\45) derived from 144 are of the opposite absolute configuration [106]. The paraherquamide family is another group of compounds containing a bicyclo[2,2,2]diazaoctane ring system; the structures of the members are shown in Fig. (33). Paraherquamide A (146) was isolated as a toxic metabolite from Penicillium paraherquei by Yamazaki and coworkers [107, 108]. In 1990, Ondeyka and coworkers reported the structural determination and antihelmintic activity of paraherquamides B (147), C (148), D (149), E (150), F (151), and G (152) isolated from the fermentation of Penicillium charlesii [109]. Paraherquamides A (146), E (150), F (151), and G (152) were also isolated from Penicillium sp. by Blanchflower and coworkers in 1991 [110]. The new paraherquamide congeners VM 55595 (153), VM 55596 (154), VM 55597 (155), and VM 55599 (156) were isolated from Penicillium sp. IMI 331995 by
o-
146, paraherquamide A R = OH 150, paraherquamide E R = H (VM54159) o.
o-
147, paraherquamide B
o-
148, paraherquamide C Fig. (33). Structures of paraherquamide family (part 1)
149, paraherquamide D
602
O'
151, paraherquamide F R = H (VM55594) 152, paraherquamide G R = OH (VM54158)
153, VM55595
o
OH
o154, VM55596
O-
155, VM55597 M HQHO
156, VM55599 157, sclerotiamide Fig. (33). Structures of paraherquamide family(part 2)
Blachflower and coworkers in 1993 [111]. Sclerotiamide (157), shown in Fig. (33), was isolated from the sclerotia of Aspergillus sclerotiorum NRRL 5167 by Whyte and Gloer in 1996 [112]. Sclerotiamide (157) causes significant mortality and unusual physiological effects against the corn earworm Helicoverpa zea. In 1997, Banks and coworkers isolated antihelmintic metabolites from Aspergillus sp. IMI 337664, and described structures of aspergillimide (VM5598) identical to asperparaline A (123), 16-keto aspergillimide (SB202327) (158) and the paraherquamides VM54159 (159), SB203105 (160), and SB200437 (161). The structures of these compounds are shown in Fig. (34) [113]. SB203105 (160) is the first example of a 4-substituted paraherquamide.
603
o-
158, 16-keto aspergillimide (SB202327) o.
160, SB203105
159,VM54159
161, SB200437
Fig. (34). Structuers of 16-keto asperlillimide and paraherquamides
CJ-17,665 (162) was isolated from the fermentation broth of Aspergillus ochraceus CL41582 by Sugie and coworkers in 2001 [114]. CJ-17,665 (162) inhibits the growth of multi-drug resistant Staphylococcus aureus, Streptomyces pyogenes, and Enterococcus faecalis. In 2002, Qian-Cutrone and coworkers isolated stephacidins A (163) and B (164) from A. ochraceus WC76466 [115]. Stephacidins A
o A
H
163, stephacidin A Fig. (35). Structures of CJ-17,665 and stephacidins
164, stephacidin B
604
(163) and B (164) show in vitro cytotoxic activity against various antitumor cell lines, but 164 exhibits more potent and selective antitumor activities, especially against testosterone-dependent prostate cancer cell line, LNCaP. The structures of 162,163, and 164 are shown in Fig. (35). Marcfortines A (165), B (166), and C (167) were isolated from the mycelium of Penicillium roqueforti by Polonsky and coworkers in 1980 (Fig. (36)) [116, 117]. Marcfortines contain a piperidine ring instead of a pyrrolidine ring in paraherquamides and sclerotiamide. Biogenetically, the basic skeleton of 165 is clearly derived from a dioxopiperazine formed from tryptophan and pipecolic acid. In 1999, Kuo and coworkers elucidated the biosynthetic pathway of the pipecolic acid moiety of 165 [118]. In 2002, Williams reviewed studies on total synthesis and biosynthesis of the paraherquamide family, with a focus on the biological Diels-Alder construction of the bicyclo[2,2,2]diazaoctane ring system [119].
165, marcfortine B R = C H 3 166, marcfortine B R = H
167, marcfortine C
Fig. (36). Structuers of marcfortines
ACKNOWLEDGEMENTS The studies reported in this review were conducted at Osaka Prefecture University, Sakai, Japan. I am deeply indebted to all my coworkers, who are cited in the references. REFERENCES [1] Vandamme, E.J.; J. Biotechnol. 1994, 37, 89-108. [2] Tamura, S.; Takahashi, N.; Miyamoto, S.; Mori, R.; Suzuki, S.; Nagatsu, J.; Agric. Biol. Chem. 1963, 27, 576-582. [3] Takahashi, N.; Suzuki, A.; Tamura, S.; Agric. Biol. Chem. 1966, 30, 1-12.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
611
CHEMISTRY AND BIOLOGICAL ACTIVITIES OF NATURALLY OCCURRING PHTHALIDES GE LIN1, SUNNY SUN-KIN CHAN1, HOI-SING CHUNG1, SONG-LIN LI2 'Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P. R. China, institute of Nanjing Military Command for Drug Control, No. 293, Zhongshan Eastern Road, Nanjing 210002, P. R. China ABSTRACT: Phthalides are widely present in plants, fungi and liverworts. Many phthalide-containing plants have been used worldwide as herbal remedies in traditional and folk medicines, dietary supplements and food flavorings. In this review, the chemical structures, classifications, and plant sources of about 137 natural phthalides of plant origins are described. The recent advances in the biological activities of these pthalides, such as actions on the central nervous system, anti-platelet aggregation and anti-thrombosis, cardiac function modulation and anti-angina, inhibition of smooth muscle cell proliferation, protection against cerebral ischemia, and smooth muscle relaxation, are also discussed. INTRODUCTION To date, most known natural phthalide compounds have been identified from plants. Phthalides are also found in fungi, bacteria and liverworts. More than 180 naturally occurring phthalide derivatives have been identified, among them about 137 phthalides isolated from a wide variety of plant species. Most of the naturally occurring phthalides have been reported to be biologically active with a wide range of pharmacological activities, including actions on the central nervous system, anti-angina, anti-platelet aggregation, anti-smooth muscle proliferation, anti-thrombosis, cardiac function modulation, and protection against cerebral ischemia. Since ancient times, phthalide-containing plants have been used worldwide as herbal remedies in traditional and/or folk medicines, dietary supplements, and food flavorings. For example, the leafstalks and fruit of Apium graveolens have a long history of use as healthy food and food flavorings in both Western and Eastern countries [1]. In traditional Chinese medicinal (TCM) practice, phthalide-containing herbs are recognized as some of the most commonly used natural medicines. Two of the best known and most commonly used phthalide-containing TCM herbs, Rhizoma Chuanxiong (Chinese name Chuanxiong) and Radix Angelicae Sinensis (Chinese name Danggui), have been used for the treatment of cerebro- and cardio-vascular diseases and female irregular menstruation for more than two thousand years [2, 3]. In Japan, the roots of Angelica acutiloba (Japanese name Toki) and the rhizomes of Cnidium officinale (Japanese name
612 Senkyu) have also been used as herbal medicines, which have similar therapeutic uses to that of Danggui and Chuanxiong, respectively [4, 5]. In addition to the general use of bioactive phthalides in their natural mixture forms, recently a few isolated phthalides have come under development as pure agents for the treatment of cerebro- and cardiovascular diseases [6, 7]. In this article, we review the chemical structures and sources of the currently known naturally occurring phthalides of plant. Recent advances related to the biological activities of the extensively investigated natural phthalides are also described. As examples, we focus on the phthalide ingredients and medical uses of three commonly used phthalide-containing TCM herbs.
PLANT Sources, chemical structures and classifications of natural phthalides Chemical Structure of Phthalide The basic core structure of phthalide is l(3H)-isobenzofuranone, which contains a benzene ring (ring A) fused with a y-lactone (ring B) between carbon atoms 1 and 3 (Fig. (1)). To date, all known natural phthalide compounds have been identified as derivatives of l(3H)-isobenzofuranone. The structures of these derivatives either have the core structure substituted with one or more groups at different positions or contain a reduced form with one, two or no double bond(s) in ring A and various substitutions at different positions. The detailed structures of the naturally occurring phthalide derivatives identified from plants are discussed in the Classifications of Natural Phthalides Section below.
Fig. (1). Chemical Structure of Phthalide (l(3H)-isobenzofuranone)
613 Plant Sources of Natural Phthalides So far, about 137 natural phthalides have been isolated from more than 202 plant species. These species belong to 23 families, namely Amaryllidaceae, Apocynaceae, Aristolochiaceae, Asteraceae (Compositae), Berberidaceae, Bignoniaceae, Fabaceae, Gentianaceae, Gramineae, Lamiaceae, Loganiaceae, Malpighiaceae, Mysinaceae, Orchidaceae, Papaeraceae, Plantaginaceae, Poaceae, Polygonaceae, Ranunculaceae, Rhamnaceae, Rosaceae, Rutaceae and Umbelliferae (Apiaceae). Most of the identified bioactive natural phthalides, except the phthalide isoquinoline type (see the following sections), are obtained from two genera Ligusticum and Angelica in the Umbelliferae family. From the genus of Ligusticum, more than 53 phthalides have been isolated from 12 species, namely L. acuminatum, L. acutilobum, L. chuanxiong, L. jeholense, L. jeholense var. tenuisectum, L. mutellina, L. officinale, L. porteri, L. sinense, L. sinense c.v. chaxiong, L. tenuissimum and L. wallichii. Most of these species are utilized as herbal medicines [1, 8-12]. For example, the TCM herb Chuanxiong, one of the most commonly used Chinese herbal medicines for the treatment of cerebro- and cardiovascular diseases, is derived from the rhizome of L. chuanxiong. On the other hand, 38 phthalide derivatives have been isolated from 9 species of Angelica, the second phthalide-enriched genus in the Umbelliferae family. These 9 species include A. acutiloba, A. acutiloba var. sugiyamae, A. carmichaeli, A. dahuricae, A. glauca, A. pubescentis, A. sinensis, A. tenuissima and A. teruata. The well-known TCM herb Danggui used for the treatment of female irregular menstruation is derived from the root of A. sinensis. Furthermore, various phthalides have been identified from several species of the Apium genus {Umbelliferae), and Apium graveolens is often consumed as healthy food worldwide [1], Details of the natural sources of phthalides identified from plants are summarized in Table 1.
Table 1. Plant Sources of Naturally Occurring Phthalides No.
Compound Type/Name1
CA registered No.2
Plant Species
Refs.
3-SUBSTITUTED PHTHALIDE TYPE
Non-alkaloid phthalide 1
Angeloylsenkyunolide F
112899-64-6
Angelica acutiloba
[5]
2
3-Butyl-5,6-dihydro1 (4H)-isobenzofuranone
141120-37-8
Pelroselinum crispum, P. crispum var. tuberosum
[13, 14]
3*
3-Butyl-4,7dihydroxyphthalide
444018-09-1
Ligusticum chuanxiong
[15]
4*
3Butylhexahydrophthalide
3553-34-2
Tragopogon porrifolius
[16]
614 5
3«,3a«,7a«-3Butylhexahydrophthalide
193742-12-0
Apium graveolens, A. graveolens var. rnpaceum; Foeniculum vulgare
[IV, 18]
6
3R,3aR,7aS-3Butylhexahydrophthalide
189504-31-2
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
7
3R,3aS,7aR-3Butylhexahydrophthalide
6431-21-6 or
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
3R,3aSJaS-3Butylhexahydrophthalide
6431-22-7 or
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
9
3S,3aR,7aR-3Butylhexahydrophthalide
153546-51-1
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
10
3S,3aRJaS-3Butylhexahydrophthalide
114883-66-8
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
11
3S,3aS,7a«-3Butylhexahydrophthalide
2550-47-2 or
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
12
3S,3aS,7aS-3Butylhexahydrophthalide
153546-49-7
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[IV, 18]
13*
3-Butyl-4hydroxyphthalide (Chuanxingol)
74459-23-7 or
Ligusticum chuanxiong, L. wallichii
[19]
14
35-Butyl-4hydroxyphthalide
74459-24-8
Ligusticum chuanxiong, L. wallichii
[9,19]
15
3Z-Butylidene-4,5dihydroxyphthalide
91652-79-8
Ligusticum wallichii
[20]
16*
3-Butylidene-4hydroxyphthalide
146946-20-5
Cnidium officinale; chuanxiong, L. wallichi
17
3Z-Butylidene-4hydroxyphthalide
102673-72-3
Meum alhamanlicum
18*
3-Butylidene-5hydroxyphthalide
146946-21-6
Cnidium officinale; chuanxiong
Ligusticum
[21]
19*
3-Butylidene-7hydroxyphthalide
103659-69-4
Angelica wallichii
Ligusticum
[25, 26]
20*
Butylidenephthalide
551-08-6
Angelica glauca; Bupleurum Chinese; Cnidium officinale; Conioselinum kamtschaticum, C. tataricum; Geum montanum; Levisticum officinale; Ligusticum acuminatum, L. acutilobum, L. porteri, L. sinense, L. sinense c. v. chaxiong, L. tenuissimum, L. wallichii; Oenanthe javanica; Perilla frutescens; Pituranthos tortuosus; Scutellaria baicalensis; Seseli indicum
[1, 12, 20, 2740]
76681-73-7
Angelica acutiloba, A. acutiloba var. sugiyamae, A. carmichaeli; Apium graveolens; Cnidium officinale;
[14, 41-47]
8
193742-11-9
193742-13-1
124600-37-9
87421-30-5
(3-Butylidenephthalide)
21
£-Butylidenephthalide
sinensis;
Ligusticum
[2123] [24]
615 Glaucosciadium cordifolium; Levisticum officinale; Ligusticum chuanxiong, L. mutellina; Lomatium lorreyi; Meum athamanticum; Petroselinum crispum. P. crispum var. tuberosum 22
Z-Butylidenephthalide
72917-31-8
Angelica sinensis, A. tenuissima; Apium graveolens, A. graveolens var. dulce, A. graveolens var. rapaceum; Cnidium officinale; Glaucosciadium cordifolium; Levisticum officinale; Lomatium torreyi; Meum athamanticum; Opopanax chironium; Petroselinum crispum; Pituranthos scoparius; Trachyspermum roxburghianum
[13, 42-44, 46-54]
23*
3-Butylidene-4,5,6,7tetrahydro-6«,7Sdihydroxyphthalide
153609-96-2
Cnidium officinale; chuanxiong
Ligusticum
[21]
24*
3-Butylidene-4,5,6,7tetrahydro-65',75'dihydroxyphthalide
153609-97-3
Cnidium officinale; chuanxiong
Ligusticum
[21]
25*
3£-Butylidene-4,5,6,7tetrahydro-6,7dihydroxyphthalide
210045-94-6
Angelica sinensis; chuanxiong
Ligusticum
[25, 55]
26
3£-Butylidene-4,5,6,7tetrahydro-6«,7Sdihydroxyphthalide
162426-23-5
Polygonum multiflorum
[56]
27
3£-Butylidene-4,5,6,7tetrahydro-65,75dihydroxyphthalide
162426-22-4
Polygonum multiflorum
[56]
28*
3-Butylidene-4,5,6,7tetrahydro-7^?-hydroxy6R-( 1 -oxobutyl)phthalide
146986-61-0
Cnidium officinale; chuanxiong, L. wallichii
Ligusticum
[21, 23]
29*
3-Butylidene-4,5,6,7tetrahydro-77?-hydroxy-65(1 -oxobuty1)phthalide
146986-60-9
Cnidium officinale; chuanxiong, L. wallichii
Ligusticum
[21, 23]
30*
Butylphthalide
6066-49-5
Anelhum graveolens, A. sowa; Angelica sinensis, A. tenuissima; Apium graveolens, A. graveolens var. dulce; Asarum canadense; Cenolophium denudatum; Chrysanthemum carinatum; Cnidium officinale; Daucus carota; Geum montanum; Levisticum officinale; Ligusticum aculilobum, L. chuanxiong, L. sinense, L. sinense c. v. chaxiong, L. wallichii; Meum athamanticum; Opopanax chironium; Petroselinum crispum, P. crispum var. tuberosum; Scutellaria baicalensis; Trifolium prafense, T. repens
(3-Butylphthalide)
[1, 9, 14, 25, 31,32, 34, 39, 47-49, 52, 5766]
616 31
3«-Butylphthalide
125412-70-6
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[67]
3413-15-8
Apium graveolens, A. graveolens var. rapaceum; Foeniculum vulgare
[67]
[21]
((+)-Butylphthalide) 32
35-Butylphthalide ((-)-Butylphthaiide)
33
35-Buty1-4,5,6,7tetrahydro-6/?,7Kdihydroxyphthalide
153609-99-5
Cnidium officinale; chuanxiong
34*
3-Butyl-4,5,6,7-tetrahydro6-hydroxy-7methoxyphthalide (NG072)
147816-36-2
Apium graveolens
[68]
35
3/?-Butyl-3aS',4,5,6tetrahydrophthalide
2550-44-9 or
Petroselinum crispum, P. crispum var. tuberosum; Tragopogon porrifolius
[13, 16]
36
351-Butyl-4,5,6«,75'tetrahydro-3,6,7trihydroxyphthalide
103238-08-0
Ligusticum chuanxiong
[69]
37*
Catalpalactone
1585-68-8
Catalpa bignonioides, C. ovata
[70]
38*
Celephthalide A
638207-88-2
Apium graveolens
[71] [71]
114923-85-2
Ligusticum
39*
Celephthalide B
638207-89-3
Apium graveolens
40
Celephthalide C
654083-69-9
Apium graveolens
[71]
41
Cnidilide
3674-03-1 or
Angelica glauca; Apium graveolens var. dulce, A. graveolens var. rapaceum; Cnidium officinale; Conioselinum tataricum; Ligusticum chuanxiong, L. jeholense, L. jeholense var. tenuisectum, L. sinense, L. tenuissimum; Opopanax chironium; Peucedanum ostruthium; Scutellaria baicalensis
[9, 12, 27, 30, 35, 39, 50, 52, 72-74]
178602-60-3
42*
6,7-Epoxyligustilide
106533-40-8
Ligusticum wallichii
[75]
43*
Z-6,7-Epoxyligustilide
210036-10-5
Angelica sinensis
[25]
44*
(6-O-P-DGlucopyranosyl)pedicelloside
110906-82-6
Genliana pedicellata
[761
45
Isocnidilide
3553-29-5 or
(Z-Neocnidilide)
124815-25-4
Anethum graveolens; Apium graveolens var. dulce, A. graveolens var. rapaceum; Petroselinum crispum, P. crispum var. tuberosum; Tragopogon porrifolius
[13, 16, 50, 57]
46
isosenkyunolide
82467-94-5
Levisticum officinale
[77]
47*
Ligustilide
4431-01-0
Angelica acutiloba, A. dahuricae
[1, 28, 29, 3537, 39, 78-83]
Angelica leruata; Bupleurum Chinese; Conioselinum kamtschaticum; Ligusticum acutilobum, L. jeholense.
617 L. jeholense var. tenuisectum, L. officinale, L. tenuissimum; Lomatium californicum; Oenanthe javanica; Perilln frutescens; Pituranthos tortuosus; Pleurospermum hookeri; Scutellaria baicalensis 48
£-Ligustilide
81944-08-3
Angelica glauca, A. sinensis; Apium graveolens, A. graveolens var. dulce, A. graveolens var. rapaceum; Cenolophium denudatum; Cnidium officinale; Geum montanum; Levisticum officinale; Ligusticum chuanxiong, L. mutellina, L. porteri; Lomatium torreyi; Petroselinum crispum; P. crispum var. tuberosum; Trachyspermum roxburghianum
[14, 25, 31, 33, 42, 45, 46, 50, 54, 55, 61, 84, 85]
49
Z-Ligustilide
81944-09-4
Anethum graveolens, A. sowa; Angelica glauca, A. sinensis, A. tenuissima; Apium graveolens, A. graveolens var. dulce, A. graveolens var. rapaceum; A. graveolens var. secalinum; Bifora lesticulata; Capnophyllum peregrinum; Cenolophium denudatum; Cnidium officinale; Coriandrum sativnum; Geum montanum; Glaucosciadium cordifolium; Helieita longifoliata; Levisticum officinale; Ligusticum chuanxiong, L. mutellina, L. porteri, L. wallichii; Lomatium torreyi; Meum athamanticum; Opopanax chironium; Petroselinum crispum, P. crispum var. tuberosum; Peucedanum ostruthium; Silaum silaus; Todaroa montana; Trachyspermum roxburghianum
[10, 14, 31, 42, 43, 45-47, 49, 50, 52, 54, 55, 58, 61, 65, 74, 8491]
50
£-Ligustilidiol
162426-22-4
Polygonum multiflorum
[56]
(3£-Butylidene-4,5,6,7tetrahydro-65,75'dihydroxyphthalide) 51
Neocnidilide
4567-33-3
Anethum graveolens, A. sowa; Angelica sinensis; Apium graveolens, A graveolens var. rapaceum; Bifora testiculata; Cenolophium denudatum; Cnidium officinale; Coriandrum sativnum; Libanotis laticalycina; Ligusticum chuanxiong, L. sinense, L. sinense c.v. chaxiong, L. tenuissimum; Peucedanum divaricata; Saposhnikovia divaricata; Scutellaria baicalensis; Seseli yunnanenese; Silaum silaus; Todaroa montana
[1, 9, 12,25, 34, 35, 39, 58, 61, 86, 89, 92, 93]
52*
Pedicelloside
94617-34-2
Genliana pedicellata
[94]
53*
Pedicellosine
110941-52-1
Gentiana pedicellata
[76]
618 54*
Pedigiucoside
55*
Pedirutinoside
56*
(-)-Sedanenolide (3-Butyl-4,5dihydrophthalide)
166733-97-7
Sedanolide
3861-62-9 or
57*
124183-39-7
Gentiana pyrenaica
[95]
104669-04-7
Gentiana pedicellata
[96]
62006-39-7 or
Angelica glauca, A. sinensis; Apium graveolens; Ligusticum chuanxiong, L. wallichii; Meum athamanticum; Petroselinum crispum, P. crispum var. tuberosum, P. sativum
[14, 25, 27, 65, 97100]
Apium graveolens var. secalinum; Asarum canadense; Aucus carota; Levisticum officinale; Petroselinum sativum
[44, 60, 64, 88, 100]
63038-10-8
Anethum graveolens, A. sowa; Angelica sinensis, A. tenuissima; Apium graveolens, A. graveolens var. rapaceum; Bifora testiculata; Cnidium officinale; Levisticum officinale; Ligusticum chuanxiong, L. sinense c.v. chaxiong, L. wallichii; Opopanax chironium; Petroselinum crispum var. tuberosum; Peucedanum osiruthium
[3, 20, 49, 52, 58, 74, 77, 86, 89, 101KB]
93236-67-0
Angelica sinensis; Cnidium officinale; Ligusticum chuanxiong, L. wallichii; Meum athamanticum
[4, 9, 20, 24, 25]
91652-78-7
Angelica sinensis; Cnidium officinale; Ligusticum wallichii;
[20, 24, 25, 104]
6415-59-4
58
Senkyunolide A (Senkyunolide)
59
Senkyunolide B (3Z-Butylidene-7hydroxyphthalide)
60
Senkyunolide C (3Z-Butylidene-5hydroxyphthalide)
Meum athamanticum
61*
Senkyunolide D
94530-82-2
Angelica sinensis; Cnidium officinale; Ligusticum wallichii
[20, 25, 104]
62*
Senkyunolide E
94530-83-3 or
(3Z-Butylidene-2hydroxyphthalide)
102734-66-7
Angelica acutiloba, A. sinensis; Cnidium officinale; Ligusticum chuanxiong. L. wallichii; Meum athamanticum
[4, 5, 20, 21, 24, 25]
£-Senkyunolide E
153546-50-0
Ligusticum chuanxiong
[9]
Senkyunolide F
94530-84-4 or
(3Z-Butylidene-4,5dihydro-2hydroxyphthalide)
102848-87-3
Angelica acutiloba, A. sinensis; Cnidium officinale; Ligusticum chuanxiong, L. wallichii; Meum athamanticum
[4, 5, 9, 20, 24,25]
Senkyunolide G
94530-85-5
Angelica sinensis; Cnidium officinale; Ligusticum sinense c.v. chaxiong, L. wallichii
[20, 25, 103, 104]
93379-53-4 or
Angelica acutiloba; Cnidium officinale; Ligusticum chuanxiong, L.
[4, 5, 9, 20,
63*
(3£-Buty1idene-2hydroxyphthalide) 64*
65*
(3-Butyl-4,5-dihydro-3hydroxyphthalide) 66
Senkyunolide H
619
67
(3Z-Butylidene-4,5,6,7tetrahydro-e/fJS1dihydroxyphthalide)
94596-27-7
sinense c. v. chaxiong, L. wallichii
103]
Senkyunolide I
88551-87-5 or
Angelica acutiloba, A. sinensis
(Z-Ligustilidiol)
94596-28-8
Cnidium officinale; Ligusticum chuanxiong, L. sinense c.v. chaxiong, L. wallichii; Meum athamanticum
[5, 9, 25, 99, 103, 105, 106]
94530-86-6
Apium graveolens; Cnidium officinale; Ligusticum chuanxiong, L. wallichii
[9, 20, 104, 107]
114569-33-4
Angelica wallichii
Ligusticum
[20, 25]
114569-34-5
Angelica acutiloba; chuanxiong, L. wallichii
Ligusticum
[20, 108, 109]
140694-58-2
Apium graveolens; chuanxiong
Ligusticum
[9, 107]
142235-81-2
Ligusticum chuanxiong
[109]
172549-37-0
Ligusticum chuanxiong
[110]
172723-28-3
Ligusticum chuanxiong
[110]
124183-38-6
Gentiana pyrenaica
[95]
(3Z-Butylidene-4,5,6,7tetrahydro-6S',7S'dihydroxyphthalide) 68
Senkyunolide J ((-)-Senkyunolide J) (3«-Butyl-4,5,6,7tetrahydro-65,75dihydroxyphthalide)
69
Senkyunolide K. ((-)-Senkyunolide K)
sinensis;
(3S-Butyl-4,5-dihydro-4«hydroxyphthalide) 70
Senkyunolide M (3Z-Butylidene-4,5,6,7tetrahydro-75'-hydroxy-6/?(1 -oxobutyl)phthalide)
71
Senkyunolide N (35'-Butyl-4,5,6,7tetrahydro-65',75'dihydroxyphthalide)
72
Senkyunolide Q (3Z-Butylidene-4,5,6,7tetrahydro-7/?-hydroxy6R-( 1 -oxobutyl)phthalide)
73
Senkyunolide R (3Z-Butylidene-4,5,6,7tetrahydro-2«,65',75trihydroxyphthalide)
74
Senkyunolide S (3Z-Butylidetie-4,5,6,7tetrahydro-2/?,6«,7#trihydroxyphthalide)
75*
6'-Vanilloylpediglucoside Alkaloid phthalide
76
Aobamidine
59614-38-9
Corydalis lutea, C. ochotensis var. raddeana; Fumaria macrosepala
[111113]
77*
Coryrutine
104736-02-9
Corydalis rutifolia subsp. kurdica
[114]
620 78
/V-Methylhydrastine
55922-35-5
Corydalis rutifolia subsp. kurdica; Fumaria densiflora, F. gaillardotii
[114116]
79
Microcarpine
93552-71-7
Fumaria microcarpa
[117]
80
Narlumicine
73710-85-7
Fumaria indica
[118]
81*
Narlumidine
73710-86-8
Fumaria indica
[118]
82
Pierardin
24282-25-5
Dendrobium pierardii
[119]
83*
Shihunine
4031-12-3 or
Banisteriopsis caapi; Behria tenuiflora; Dendrobium loddigesii. D. lohohense, D. pierardii
[120123]
34413-11-1 NON-3-SUBSTITUTED PHTHALIDE TYPE 84
Anaphatol
76382-73-5
Anaphalis contorta
[124]
85
Araneophthalide
109028-44-6
Anaphalis araneosa
[125]
86
Aranochromanophthalide
109028-47-9
Anaphalis araneosa
[125]
87
Arenophthalide A
57429-87-5
Helichrysum arenarium
[126]
88
5,7-Dihydroxyphthalide
27979-58-4
Anaphalis contorta; Helichrysum arenarium; Polygonum cuspidatum
[124, 127, 128]
89
4,6-Dimethoxy-5hydroxyphthalide
61052-37-7
Ligusticum wallichi
[23]
90
5,7-Dimethoxyphthalide
3465-69-8
Anaphalis contorta; Helichrysum arenarium, H. italicum;
[124, 127, 129]
91
Djalonensin
62512-04-3
Antholeista djalonensis
[130]
92
4-Hydroxyphthalide
13161-32-5
Avenafatua, A saliva
[131]
93
5-Methoxy-7hydroxyphthalide
24953-77-3
Anaphalis contorta; Helichrysum arenarium, H. italicum, H. plicatum, H. polyphyllum; Myrsine Africana; Rhamnus procumbens; R. serrata, R. virgata, R. wighlii
[124, 129, 132139]
94
Phthalidochromene
109028-43-5
Anaphalis araneosa
[125]
95
Platypterophthalide
109028-45-7
Helichrysum platypterum
[125]
PHTHALIDE TYPE
DIMER
96*
Angelicide
92935-94-9
Angelica sinensis
[25]
97
Angelicolide
90826-58-7
Angelica glauca
[140]
98
Angeolide
81957-73-5
Angelica glauca; Levisticum officinale
[84, 91]
99*
8-Butyl-l-butylidene3a,4,5,5a,6,7,8,10b-
117034-08-9
Ligusticum porteri
[10]
621 octariydro-lH-5,10cethanonaptho[l,2-c:7,8c']difuran-3,10-dione 100*
3-Butyl3,4,4\5,5',5a,6,6',7',7adecahydro-6-propylspiro[cyclo[e]isobenzofura
117005-25-1
Ligusticum porteri
[10]
117005-26-2
Ligusticum porteri
[10]
128718-46-7
Angelica sinensis
[2]
isobenzofuran]-l,3'-dione 101*
3-Butyl-3,4,5,5a,6,7ahexahydro-6-propylspiro[cyclo[e]isobenzofura isobenzofuran]-l ,3'-dione
102
3-Butylidene-6',7'dihydro-4-propylspiro[3H-3a,6etlianoisobenzofuran5(4H),1'(3'H)isobenzofuran]l,3'(6H)dione (E232)
103*
3-Butylidene-3,4,5,6,6',7'hexahydro-6-propylspiro[cyclobut[e]isobenzof uran-7(lH),l'(3'H)isobenzofuran]-l,3'-dione
144379-43-1
Bupleurum sibiricum
[141]
104
1£,8£-Dibutylidene55,5a«,6,7,8,10b«hexahydro-1H-5,1 OcSethanonaphtho[l ,2-c:7,8c']difuran-3,10-dione
189576-99-6
Angelica sinensis
[142]
105
l£,8Z-Dibutylidene-
189576-97-4
Angelica sinensis
[142]
hexahydro-lH-5,10c5ethanonaphtho[l,2-c:7,8c']difuran-3,10-dione 106
lZ,8Z-Dibuty1idene55,5a/?,6,7,8,10b/?hexahydro-lH-5,10c5'ethanonaphtho[l ,2-c:7,8c']difuran-3,10-dione
189576-98-5
Angelica sinensis
[142]
107
3'ff,8'-Diliydrodiligustilide
117005-27-3
Ligusticum chuanxiong, L. porteri
[10, 98]
108*
Z'-3,8-Dihydro-6.6',7.3a'diligustilide
Ligusticum porteri, L. sinense c.v. chaxiong, L. wallichii
[10, 11,75]
205870-37-7 106533-38-4 or 117065-81-3
109*
Z,Z'-3.3',8.8'-Diligustilide
210045-96-8
Angelica sinensis
[25]
110*
Z.Z'-6.8',7.3'-Diligustilide
210045-95-7
Angelica sinensis
[25]
622 111
Levistolide A
88182-33-6 or
(Diligustilide)
205673-83-2
(Z,Z'-Diligustilide)
Angelica acutiloba, A. sinensis; Bupleurum sibiricum; Conioselinum vaginatum; Levisticum officinale; Ligusticum chuanxiong, L. porteri, L. sinense c. v. chaxiong, L. wallichii
[5, 10, 11, 25, 91, 141, 143145] [91]
112
Levistolide B
89708-24-7
Levisticum officinale
113
Riligustilide
89354-45-0 or
Angelica sinensis; chuanxiong, L. porteri
189576-96-3
Ligusticum
L. wallichii
[10, 25, 75, 144]
114
Senkyunolide 0
142797-35-1
Ligusticum chuanxiong
115
Senkyunolide P
142864-23-1
Cnidium officinale; chuanxiong
116*
Tokinolide A
112899-62-4
Angelica acutiloba
[5]
117
Tokinolide B
112966-16-2
Angelica acutiloba; Cnidium officinale; Ligusticum chuanxiong
[5, 144, 146]
118
Wallichilide
93236-64-7
Ligusticum wallichii
[8]
550-49-2
Corydalis caucasica, C. decumbens, C. gigantean, C. gortschakovii, C. hsuchowensis, C. incise, C. lutea, C. nobilis, C. ochotensis, C. ochotensis var. raddeana, C. ochroleuca, C. paniculigera, C. ramose, C. remota, C. rosea, C. sibirica, C. stewariii, C. stricta, C. thalictrifolia, C. thyrsiflora, C. vaginans; Fumaria bracteosa, F. indica, F. judaica, F. macrosepala, F. parviflora, F. vaillantii; Glaucium flavum
[112, 113, 147168]
524-46-9
Adlumia fungosa; Corydalis gigantean, C. lineariodes, C. marschalliana, C. paniculigera, C. remota, C. rosea. C. sempervirens, C. stricta, C. vaginans; Fumaria agrarian, F. bastardii, F. bellam, F. capreolata, F. densiflora, F. faurei, F. macrocarpa, F. macrosepala, F. officinalis subsp. officinalis, F. parviflora, F. petteri subsp. calcarata, F. rostellata, F. sepium, F. vaillantii
[149, 157, 160, 167, 169176]
Bicuculline
485-49-4 or
((+)-Bicuculline)
56083-00-2
Adlumia fungosa; Corydalis aurea, bastardii, C. bulbosa, C. bungeana, caucasica, C. crystalline, decumbens. C. densiflora, gigantea, C. gortschakovii, govaniana, C. hsuchowensis,
[113, 116, 147, 149, 151, 157,
[144] Ligusticum
[144, 146]
PHTHALIDE ISOQUINOLINE TYPE 119
Adlumidine ((+)-Adlumidine) (rf-Adlumidine)
120
Adlumine ((+)-Adlumine) (rf-Adlumine)
121
(rf-Bicuculline) (Bucuculline)
C. C. C. C. C. C.
623
122
humosa, C. majori, C. mucronifera, C. nobilis, C. ochroleuca, C. omeiensis, C. paniculigera, C. plalycarpa, C. pseudoadunca, C. remota, C. repens, C. rosea, C. rutifolia, C. semenovii, C. sempervirens. C. solida, C. stricta, C. suaveolens, C. taliensis, C. thyrsiflorn, C. vaginans; Dactylicapnos torulosa, D. cucullaria. D. peregrine, D. spectabilis; Fumaria asepala, F. bella, F. bmcteosa, F capreolata, F. densiflora, F. gaillardotii, F. indica, F. judaica, F. macrocarpa, F. macrosepala, F. muralis, F. parviflora, F. schleicheri, F. vaillantii
160, 162, 163, 167, 171, 173, 174, 177209]
19730-80-4
Corydalis decumbens, C. esquirolii, C. ramose, C. sewerzowi
[158, 210212]
Capnoidine
485-50-7 or
((-)-Capnoidine)
64397-08-6
Corydalis bulbosa, C. cava, crystalline, C. decumbens, densiflora, C. gigantea,
[149, 156, 170, 180, 182, 185, 188, 200, 202, 203, 207, 210, 213216]
(-)-Bicuculline (/-Bicuculline)
123
(/-Capnoidine)
C gortschakovii, C. majori, C. marschalliana, C. remota, C. rosea, C. sempervirens, C. tuberose, C. vaginans; Dactylicapnos torulosa; Fumaria asepala, F. capreolata, F. incise, F. muralis, F. vaillantii
((-)-Adlumidine) (/-Adlumidine)
124
Carlumine
C. C.
485-51-8
Corydalis decumbens, C. esquirolii, C. govaniana, C. meifolia, C. nobilis, C. omeiensis, C ramose, C. scouleri. C. sewerzowi, C. thyrsiflora
[158, 162, 186, 189, 191, 211, 212, 217219]
58031-32-6
Corydalis ledebouriana;
[220222]
((+)-Carlumine) (Corlumine) ((+)-Corlumine)
125
Corledine
Fumaria parviflora; F. vaillantii
((-)-Corledine) 126
Corlumidine
25344-54-1
Corydalis decumbens, C. lineariodes, C. scouleri; Fumaria parviflora
[160, 183, 221, 223]
((+)-Corlumidine)
127*
Decumbenine
76733-83-0
Corydalis decumbens
[183]
128
5'-0-Demethylnarcotine
152503-33-8
Papaver somniferum
[224]
129
(+)-a-Hydrastine
53950-51-9
Corydalis rutifolia, C. solida; Fumaria bracteosa, F. densiflora, F.
[163, 197, 203,
(rf-a-Hydrastine)
624 schleicheri, F. vaillantii
216, 225 226]
Corydalis stricta; Dactylicapnos torulosa; Fumaria parviflora; Hydrastis canadensis; Stylomecon heterophylla
[200, 221, 227229]
Corydalis caucasica, pseudoadunca, C. Dactylicapnos torulosa
C. stricta;
[147, 193, 200, 230]
118-08-1
Berberis Inurina, B. vulgaris; Corydalis stricta; Fumaria bastardii. F. indica, F. parviflora, F. vaillantii; Hydrastis canadensis
[179, 208, 221, 231235]
Hypecoumine
100163-16-4 or
(Decumbenine C)
117772-89-1
Corydalis decumbens; leptocarpum
[236, 237]
134
(+)-A'-Methylnarcotine
51606-51-0
Papaver cylindricum
[238]
135
Narcotine
128-62-1
Fumaria parviflora; Papaver armeniacum, P. cylindricum, P. ecaisnei, P. fugax, P. oreophilum, P. paeoniflorum, P. pericarpium, P. rhoeas, P. rhopalothece, P. setigerum, P. somniferum, P. auricola, P. triniifolium; Plantago arenaria; Rauwolfia heterophylla
[221, 239252]
521-40-4
Papaver somniferum
[253]
125263-86-7
Fumaria indica
[254]
((+)-Stylophylline)
130
(-)-a-Hydrastine
4370-85-8 or
(7-a-Hydrastine)
60827-73-8
(Stylophylline) 131
(+)-(5-Hydrastine
29617-43-4 60594-55-0
or
((+)-Hydrastine) (rf-Hydrastine) (Isocoryne) 132
(-)-P-Hydrastine ((-)-Hydrastine) (/-Hydrastine)
133*
(Noscapine) ((-)-Narcotine) (a-Narcotine)
136
Narcotoline
Hypecoum
((-)-Narcotoline) (Desmethylnarcotine) 137
Papraine ((+)-Papraine)
'Other name(s) are indicated in the parentheses. 2
In case more than one CA registered number has been given to the same phthalide, all numbers are listed.
*The stereochemistry is unknown.
Classification of Natural Phthalides In this review, the natural phthalides of plant origin are classified into four types based on chemical structure (Fig. (2-6)): 1) 3-substituted phthalides; 2) non-3-substituted phthalides; 3) phthalide dimers; and 4) phthalide isoquinolines. The 3-substituted phthalide derivatives are further classified into two subtypes, namely non-alkaloid phthalides (Fig. (2)) and alkaloid phthalides (Fig. (3)). Phthalide alkaloids without the isoquinoline structure are always 3-substituted and are accordingly subclassified as the
625 alkaloid phthalide subtype (Fig. (3)). Phthalide compounds without any 3-substitutions belong to the non-3-substituted phthalide type (Fig. (4)). Phthalide dimers have two phthalide moieties joined (Fig. (5)), while compounds containing both phthalide and isoquinoline moieties are classified as phthalide isoquinolines (Fig. (6)). Furthermore, several natural compounds, which are claimed to be phthalide isoquinoline derivatives in various review articles published yearly in Natural Product Reports [255-274], are not included in the phthalide isoquinoline type in this review because these natural products no longer have the intact phthalide and/or isoquinoline core structure. Nevertheless, they contain structures related to phthalide and/or isoquinoline, such as a reduced carbonyl group in the phthalide y-lactone (ring B) and/or an opened ring in the isoquinoline moiety. The detailed structures, natural sources, and biological activities of this group of phthalide isoquinoline related compounds have been reviewed previously [255-274]. Various phthalides are either stereochemical isomers or enantiomers, and their stereochemical structures are clearly illustrated in Figs. (2-6). Those with unresolved stereochemical structures are summarized in Table 1 with an indication of unknown stereochemistry, and their structures without the confirmation of stereochemistry are also shown in Figs. (2-6). The details of the classifications and chemical structures of four types of natural phthalides are illustrated in Figs. (2-6). Most of the isolated natural phthalides belong to the 3-substituted phthalide type, which accounts for about 61% of the total known naturally occurring phthalides, and of these, non-alkaloid phthalides constitute the most important subtype due not only to their abundance in nature (75 compounds identified) but also their extensively reported pharmacological activities. The pharmacological activities of individual phthalides are discussed in the following Biological Activity Section.
626
10
13 OH
CH(CH2)2CH3
16
18
v^J
LZ9
628
CH2OH
629
O
OH
630
Fig. (2). Structures of phthalides of the non-alkaloid phthalide subtype
80
82
Fig. (3). Structures of phthalides of the alkaloid phthalide subtype
83
631
Fig. (4). Structures of phthalides of the non-3-substituted phthalide type
632 CH(CH2)2CH3
CH(CH2)2CH3
102
633
in
634
115
0
116
118 Fig. (5). Structures of phthalides of the phthalide dimer type
117
635
125
\
/
126
636
133
134
637
136
137 Fig. (6). Structures of phthalides of the phthalide isoquinoline type
Biological Activities of Natural Phthalides With the growing interest in natural products and herbal remedies globally, the awareness of biological activities of naturally occurring phthalides has also increased. Most pharmacological studies have been conducted using isolated pure phthalide derivatives even though most of the clinically used natural phthalides are prescribed as mixed herbal extractions and/or raw herbal materials. Early work was focused mainly on the phthalide isoquinolines. Among 19 naturally occurring phthalide isoquinolines, the pharmacological actions of bicuculline (121), (+)-hydrastine (131) and narcotine (135) have received much attention. Bicuculline is a well-defined y-aminobutyric acid A (GABAA) antagonist and has been extensively utilized as a pharmacological tool to delineate the properties of the GABAA receptors [275-277]. (+)-Hydrastine is another GABAA antagonist and has been reported to be more potent than bicuculline [278]. Narcotine is a well-known clinically used anti-tussive agent. Its mechanism of action is poorly understood but is thought to involve cr-opioid receptor antagonism [279]. Other potential therapeutic uses of narcotine have been reported recently, including the treatment of stroke [280] and cancer [281]. Several excellent reviews on the pharmacological properties of bicuculline and (+)-hydrastine are available [282-284], and the pharmacological effects of phthalide isoquinolines have been reviewed yearly since 1984 [255-274], thus will not be described in detail here. With the growing interest in TCM research, most recent studies focus on the phthalides of TCM origin, such as butylidenephthalide (20) and butylphthalide (30). A
638 wide range of pharmacological activities of these natural phthalides has been reported, ranging from local inhibition of smooth muscle contraction to central anticonvulsant action (Table 2). In general, the biological activities of phthalides can be classified into six main categories: actions on the central nervous system, inhibition of platelet aggregation and anti-thrombosis, cardiac function modulation and anti-angina, inhibition of smooth muscle cell proliferation, protection against cerebral ischemia, and smooth muscle relaxation. The details of each of these biological activities are described in the following sections.
Table 2. Summary of the Biological Activities of Naturally Occurring Phthalides of Plant Origin Biological Activities
Phthalide
Anti-angina
Butylidenephthalide (20), Z-Butylidenephthalide (22)
Anti-convulsion
Anti-platelet aggregation and anti-thrombosis
Sedanenolide (56), 3«-Butylphthalide (31), 35Butylphthalide (32) Butylidenephthalide (20), 3#-Butylphthalide (31), 3SButylphthalide (32), Z-Ligustilide (49), Senkyunolide A (58)
Blood viscosity reduction
Butylphthalide (30), Cnidilide (41), Senkyunolide A (58), Senkyunolide P (115), Levistolide B (117)
Cardiac function modulation
Butylidenephthalide (20), Butylidenephthalide (22), Ligustilide (47), Senkyunolide A (58)
Inhibition of learning and memory impairment
Butylidenephthalide (20), Butylphthalide (30), 3RButylphthalide (31), 35-Butylphthalide (32) Butylidenephthalide (20), Butylphthalide (30),
Inhibition of smooth muscle cell proliferation
Protection against cerebral ischemia
Sedation and sleep enhancement
Smooth muscle relaxation
Cnidiline (41), Ligustilide (47), Senkyunolide A (58), Senkyunolide H (66), Senkyunolide I (67), Senkyunolide .1 (68) Butylphthalide (30), 37?-Butylphthalide (31), 3SButylphthalide (32) Butylidenephthalide (20), Butylphthalide (30), Ligustilide (47), Sedanenolide (56) Butylidenephthalide (20), Z-Butylidenephthalide (22), Butylphthalide (30), Levistolide A (111), Ligustilide (47), Riligustilide (113), Senkyunolide P (115), TokinolideB(117)
639 Actions on the Central Nervous System Several phthalides have been shown to affect the central nervous system. Matsumoto and colleagues used a pentobarbital-induced animal sleep model to test butylidenephthalide (20) [285]. In their experimental design, mice were either housed together or individually for 4 weeks prior to pentobarbital treatment. Comparing the control mice in these two groups, the pentobarbital-induced sleep time was significantly shorter in the individually housed mice. However, after a single intraperitoneal administration of phthalide 20 at a dose of 20 mg/kg to the individually housed mice, the sleep time in this group became similar to that in the group-housed control animals, and this effect was dose-dependent at the dose range from 10 to 30 mg/kg [285]. Furthermore, in the group-housed mice, pentobarbital-induced sleep time was significantly reduced after treatment with methoxamine (an a r adrenoceptor agonist), yohimbine (an <x2-adrenoceptor agonist) and FG7142 (a GABAA inverse agonist). Phthalide 20 at an intraperitoneal dose of 20 mg/kg was able to reverse the reduced sleep time caused by all these three agents. The results suggested that the effects of phthalide 20 on pentobarbital-induced sleep in mice might involve central adrenergic and/or GABAA systems [285]. In an avoidance performance rat model [286], avoidance impairment was induced by intracisternal administration of AF64A (a central cholinergic neurotoxin), scopolamine (a muscarinic receptor antagonist) or mecamylamine (a nicotinic receptor antagonist), and the combination of scopolamine and mecamylamine exhibited a synergistic effect. Phthalide 20 at an intraperitoneal dose of 50 mg/kg markedly reversed avoidance impairment induced by these three antagonists used separately or concurrently with scopolamine and mecamylamine. The inhibitory effect of 20 on scopolamine-induced avoidance impairment was not blocked by scopolamine methylbromide, a peripheral muscarinic receptor antagonist. Therefore, the authors suggested that the action of 20 on the impairment of avoidance performance was related to the central cholinergic pathway via muscarinic and/or nicotinic receptors [286]. However, further investigations are warranted to elucidate these mechanisms. A single intraperitoneal administration of butylphthalide (30) (50-100 mg/kg) prolonged pentobarbital-induced sleep time in male Albino mice [287]. A similar effect was also observed after inhalation of 30 at 0.5-lmg/3L air/min [288-290]. Furthermore, in a rat chronic seizure model induced by coriaria lactone [291-295], significant damage was found in the cerebral neurons and cerebellar Purkinje cells in control animals after seizure induction, but not in rats orally pretreated with 700 mg/kg of phthalide 30. In addition, compound 30 appeared to antagonize learning and memory impairment in the same animal model. Intraperitoneal injection of 35-butylphthalide (32) protected both mice and rats against seizure development induced by audio- and electro-shock, and convulsive agents such as metrazole or coriaria lactone [292-295]. However, controversial results were obtained in a separate study reported by Dong and co-workers [296]. In their murine model, subcutaneous administration the /?-enantiomer 31 and its racemic mixture
640 moderately inhibited metrazole-induced seizure, but not the S-enantiomer 32. Furthermore, both R- (31) and 5-enantiomers (32) as well as their racemic mixture inhibited electro-shock seizure in a dose-dependent manner with ED50 values of 83.4, 104.8 and 73.1 mg/kg, respectively [296]. Using a similar electro-shock seizure model, Guo [297] examined structure-activity relationships by testing 31 and fourteen other structurally related synthetic derivatives. The results revealed that the anti-convulsive potency of the 3-substituted phthalides, all containing an aromatic ring A, depends upon various factors, including the configuration of 3-substitution, the lipophilicity, and the electron density at the 6 position in the aromatic ring. Phthalides having an R configuration at position 3 are significantly more potent than those with an S configuration, and the more lipophilic compounds exhibit greater potency. Moreover, the electron withdrawing substituent at position 6 seems to enhance anti-convulsive activity. In this study, only a limited number of phthalides, all having an aromatic ring A, was examined, however. Further studies on different types of phthalides are required for the elucidation of a complete structure-anti-convulsive activity relationship of this group of compounds. In a pentobarbital-induced sleep experiment conducted by Matsumoto and colleagues [285], the reduction of pentobarbital-induced sleep time in individually housed mice (as compared to that in group-housed mice) was significantly attenuated in a dose-dependent manner after single intraperitoneal doses of ligustilide (47) ranging from 5 to 20 mg/kg. Compound 47 (20 mg/kg i.p.) also attenuated yohimbine, methoxamine or FG7142mediated pentobarbital-induced sleep time reduction in group-housed mice. These results suggested that the central effects of 47 might also involve adrenergic and/or GABAA pathways [285]. In male Albino mice, intraperitoneal pretreatment with 50 mg/kg of sedanenolide (56) significantly prolonged pentobarbital-induced sleep time. On the other hand, when 56 was administered immediately after recovery from pentobarbital-induced sleep, the animals fell asleep again. However, 56 did not affect ethanol-induced sedation in mice [287]. Phthalide 56 was also reported to be effective against seizure in different animal seizure models [292]. In an electro-shock seizure test, a single intraperitoneal dose of compound 56 at higher than 100 mg/kg in rats or 150 mg/kg in mice prevented seizure [292]. After a 250 mg/kg intraperitoneal dose of 56 over 30-min, the induction threshold for seizure was significantly elevated, and the percentage of rats developing seizures after subcutaneous pretreatment of metrazol decreased significantly from 97% (control) to 20% [292]. Furthermore, intraperitoneal administration of 250 mg/kg of 56 significantly protected rats against audiogenic seizure, and at 30 min after dosing, none of the five animals seizured, while all five rats in the control group developed seizure [292].
641 Anti-Platelet Aggregation and Anti-Thrombosis Various isolated natural phthalides have been investigated for their anti-platelet aggregation and anti-thrombosis activities. As summarized in Table 2, five 3-substituted phthalides, namely butylidenephthalide (20), 3/?-butylphthalide (31), 35-butylphthalide (32), Z-ligustilide (49) and senkyunolide A (58), were found to be effective against platelet aggregation. Teng et al. [298] have investigated the effects of phthalide 20 on platelet aggregation caused by various inducers. In washed rabbit platelets, phthalide 20 dose-dependently inhibited arachidonic acid- (AA) and collagen-induced aggregation. The inhibitory potency was significantly higher in AA-induced aggregation (IC50 70 uM) than collagen-induced aggregation (IC50 120 (J.M). Moreover, phthalide 20 inhibited ATP release caused by both AA and collagen, and its inhibitory potency on ATP release was greater (AA: IC50 40 uM, collagen: IC50 60 uM) than that against aggregation. Compound 20 also significantly inhibited platelet aggregation induced by platelet-activating factor (PAF) and ADP, but not that induced by thrombin or ionophore A23187. In addition, compound 20 markedly inhibited thromboxane B 2 (TXB2) formation caused by collagen, AA, thrombin, and ionophore A23187. In a study using human platelet-rich plasma (PRP), phthalide 20 at a concentration of 100 uM abolished the secondary phase of platelet aggregation and ATP release induced by epinephrine [298]. Teng et al. claimed that compound 20 inhibited the aggregation of ADP-refractory, thrombin-degranulated, and chymotrypsin-treated washed rabbit platelets, suggesting that the inhibitory actions of compound 20 on platelet aggregation was not due to a direct blockade of substrates binding to ADP receptors [298]. Furthermore, similarly to the inhibitory effects of indomethacin and aspirin which are cyclooxygenase inhibiters, the formation of prostaglandin E2 induced by the incubation of cyclooxygenasecontaining guinea-pig lung homogenate with AA was inhibited by compound 20 [298]. Based on the available results, Teng and his colleagues [298] suggested that phthalide 20 inhibits platelet aggregation mainly by inhibiting cyclooxygenase leading to the reduction of thromboxane A2 formation. Nevertheless, there are no studies definitely proving its inhibition of cyclooxygenase, thus the current data are not sufficient to support this mechanism. We observed recently that a relatively high concentration (300 uM) of phthalide 20 in rat PRP significantly inhibited platelet aggregation induced by collagen but not by ADP and U-46619 (a thromboxane A2 receptor agonist) [299]. The inconsistency between the previously reported data and our results may be due to the different species (rat versus rabbit) and preparations (platelet-rich plasma versus washed platelets) used. Xu and Feng investigated the effects of racemic butylphthalide and its enantionmers (S-, 32) and (R-, 31) on platelet aggregation in rat PRP [300]. Both enantiomers and the racemic mixture (3-100 uM) dose-dependently inhibited platelet aggregation in rat PRP induced by ADP, collagen and AA, but did not affect thrombin-induced platelet aggregation. Furthermore, the 5-enantiomer 32 and racemic mixture (10-100 uM) increased intracellular cAMP concentration in a dose-dependent manner. On the other
642 hand, only the S-enantiomer 32 (1-100 uM) significantly inhibited serotonin (5-HT) release from platelets, and at a high concentration (100 uM) it reduced platelet thromboxane A2 (TXA2) levels. In the in vivo study with an inrraperitoneal dose ranging from 5 to 20 mg/kg, the S-enantiomer 32 and racemic mixture dose-dependently inhibited thrombus formation in rats, whereas the .ft-enantiomer 31 did not exhibit such an activity. Based on these results, Xu and Feng concluded that the S-enantiomer 32 most effectively inhibited platelet aggregation and thrombus formation possibly through regulation of cAMP levels and 5-HT release [300], and have claimed these findings in their Chinese patent [6]. Our recent study conducted in rat PRP demonstrated that similar to the effect of phthalide 20, senkyunolide A (58) at a high concentration of 300 uM also significantly inhibited platelet aggregation induced by collagen but not those induced by U-46619 and ADP [299]. Z-ligustilide (49) at a concentration of 150 uM almost completely blocked platelet aggregation induced by collagen and U-46619, and also inhibited ADP-induced platelet aggregation by 40%. However, its mechanism was unknown and needs further investigations. Naito et al. investigated reduction of blood viscosity by nine phthalides [301]. Among the compounds tested, two phthalide dimers: tokinolide B (118) and senkyunolide P (116), and three 3-substituted phthalides: butylphthalide (30), cnidilide (41) and senkyunolide A (58), significantly reduced blood viscosity in Wistar rats. The blood viscosity reduction was 7.3%, 9.4%, 9.4%, 18.5% and 12.8% for phthalides 30, 41, 58, 115 and 117, respectively. The other four phthalides 20, 47, 111 and 113 did not significantly affect blood viscosity. Cardiac Function Modulation and Anti-Angina Z-Butylidenephthalide (22) and its racemic mixture were suggested as anti-anginal agents by Ko et al. [302, 303]. Employing an experimental variant angina model in anesthetized dogs [302] and conscious rats [303], butylidenephthalide (20) and Zbutylidenephthalide (22) abolished pituitrin-induced T-wave lowering, which acts as an electrocardiographic indicator of myocardial injury. This finding was claimed to be quite promising as the result was comparable to those of the two clinically used antianginal agents, nitroglycerin and verapamil [303]. Nevertheless, the angina model remains to be validated since angina pectoris and myocardial injury are not the sole explanations for T-wave lowering [304]. In a study using isolated guinea pig hearts, phthalide 20 exhibited negative chronotropic and inotropic responses and attenuated the decrease in coronary flow induced by pituitrin. This demonstrated the preload- and afterload-reducing properties of 20, which may contribute to its anti-anginal effect in vivo [305]. This speculation, however, was challenged by the results in that 20 decreased the heart rate in renal hypertensive anesthetized rats [302] but its Z-isomer 22 did not affect the heart rate in normotensive conscious rats [303]. The impacts of phthalides 20 and 22 on cardiac function and angina pectoris/myocardial ischemia remain uncertain.
643 Other phthalides have also been investigated for their effects on cardiac function. Ligustilide (47) and senkyunolide A (58) exerted negative inotropic actions without changing atrial contraction rate in the isolated guinea pig atria [306]. Inhibition of Smooth Muscle Cell Proliferation The anti-smooth muscle cell proliferative activities of a number of phthalides derived from herbs Cnidium rhizome and Angelica root have been investigated by Kimura et al. [307-309], because a Sino-Japanese medicine called "Shimotsu-to" consisting of these two herbs plus two other Japanese medicinal herbs has been clinically used for the treatment of atherosclerosis, and atherosclerotic plaques formation is initiated by abnormal vascular smooth muscle cell proliferation [307-309]. In the parallel studies, the anti-proliferative activities of each of the isolated pure phthalides and the crude extracts of these two herbs were compared. The results demonstrated that phthalides 20, 30, 41, 47, 51, 58, 66, 67 and 68 exhibited anti-proliferative activity with different potencies in primary cultures of mouse aorta smooth muscle cells (Table 3, Fig. (7)). These active phthalides were all found in Cnidium rhizome, while compounds 20, 47, 51, 58 and 67 were also present in Angelica root. Therefore, the active phthalides present in both Cnidium rhizome and Angelica root were suggested to contribute at least partly to the preparation's anti-atherosclerotic action in the clinical setting. Furthermore, 66 was found to be the most potent of 7 phthalides (Table 3) based on IC50 valus for cell proliferation induced by 10% fetal bovine serum (FBS) [307-309].
Table 3. The Anti-Proliferative Activity of Different Phthalide Derivatives in the Primary Culture of Mouse Aorta Smooth Muscle Cells Phthalide
IC50 (ng/mL)
References
Butylidenephthalide (20)
3.25
[307-309]
Butylphthalide (30)
>20
[308]
Cnidilide (41)
>20
[308]
Ligustilide (47)
1.68
[307-309]
Neocnidilide (51)
6.22
[307-309]
Senkyunolide A (58)
1.52
[307-309]
Senkyunolide H (66)
0.1
[308, 309]
Both competence and progression phases of the anti-proliferative effects of these phthalides were further investigated by the same research team [308, 309]. Competence factors initiate the proliferation of smooth muscle cells. This makes them competent to synthesize DNA, allowing the cells to progress through the GtJG\ phase of the cell cycle
644 followed by DNA synthesis mediated by progression factors. In these studies, the time to initiation of smooth muscle cell proliferation and cell doubling time were measured and used as the indices of the 10% FBS-induced competence and progression phases of the cell cycle, respectively. Although all of the anti-proliferative phthalides inhibited the competence phase with relatively high potencies, the patterns of inhibition differed. Phthalide 66 inhibited both competence and progression phases, whereas phthalides 20 and 67 more potently inhibited competence and progression, respectively. Furthermore, the inhibition of smooth muscle cell proliferation was reversible and not due to cell damage and/or toxicity [308, 309]. Kobayashi and colleagues further investigated the structure-activity relationship of ten naturally-occurring phthalides [307-309]. Using the same smooth muscle cell culture model, senkyunolides D (61), E (62), F (64) and G (65), which have either 3-hydroxy or 3-hydroxylated side chain substitution on phthalide ring A were found to be inactive. The competence and progression inhibition potencies of the active anti-proliferative derivatives were in the following order: 66 > 68 > 67 > 47 = 58 > 20 [309]. The results suggested that phthalides having 6,7-disubstitutions at ring A were more potent than those containing a double bond at the 6 and 7 positions. Moreover, in order to study the influence of 6,7-disubstitutions at ring A on the anti-proliferative activity, 6-hydroxy-7chloro-6,7-dihydroligustilide, a phthalide senkyunolide L (Fig. (7)), believed to be formed not naturally but during the extraction and purification of phthalides from Cnidium rhizome, was also tested. The results indicated that this artificially formed compound was much more potent than the most potent natural phthalide 66. Although it was not clearly explained by the authors, based on the results of this structure-activity study, we proposed a relationship between the structure and the anti-proliferative activity of phthalides as illustrated in Fig. (7). A 3-alkyl side chain substitution at site 1 of the phthalide core structure seems essential for the anti-proliferative effect, because all identified active phthalides contain a 3-alkyl side chain substitution; whereas in the cases of phthalides 61, 62, 64 and 65; once the 3-substitution is replaced by 3-hydroxy and/or 3-hydroxylated side chain substitution, they become inactive. Moreover, 6,7disubstitutions at ring A (site 2 in Fig. (7)), for instance 6,7-dihydroxy in the most potent natural phthalide 66 and 6-hydroxy-7-chloro in the potent non-natural senkyunolide L, significantly contribute to the anti-proliferative activity. In addition, the degree of unsaturation in ring A may also affect the potency of anti-proliferative activity because compounds 66, 67 and 68 are more potent than 47 and 58, and the former three all have only one double bond while the later two have two double bonds in ring A. Phthalide 20 with an aromatic ring A in the structure has the lowest potency. However, only a limited number of phthalides and only the Z-isomers of the 3-butylene substituted derivatives were investigated. Further extensive and systematic studies of different phthalide derivatives are warranted to elucidate the structure-anti-proliferative activity relationships of phthalides.
645 Site 1
O Site 2 Phthalide
R3
R3'
R4
R5
R6
Senkyunolide L
H
H
—Cl
Senkyunolide H (66)
H
H
"OH
Senkyunolide I (67)
H
H
'"OH
Senkyunolide J (68)
H
H
Ligustilide (47)
H
H
Senkyunolide A (58)
H
H
R7
Activity
—OH
++++
—OH
++++
"OH
•OH
+++
•OH
+++
Butylidenephthalide (20) O. Senkyunolide D(61)
OH
H
OH
H
Senkyunolide E (62)
Senkyunolide F (64)
Senkyunolide G (65)
H
Based on the results of reference [309], arbitrary units are used to express the anti-proliferation activity, ++++: significantly active; +++: very active; ++: active; +: slightly active; -: inactive.
Fig. (7). The Proposed Structure-Anti-Proliferative Activity Relationships of Phthalides
646 Protection against Cerebral Ischemia The first indication that butylphthalide (30) and its two enantiomers 31 (3R-) and 32 (3S) have anti-stroke properties was based on their abilities to delay stroke occurrence and to prolong post-stroke life span in spontaneous hypertensive stroke-prone rats [310]. Adopting the middle cerebral artery occlusion (MCAO)-induced focal ischemia rat model, Feng et al. demonstrated that 30, 31 and 32 decreased infarct size [311, 312]. This finding may be related to their abilities to increase regional cerebral blood flow [313-315] and arteriole diameter [315]. Phthalides 30, 31 and 32 lessened MCAOinduced inflammation [316], and reduced cerebral edema [317, 318] and blood-brain barrier permeability [318]. In addition, these three phthalides caused a beneficial postMCAO learning improvement in an active avoidance test [319]. The protective activities of 30, 31 and 32 on cerebral injuries are summarized in Table 4. Heat shock protein 70 and c-fos mRNA expression which were escalated during cerebral ischemia were reduced by phthalide 30 [320]. Moreover, 30, 31 and 32 diminished apoptotic neuronal cell death [321], while ameliorating the activities of mitochondrial respiratory chain complexes [322] and choline acetyltransferase (which is related to learning) [323], lending further support for the cerebro-protective potential of these three phthalides against focal ischemia. In addition to the MCAO-induced focal ischemia model, other in vivo experimental cerebral ischemia models were employed to examine the activities of 30. Compound 30 was protective against cerebral damage induced by 4-vessel occlusion (experimental cerebral ischemia secondary to cardiac failure) [324, 325], physical closed head injury [326, 327], and subarachnoid hemorrhage [328-330] (Table 4). In an attempt to elucidate the mechanism of actions of 30, Feng et al. reported that it decreased the release of AA [326] while increasing the 6-keto-PGFia (a stable metabolite of PGI2) / TXB2 (a stable metabolite of TXA2) ratio in the MCAO model [331]. As PGI2 is a potent vasodilator while TXA2 a potent vasoconstrictor, the authors speculated that an augmented ratio was involved in the phthalide 30-mediated increase in regional cerebral blood flow, which in turn was thought to lead to protection against focal cerebral ischemia. However, in these studies the regional cerebral blood flow and infarct size were not simultaneously determined and no correlation analysis was performed to support this hypothesis. Feng et al. further demonstrated that intracellular Na+ concentration increased while K concentration decreased after 24 hours of MCAO, and compound 30 was able to attenuate these changes [317]. In general, the rise in intracellular Na+ concentration together with the decline of ATP during ischemia hampers Na+/Ca2+-ATPase, which results in intracellular Ca2+ overload. One of the detrimental consequences of Ca2+ overload is the activation of calcineurin and calpain, causing ischemic injury [332]. Phthalides 30 and 31 were demonstrated to suppress the enhanced activities of both calcineurin and calpain [333], although it did not alter the elevation of Ca2+ level mediated by focal ischemia [312]. +
647 Phthalides 30, 31 and 32 were also studied for their effects on the activities of various anti-oxidative enzymes and ATPases whose involvement in ischemia is well known [334]. Focal ischemia-mediated reduction of the activities of superoxide dismutase and glutathione peroxidase (both antioxidative enzymes) during focal ischemia was ameliorated by 30 and 32 but not the 3^-enantiomer 31 [334]. ATPase activities responded similarly [334]. Compounds 30 and 32 improved the suppressed Na+/K+-ATPase and Ca2+-ATPase activities in an inversely dose-dependent manner; the lowest dose tested causing the highest degree of response [334]. It is not known whether the dosages examined exhibited any toxicity as toxicological data were not reported. Further investigations are warranted. Mechanisms of action of 30, 31 and 32 were probed in various cell lines. Phthalides 30, 31 and 32 prevented cortical neuronal cell death and inhibited the release of several injury surrogate biomarkers induced by KC1 [335], A'-methyl-Z)-aspartate (NMDA) [335], AA [336] and hypoxia/hypoglycemia [337-339]. These effects appeared to be related to an increase in NO and PGI2 release from neuronal [340, 341] and cerebral endothelial cells [342]. Phthalides 30, 31 and 32 reduced superoxide anion production in a xanthine-xanthine oxidase reaction system [343]. The three phthalides also decreased intracellular Ca2+ level in cortical neuronal cells [344]. Furthermore, phthalides 30, 31 and 32 ameliorated the abnormal activities of several mitochondrial respiratory chain complexes induced by MCAO [322] and those of mitochondrial ATPase induced by hypoxia/hypoglycemia in cortical neuronal cells [345]. Despite the extensive effort of Feng et al. [310-331, 333-345], the mechanisms underlying the cerebro-protective actions of phthalides 30, 31 and 32 remain largely unclear. One of the possible reasons is that the authors often failed to demonstrate any causation or even correlation between the butylphthalide-mediated cerebro-protective responses and the various mechanisms proposed; for example, direct evidence of protection such as reduction of infarct size was not reported in the mechanistic studies. The lack of consistency in the experimental protocols employed between the antiischemia and mechanism studies, such as 24-hour MCAO without reperfusion versus 2hour MCAO with 24-hour reperfusion, also renders direct comparisons and correlations difficult (see Table 4). Moreover, some ambiguous data presentations and inadequate statistical analysis further complicated the interpretation of results in a number of studies. Nevertheless, these phthalides remain as potential therapeutic agents for the treatment of cerebral ischemia, and phthalide 30 was claimed to be effective for the treatment of cerebrovascular disease in a Chinese patent [7].
648 Table 4. Protective Effects of Butylphthalide (30) and Its Two Enantiomers 3R- (31) and 35- (32) Against Cerebral Injuries Established by Various Experimental Models
Administration Route and Time 1
Model
Ischemia Protocol2
3
route
pre-ischemia
Post-ischemia
(in minutes)
(in minutes)
Effects4
Refs.
MCAO
24hrl
i.p./ p.o.
--
15
4-Infarct size
[311, 312]
MCAO
24hrl
p.o.
--
15
4-Edema
[317]
MCAO
24hrl
i-g-
--
15
TActive avoidance response
[319]
MCAO
3 hrl
i.g./ i.p.
--
10
trCBF
[313, 314]
MCAO
2hrl
i.p.
60
--
tBlood flow, [315] tArteriole diameter tBlood flow, MCAO
2 hrl
i.p.
--
20
--
60
3 hrl/
4-BBB permeability, i.p.
MCAO
[315] tArteriole diameter
3hrR
[318] ^Edema
1 hrl/ MCAO
i.p.
--
10&60
^Inflammation
[316]
i.p.
30
--
^Cerebral injury
[324]
5&60
4*BBB permeability,
--
(post-injury)
^Edema
[326, 327]
24hrR 4-Vessel occlusion
20 min I / 24hrR
Closed head injury
--
i.p.
Subarachnoid hemorrhage
3hr
i.g./ i.p.
5
-
trCBF
[329]
Subarachnoid hemorrhage
3hr
i.p.
--
5
trCBF
[330]
'MCAO: middle cerebral artery occlusion. 2
I: ischemia; R: reperfusion.
3
i.g.: intragastrical; i.p.: intraperitoneal; p.o.: per os (oral).
4
BBB: blood-brain barrier; rCBF: regional cerebral blood flow.
649 Smooth Muscle Relaxation The anti-spasmodic response is one of the most recognized properties of several phthalides (Table 5). Among them, butylidenephthalide (20) and its Z-isomer (22) are well-documented relaxants tested in various isolated vascular [302, 346, 347], intestinal [348, 349], respiratory, [350] and reproductive smooth muscle preparations [348]. Ko et al. reported that phthalides 20 and 22 inhibited KC1- and phenylephrineinduced precontraction in a non-competitive and endothelium-independent manner with similar potencies in several isolated blood vessels [346, 347]. Compound 20 equally reduced cumulative KC1- and phenylephrine-mediated vasoconstriction, but significantly more potently antagonized cumulative high K+-depolarized Ca2+-induced contraction [346, 347]. Compound 22 behaved similarly [346, 347]. The authors thus hypothesized that 20 and 22 inhibited Ca2+ mobilization via the voltage-operated Ca2+ channel less selectively than other contraction mechanisms mediated by KC1 and phenylephrine [346]. Nonetheless, the reported IC50 values of 20 and 22 in the above experiments were quite high (>100 uM), so it was very likely that these phthalides exhibited vasodilatation via other mechanisms. In fact, our laboratory has recently demonstrated that nifedipine, an L-type voltage-operated Ca2+ channel antagonist, failed to affect both the potency and efficacy of compound 20 in producing relaxation against U-46619-induced precontraction in the rat aorta [351]. In the same preparation, phthalide 20 as well as SKF 96365, a non-selective cation channel (NSCC) and store-operated Ca2+ channel (SOCC) blocker, but not LOE 908, a NSCC blocker, fully relaxed thapsigargin- (a sarcoplasmic reticulum Ca +-ATPase inhibitor) mediated, nifedipine-insensitive contraction in a Ca2+ re-addition protocol [351], Thus, phthalide 20 may antagonize SOCC. Nevertheless, further studies adopting more sophisticated strategies such as patch-clamp recording and intracellular Ca2+ imaging are required to improve our understanding of Ca2+ mobilization. In another attempt, Ko et al. discovered that propranolol (a B-adrenoceptor antagonist), glibenclamide (a KATP-channel inhibitor), and charybdotoxin (a BKcachannel inhibitor) were ineffective against phthalide 20-induced vasorelaxation in various dog blood vessels [347]. Moreover, compound 20 did not alter the vasodilatory response of 3-isobutyl-l-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor, but augmented those of sodium nitroprusside and forskolin [347, 352]. The authors speculated that this effect was due to cAMP- and cGMP-dependent inhibition of PDEs [347]. However, although 20 was reported to cause mild inhibition of various PDEs, 2',5'-dideoxyadenosine (an adenylate cyclase inhibitor) and methylene blue (a soluble guanylate cyclase inhibitor) failed to antagonize vasodilatation caused by 20 [347]. Whether the relationship between phthalide 20-mediated PDEs inhibition and vasodilatation is correlation or causation remains elusive. Our results failed to support the findings of Ko et al. in that endothelium-removal, methylene blue and lH-[l,2,4]oxadiazolo- [4,3-a]quinoxalin-l-one (ODQ, a soluble guanylate cyclase inhibitor) all right-shifted the phthalide 20 dose-response curve against U-46619-induced vasoconstriction [352]. These right-shifts may be explained either by
650 20 releasing NO or by basal NO augmenting the vasodilatory activity of the phthalide [352]. Direct measurement of NO levels may shed light on this issue. On the other hand, the failure of another adenylate cyclase inhibitor, SQ 22536, to inhibit phthalide 20-mediated vasodilatation further supports the involvement of a mechanism other than adenylate cyclase activation [352]. Based on the above reported results, we propose, as shown in Fig. (8), that the mechanisms by which compound 20 causes smooth muscle relaxation may involve the inhibition of (i) VOCC; (ii) SOCC; (iii) Ca2+ release from internal stores; and (iv) PDEs, and also the activation of NO synthesis and/or basal NOphthalide 20 synergism. Endotheliol ce
©1 ---
Butylidenephthalide
Smooth muscle cell
Fig. (8). Proposed Mechanisms Underlying the Smooth Muscle Relaxation Effect of Buiylidctiephttialide (20)
NOs: NO synthase; PDEs: phosphoiliesteinses; sGC; soluble guanylate cyclase; SOCC. stote-opeiateil calcium channel; VOCC: voltage-operated calcium channel; (+): activation; (-): inhibition.
Ko et al. explored how phthalides 20 and 22 mediate the translation from vasodilatation to anti-hypertensive potential. While phthalides 20 and 22 had no effect on the blood pressure of normotensive anesthetized [302] and conscious animals [303], 20 reduced both systolic and diastolic blood pressure in renal hypertensive anesthetized rats [302], Phthalide 20 also lowered coronary arterial pressure without affecting mean arterial blood pressure in anesthetized dogs [302].
651 Liu and Feng demonstrated that butylphthalide (30) competitively right-shifted the cumulative dose-response curve of noradrenaline-induced contraction while noncompetitively right-shifting that of KC1 in isolated rat tail artery [353]. Phthalide 30 also inhibited noradrenaline-sensitive internal Ca2+ stores more selectively than noradrenaline-mediated external Ca2+ entry [353]. Phthalide 30 dose-dependently relaxed phenylephrine- and KCl-induced precontractions in aortas isolated from spontaneous hypertensive rats [354]. Neither endothelium removal nor A^-nitro-L-arginine methyl ester (L-NAME) affected the vasodilatory response of 30 [354]. These results were not in line with Xu's observation that 30 stimulated NO release from bovine aortic endothelial cells [342]. Phthalide 30 also non-competitively right-shifted cumulative phenylephrine and high K+-depolarized Ca2+ dose-response curves but did not affect caffeine-induced Ca2+ release from internal stores [354]. The discrepancies among experiments may have been due to the differences in the choice of animals and preparations utilized. Phthalide 30 did not affect mean arterial blood pressure in normal anesthetized rats [313] or subarachnoid hemorrhaged rats [329, 330]. In spontaneous hypertensive rats, however, 30 elicited a transient reduction in systolic blood pressure without affecting the activities of plasma and tissue angiotensin converting enzymes or urine output, which were diuresis indicators [354]. Reports that ligustilide (47) produces relaxation in uterine [355], respiratory [356], and micro-vascular smooth muscle preparations [357] were ambiguously presented, therefore, follow-up validation studies are required. The vasodilatory actions of various phthalide dimers were studied in several isolated rat preparations [301]. While they did not alter methoxamine-induced perfusion pressure of mesenteric arteries, the phthalide dimers relaxed KCl-induced vasoconstriction in rat aorta with decreasing potency according to the following order: tokinolide B (117) > levistolide A (111) > senkyunolide P (115) > riligustilide (113) [301]. Subsequently these four phthalide dimers were claimed in a Japanese patent to produce vasodilatory effects on KC1 and noradrenaline contracted rat mesenteric arteries [146]. Nevertheless, in general, the anti-spasmodic potencies of several phthalides in different isolated tissue preparations are relatively weak and their vasorelaxing properties in vitro often fail to translate to hypotensive effects in vivo. The potential benefits of phthalides as anti-hypertensive agents remain to be verified.
652 Table 5. Vasorelaxing Effects of Phthalides in Various Isolated Vascular Preparations
Phthalide
Butylidenephthalide (20) Butylidenephthalide (20) Butylidenephthalide (20) Butylidenephthalide (20) Z-Butylidenephthalide (22)
Butylphthalide (30)
Animal
Dog
Dog
Dog
Dog
Rat
SHRsp1
Tissue
Contractile Agent
EC 5 0
References
(jiM)
Coronary artery
PGF2(<
179
K.CI
168
Mesenteric artery
Phenylephrine
403
KC1
764
Femoral artery
Phenylephrine
373
KC1
369
Phenylephrine
78
KC1
69
Phenylephrine
125
KC1
219
Phenylephrine
73
KC1
122
[347]
[347]
[347]
[347]
Femoral vein
[346]
Aorta
[354]
Aorta
Levistolide A (111)
Rat
Aorta
KC1
4.3
[301]
Riligustilide (113)
Rat
Aorta
KC1
26
[301]
SenkyunolideP(115)
Rat
Aorta
KC1
18
[301]
TokinolideB(117)
Rat
Aorta
KC1
2.2
[301]
'SHRsp: spontaneous hypertensive stroke-prone rat
Three commonly used phthalide-containing Traditional Chinese MEdicinal herbs Radix Angelicea Sinensis (Chinese name Danggui) derived from Angelica sinensis, Rhizoma Chuanxiong (Chinese name Chuanxiong) derived from Ligusticum chuanxiong, and Rhizoma Ligustici (Chinese name Gaoben) derived from both Ligusticum sinense and L. jeholense are three widely prescribed phthalide-containing TCM herbs used in the treatment of a wide range of diseases. As summarized in Table 6, phthalides present in these herbs belong to either the 3-substituted phthalide type or the phthalide dimer type. To date, about 36 phthalides have been isolated and identified from Chuanxiong [9, 15, 19, 21, 41, 55, 69, 98, 109, 110, 144, 358], 27 phthalides from
653 Danggui [2, 25, 48, 87, 142], and 7 phthalides from Gaoben [12, 30, 32, 35]. Eight phthalides 20, 30, 47, 66, 67, 96, 109 and 111 were found as the main components in Radix Angelicea Sinensis [25]. Our recent HPLC-DAD-MS analysis revealed that seven phthalides 30, 47, 58, 66, 67, 111 and 112 are the main ingredients present in Rhizoma Chuanxiong [3]. Moreover, in comparison with the Japanese medicinal herb Senkyu, which is derived from the rhizomes of Cnidium officinale and has a similar clinical use as Chuanxiong, the quantities of 47, 58, 66, 67, 111 and 113 in Chuanxiong were reported to be about 3 times higher than those in Senkyu. On the other hand, the content of cnidilide (41) in Senkyu was about 4 times of that in Chuanxiong [358]. Such differences in chemical profiles of these two herbs may lead to their different therapeutic outcomes. Table 6. Phthalide Ingredients Present in Three Commonly Used Phthalide-Containing TCM Herbs
Name of TCM herb
Plant source(s) Phthalide ingredients'
(Chinese name) Rhizoma Chuanxiong (Chuanxiong)
Ligusticum chuanxiong
36 phthalides including:
3-Butyl-4,7-dihydroxyphthalide (3), 3-Butylidene-4hydroxyphthalide (13), 3S-Butyl-4-hydroxyphthalide (14), EButylidenephthalide (21), 3-Butylidene-4,5,6,7-tetrahydro-6«,7Sdihydroxyphthalide (23), 3-Butylidene-4,5,6,7-tetrahydro-65',75'dihydroxyphthalide (24), 3£-Butylidene-4,5,6,7-tetrahydro-6,7dihydroxyphthalide (25), 3-Butylidene-4,5,6,7-tetrahydro-7«hydroxy-6/?-(l-oxobutyl)phthalide (28), 3-Butylidene-4,5,6,7tetrahydro-7/?-hydroxy-6S'-( 1 -oxobutyl)phthalide (29), Butylphthalide (30), 3,S-Butyl-4,5,6,7-tetrahydro-6.ff,7«dihydroxyphthalide (33), 3S-Butyl-4,5,6,7-tetrahydro-3,6/?,75'trihydroxyphthalide (36), Cnidilide (41), £-Ligustilide (48), ZLigustilide (49), Neocnidilide (51), (-)-Sedanenolide (56), Senkyunolide A (58), Senkyunolide B (59), Senkyunolide E (62), ESenkyunolide E (63), Senkyunolide F (64), Senkyunolide H (66), Senkyunolide I (67), Senkyunolide J (68), Senkyunolide M (70), Senkyunolide N (71), Senkyunolide Q (72), Senkyunolide R (73), Senkyunolide S (74), 3'#,8'-Dihydrodiligustilide (108), Levistolide A (111), Riligustilide (113), Senkyunolide O (114), Senkyunolide P (115), TokinolideB(117) Rhizoma Ligustici (Gaoben)
Ligusticum sinense and L. jeholense
7 phthalides including:
Butylidenephthalide (20), Butylphthalide (30), Cnidilide (41), Ligustilide (47), Z-Ligustilide (49), Neocnidilide (51), (-)Sedanenolide (56)
654 Radix Sinensis
Angel icae Angelica
(Danggui)
27 phthalides including:
3-Butylidene-7-hydroxyphthalide (16), Z-Butylidenephthalide (22), 3£-Butylidene-4,5,6,7-tetrahydro-6,7-dihydroxyphthalide (25), Butylphthalide (30), Z-6,7-Epoxyligustilide (43), £-Ligustilide (48), Z-Ligustilide (49), Neocnidilde (51), (-)-Sedanenolide (56), Senkyunolide A (58), Senkyunolide B (59), Senkyunolide C (60), Senkyunolide D (61), Senkyunolide E (62), Senkyunolide F (64), Senkyunolide G (65), Senkyunolide I (67), Senkyunolide K (69), Angelicide (96), 3-Butylidene-6',7'-dihydro-4-propyl-spiro[3H3a,6-ethanoisobenzofuran-5(4H),l'(3'H)-isobenzofuran]l,3'(6H)dione (102), l£,8£-Dibutylidene-5S,5a/J,6,7,8,10b/?-riexahydro-l H5,10cS'-ethanonaphtho[l,2-c:7,8-c']difuran-3,10-dione (104), 1£,8ZDibutylidene-5S,5a/?,6,7,8,1 Obff-hexahydro- 1H-5,1 OcSethanonaphtho[l,2-c:7,8-c']difuran-3,10-dione (105), 1Z,8ZDibutylidene-5S,5ai?)6>7>8>10b/?-hexahydro-lH-5,10c5ethanonaphtho[l,2-c:7,8-c']difuran-3,10-dione (106), Z,Z'3.3\8.8'-Diligustilide (109), Z,Z'- 6.8',7.3'-Diligustilide (110), Levistolide A (111), Riligustilide (113)
"Please refer to Table 1 for references
Clinical Indications As documented in the Chinese Pharmacopoeia, Danggui and Chuanxiong are mainly used to relieve pain induced by the so-called "blood stagnation syndrome" [359]. The manifestation of their analgesic actions, according to the TCM theory, is related to the facilitation of blood circulation and removal of blood stasis [359]. In TCM practice, Danggui is primarily prescribed for the treatment of gynaecological disorders such as irregular menstruation, amenorrhea, and dysmenorrhea, while Chuanxiong is commonly used for the treatment of migraine and headache [360, 361]. These two herbs are also used as remedies for asthma, stroke and angina pectoris [360, 361]. Gaoben is usually used as a pain-killer for headache-like symptoms [362]. Clinical Evaluations Despite the growing interest in the pharmacology of naturally occurring phthalides, to date butylphthalide (30) is the only single phthalide rather than in a mixture being studied in clinical trials. In late 2002, a phase III clinical trial was conducted in China to investigate the therapeutic potential of butylphthalide (30) in treating cerebral ischemia [321, 363]. The details of the clinical trial have not been published yet, but the investigators claimed that phthalide 30 elicited "obvious therapeutic effects with minimal adverse reaction in 590 patients with acute cerebral ischemia" [363].
655 Other clinical studies, which include only a few case reports, were all carried out using the extracts of phthalide-containing TCM herbs [364-368]. However, in most of these studies, the general and fundamental clinical trial designs employing features such as randomization, double blinding, placebo control, and proper statistical analysis were rarely employed. Rhizoma Chuanxiong (3 g/day for 14 days) was claimed to ameliorate symptoms of acute cerebral infarction [364]. In a separate study by the same group of investigators in China, Rhizoma Chuanxiong (1 g/day for 1 -2 years) was reported to improve symptoms of transient cerebral ischemic attack [365]. Nevertheless, which symptoms were examined and the details of the scoring system adopted were not documented in either study. Crude oil extract from Radix Angelicae Sinensis (10-30 mg) was reported to alleviate spasm-mediated abdominal pain under various clinical conditions in 151 out of 162 patients (93%) in China, and this effect was comparable to that of 0.3 mg atropine (34 out of 35 patients, 97%) [366]. An in-patient study was conducted in China to study the effect of Radix Angelicae Sinensis (62.5 g, i.v./day for 10 days) to treat chronic obstructive pulmonary diseases [367]. Despite the decrease in the plasma levels of several detrimental factors (such as angiotensin-II and digitalis-like factor), pulmonary function as determined by PaO2 (arterial oxygen tension), PaCO2 (arterial carbon dioxide tension) and SaO2 (arterial oxygen saturation) did not improve. The potential estrogenic effects of Radix Angelicae Sinensis (4.5 g/day for 24 weeks) were investigated in 71 postmenopausal women in a randomized, double-blinded, placebo-controlled clinical trial conducted in the United States of America [368]. Endometrial thickness and menopausal symptoms as measured by the Kupperman index were improved equally in the herb-treated and placebo groups. Maturation value and number of superficial cells in vaginal smears were not affected in either group. The authors concluded that Radix Angelicae Sinensis, when used alone, was not more effective than placebo in treating menopausal symptoms. Concluding Remarks Although from the modern medical science perspective, phthalide-containing herbs remain to be established as clinically effective, the parallels between the pharmacological actions of different phthalides and the traditional clinical indications of these herbs underscore their potential therapeutic benefits. Structurally simple and biologically active with diverse beneficial properties, the phthalides constitute an ideal lead class of compounds for chemical modification to enhance their promising pharmacological activities. On the other hand, we enthusiastically anticipate that with the recent growing interest in natural products and herbal medicines, the therapeutic benefits of phthalides and phthalide-containing herbs will soon be established scientifically, and these ancient traditional medicines will make important contributions to modern medicinal science.
656 ACKNOWLEDGEMENTS The authors greatly acknowledge Dr. Hugh A. Semple (Director, Scientific and Regulatory Affairs, Kinetana Group Inc., Edmonton, Alberta, Canada, and Adjunct Professor in the College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada) for his critical comments and improvements to the language proficiency in the preparation of this article. Abbreviations 5-HT
Serotonin (5-Hydroxytryptamine)
6-keto-PGF 2 a
6-Keto-prostaglandin F 2 a
AA
Arachidonic acid
BBB
Blood-brain barrier
BK C a
Large-conductance calcium-activated potassium channel
EC 5 0
Effective concentration to achieve 50% of maximal response
ED50
Effective dose to achieve a response in 50% of the population
GABA
y-Aminobutyric acid
IB M X
3-Isobutyl-l-methylxantine
IC50
Effective concentration to achieve 5 0 % inhibition of a response
i.c.v.
Intracerebroventricular
i.g.
Intragastrical
i.p.
Intraperitoneal
i.v.
Intravenous
K ATP
ATP-activated potassium channel
L-NAME
A^-nitro-L-arginine methyl ester
MCAO
Middle cerebral artery occlusion
NMDA
jV-Methyl-£>-aspartate
NOs
N O synthase
NSCC
Non-selective cation channel
ODQ
l-H[l,2,4]Oxadiazolo[4,3-a]quinoxalin-l-one
PAF
Platelet-activating factor
PaCO 2
Arterial carbon dioxide tension
PaO 2
Arterial oxygen tension
657 PDE
Phosphodiesterase
PGI2
Prostacyclin
p.o.
Per os (oral)
PRP
Platelet-rich plasma
rCBP
Regional cerebral blood flow
SaO2
Arterial oxygen saturation
sGC
Soluble guanylate cyclase
SHRsp
Spontaneous hypertensive stroke-prone rat
SOCC
Store-operated calcium channel
TCM
Traditional Chinese medicine
TXA2
Thromboxane A2
TXB2
Thromboxane B2
VOCC
Voltage-operated calcium channel
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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CHEMISTRY AND BIOLOGICAL ACTIVITY OF POLYISOPRENYLATED BENZOPHENONE DERIVATIVES OSMANY CUESTA-RUBIot, ANNA LISA PICCINELLI§, LUCA RASTRELLI § ' Instituto de Farmacia y Alimentos (IFAL), Universidad de La Habana, Ave. 23, No. 21425, CP 13600 La Lisa, Ciudad de La Habana, Cuba. *Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084, Fisciano, Salerno, Italy. ABSTRACT: Plants of the family Clusiaceae or Guttiferae, in particular those belonging to the genera Clusia, and Garcinia, produce a series of oxidized and polyisoprenylated benzophenone derivatives, some of which are structurally complex and biologically active. From a biogenetic point of view, these compounds may be considered as benzophenones in which the acetate derived benzene ring is modified by intervention of isoprenyl groups. Several compounds belonging to this class have shown a wide range of biological activity such as antimicrobial, antifungal, anticarcinogenic and anti-HIV inhibitory activities. In this chapter we shall review the chemistry and biological activity of the polyisoprenylated benzophenone derivatives isolated from the genera Clusia, Garcinia, Vismia, Allanblackia, Moronobea, Symphonia, Hypericum, Tovomita, Tovomiptosis and Ochrocarpus.
INTRODUCTION Clusiaceae (Guttiferae) is a family almost exclusively tropical in distribution and comprises about 40 genera and 1200 species most of which are woody [1]. Extensive phytochemical studies have shown Clusiaceae to be a rich source of secondary metabolites including xanthones, triterpenoids, flavonoids, lactones and organic acids. In addition plants of this family produce a series of oxidized and polyisoprenylated benzophenones (PBDs), some of which are structurally complex and biologically active. From a biogenetic point of view, these compounds are thought to be of mixed shikimate and acetate biosynthetic origin in which the acetate
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derived benzene ring is modified by intervention of prenyl groups. Structural modifications and alkylation of this ring results in the formation of a complex bridged bicyclic or tricyclic system. This situation has permitted to obtain both simple (SBDs) and complex molecules as bicyclo[3.3.1]-nonane and adamantyl derivatives. Genera Garcinia and Clusia have been reported as the main sources of PBDs but these compounds have been also isolated from other genera including Vismia, Tovomita, Allanblackia, Symphonia, Hypericum, Moronobea , Tovomiptosis and Ochrocarpus recently. Bronianone, a yellow pigment present in the stem wood of G. hombroniana was the first member of polyisoprenylated benzophenone derivatives isolated from Clusiaceae [2]. This compound contains maclurin moiety, the 2,4,6,3',4'-pentahydroxy benzophenone (2). The structure proposed initially was revised and suggested as (1) finally. OH HO.
OH
(2) Maclurin
(1) Bronianone MeO
O
OH
(3) Marupone Fig.(l). Simple benzophenone derivatives and maclurin.
PBDs seem to be derivatives of maclurin or another benzophenone derivative modified in the A ring by intervention of prenyl groups. B ring can be unsubstituted or contain up to 2 phenolic groups. PBDs have shown different biological properties but, probably the three most important are the cytoprotection against HIV-1 in vitro of guttiferones [3,4], antimicrobial properties [5-11] and cytotoxic activity found in diverse nucleus [12-16]. The presence of keto-enolic equilibrium in most of them seems to play an important role on account of when this possibility disappears, a lower potential is often observed.
673
Occurrence About 100 PBDs have been isolated from different genera of the family Clusiaceae. Initially, these products were associated with genera Clusia and Garcinia, nowadays their distribution is associated with other 8 genera. Simple and complex structures have been isolated from genera Vismia, Symphonia, Hypericum, Tovomita, Allanblackia, Moronobea, Tovomiptosis and Ochrocarpus too. Resin is a rare reward limited to a few tropical genera like Clusia whose flowers produce floral resins in many species. The viscous liquid is collected by bees and used as a nest construction material. In this context some PBDs have been reported in tropical propolis [11,17]. Investigation of the chemistry of the floral resins revealed that they are composed of almost pure polyisoprenylated benzophenones [18,19]. The analysis quantitative by RP-HPLC of 11 floral resins of Clusia species belonging to the sections Chlamydoclusia, Cordylandra, Phloianthera and Polythecandra, by Porto et al. [20] described the correlation between the chemical composition and the taxonomic sections. In the sections Chlamydoclusia and Polythecandra, the floral resins have bicyclo-[3.3.1]nonane benzophenone derivatives as major constituents and simple benzophenone derivatives as minor components, while section Phloianthera floral resins have these two compound types in almost equal amounts. In section Cordylandra were isolated only compounds possessing the bicyclo-[3.3.1]-nonane benzophenone structure. It is very interesting to note the high percent of prenylated benzophenone derivatives in some natural sources. Floral resins, latex, leaves, and fruits can contain up to 70 % of these compounds [19,20]. Simple benzophenone derivatives (SBDs) Some PBDs show up to 6 isopentenyl groups although no complicated arrangements are observed in these structures. Many of them are true examples of polyisoprenylated benzophenones because the acetate derived ring shows aromatic properties. We shall consider this group as simple benzophenone derivatives and most of them are included in Table 1. Marupone (3), isolated from trunk wood of Moronobea pulchra Ducke, seems to be the first example of SBDs in genus Moronobea [21]. Its structure was deduced on spectral and chemical evidences. The allocation of the geranyl group to C-3 was based on UV spectroscopy data
674 Table 1. Simple polyisoprenylated benzophenones isolated from Compounds Bronianone (1) Marupone (3) Vismiaphenone A (4) Vismiaphenone B (5) Vismiaphenone C (6) Vismiaphenones D-G (7-10) Iso- vismiaphenone B (11) Myrtiaphenones A-B (12-13) Clusiaphenone A (14) Clusiaphenone B (IS) Clusiaphenone C-D (16-17) Kolanone(18) Tovophenones A-B (19-20) Tovophenone C(21) Grandone (22) Machuone (23) Weddellianones A (24)
Weddellianones B (25) Lanceolatone (26)
Hilarianone (27) Vismiaguianones A-E (28-32) Pseudoguttiaphenone A (33) Nemorosinic acid A (34) 3-geranyl-2,4,6-trihydroxybenzophenone (36) 4,6,4'-trihydroxy-2,3'-dimethoxy-3-prenylbenzophenone (37) Garciosaphenone (38) Cudraphenones A-D (39-42)
plants of Clusiaceae Sources G. hombroniana M. pulchra V. decipiens V. guaraminangae V. decipiens C.ellipticifolia G. myrtifolia V. guaraminangae G. pseudoguttifera V. cayennensis V. decipiens C.ellipticifolia G. myrtifolia G. pseudoguttifera C.ellipticifolia Csandiensis C.sandiensis C.ellipticifolia G .kola Tovomita mangle Tovomita brevistaminea Tovomita brevistaminea C. grandiflora C.sandiensis C. weddelliana C. lanceolata C. pana-panari C. burchellii C. fluminensis C. hilariana C. paralicola C. pernambucensis C. weddelliana C. pana-panari C. lanceolata C. burchellii C. fluminensis C. hilariana C. pana-panari C. paralicola C. pernambucensis C. hilariana V. guianensis G. pseudoguttifera C nemorosa Tovomita krukovii G. multiflora G. speciosa Cudrania chinchinensis
References 2, 21 22 23 22
26,28 24 23 31 25 22
26,28 24 31
26,28 27 27 26 5 29 30 30 19 27 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 12 31 32 6 33 34 13
considering the absence of UV AICI3 shift and the formation of two cyclization products upon acid treatment, which requires the presence of the geranyl group ortho to both hydroxyls.
675
14 SBDs closely related have been reported from the genera Vismia, Garcinia, and Clusia: vismiaphenones A-G (4-6), isovismiaphenone B (11), myrtiaphenones A-B (12,13) and clusiaphenones A-D (14-17). All of them can be considered as 2,4,6-trihydroxy-3,5-diisopentenyl benzophenone or 2,4,6-trihydroxy-3,5-diisopentenyl-4-hydroxy benzophenone derivatives. Three compounds named vismiaphenone A (4) and B (5) and isovismiaphenone B (11) were isolated from the berries of V. decipiens Schlecht-Clam [22]. Their structures are very closely related and all of them showed one unsubstituted aromatic ring. Structures were deduced from their spectral and chemical data. The proposed structure for vismiaphenone A was confirmed by acid-catalysed cyclization and cyclodehydrogenation with DDQ (2,6-dichloro-3,5-dicyanbenzoquinone). The presence of a 2,2-dimethylchromene ring and the loss of one hydroxyl group in the NMR spectra of vismiaphenone B and iso-vismiaphenone B indicated cyclization to a pyran ring of the prenyl group. Vismiaphenone C (6) was isolated from the root bark of V. guaraminangae together with the known vismiaphenone A (4). The UV, MS (m/z 105) and NMR data indicated one unsubstituted ring, two equivalent chelated hydroxyl groups, and a methoxy group. Consequently vismiaphenone C was considered an isomer of vismiaphenone A. The structure was confirmed by acid-catalysed cyclization of the side chains [23]. Myrtiaphenones A (12) and B (13) were isolated from hexane extract of G myrtifolia [24]. NMR data of myrtiaphenone A showed the presence of two OMe groups, a hydrogen-bonded phenolic OH group (8 10,92), two isoprenyl groups and a benzoyl group also identified from the intense peaks at m/z 105 and 77. The structure of 6-hydroxy-2,4-dimethoxy-3,5diprenyl benzophenone was assigned to myrtiaphenone A. This compound was synthesized previously but, it was not identified as a natural product. Myrtiaphenone A can be considered as a methyl derivative of the known compound vismiaphenone C. All NMR data of myrtiaphenone B (13) were in agreement with the structural characteristics of prenylated benzophenones. The resonances at 8 6.40 and 5.32 shared a J constant of 10 Hz, typical of a cis double bond, and a gem-dimethyl group indicated the presence of a chromene ring. Myrtiaphenones are very closely related from a structural point of view. Myrtiaphenone A can be easily converted in myrtiaphenone B by cyclo dehydrogenation with DDQ.
676 OR, (4) Vismiaphenone A, R1=R2=H, R3=CH3, R4=H (6) Vismiaphenone C, R,=R3=H, R2=CH3, R4=H (7) Vismiaphenone D, R,=R3= H, R2=CH3, R4= OH (12) Myrtiaphenone A R,=R2=CH3, R3=H, R4=H (15) Clusiaphenone B, R,=R2= R3=R4=H
OR,
(5) Vismiaphenone B, R,=R2=R3=H (8) Vismiaphenone E, R,= R2=H, R3=OH
(11) Isovismiaphenone B, Ri=R2=H
(9) Vismiaphenone F, Ri=CH3, R2=H, R3=OH
(13) Myrtiaphenone B, Ri=H, R2=CH3
O
O
(10) Vismiaphenone G
OH
(14) Clusiaphenone A OH
OH
OH (16) Clusiaphenone C Fig. (2). Simple benzophenone derivatives closely related
OH
O
(17) Clusiaphenone D
Fuller et ah, following anti-HIV bioassay-guided fractionation isolated four new prenylated benzophenones, vismiaphenones D-G (7-10), from extracts of leaves of V. cayennensis [25]. All these compounds showed to be very similar to vismiaphenones A-B (4, 5) and myrtiaphenones A-B
677
(12, 13). The main difference was associated with the benzoyl group. It was identified as a p-disubstituted aromatic ring on the basis of evidences observed in the NMR spectra and mass spectral fragmentation (m/z 93, 121). Vismiaphenone F (9) and myrtiaphenone A (12) showed a common characteristic in their 'H NMR spectra: the anomalous chemical shifts of the aryl methoxyl groups (8 3.32 and 3.19 respectively) vicinal to the benzoyl group. Authors have suggested that this feature is likely due to anisotropic shielding produced by benzoyl group. A similar effect was detected in myrtiaphenone B where the gem-dimethyl group falls in the shielding zone of the benzoyl group. The proton resonance spectrum of vismiaphenone G (10) revealed that one prenyl group had undergone epoxidation of the olefinic bond (ABX spin system). This uncommon structural characteristic among natural prenylated benzophenone derivatives is the sole difference between vismiaphenone G and vismiaphenone D (7). Clusiaphenone A-D (14-17), isolated from the genera Clusia, are SBDs closely related [26-28]. All these compounds presented one unsubstituted aromatic ring. Clusiaphenone A, isolated from the fruits of C. ellipticifolia and C. sandiensis, and clusiaphenones C-D, isolated from the fruits of C. ellipticifolia, have the two isoprenyl group ciclizated. The marked diamagnetic shift (A5= - 0.4 ppm) displayed by the a-chromene proton in the *H NMR spectra of clusiaphenones C-D agrees with a peri relationship between the phenolic hydroxyl and the chromene ring. From the biogenetic point of view, clusiaphenones A-D may derive from the same precursor (2,4,6-trihydroxybenzophenone) by attachment of two isopentenyl chains and successive cyclodehydrogenation and/or cyclization. The dried and powered fruit pulp of G. kola was extracted with light petroleum (bp 40-60 °C) [5]. A yellow-brown precipitate, kolanone (18), was obtained after standing. The base peak at m/z 105 (C7H5O) observed in the mass spectrum suggested that one of the benzophenone rings was unsubstituted. NMR data evidenced the presence of two 3-methyl-2butenyl groups and a geranyl unit, confirmed by mass spectrum which showed ions for the loss of C9H17 and C10H17 as well as for the loss of C 5 H 8 and C5H9. Three polyisoprenylated benzophenones closely related and named tovophenones A-C have been reported in Tovomita species. Spectral data of these compounds were consistent with a polyisoprenylated benzophenone with one w-hydroxyphenyl group and a lavandulyl chain (C10H17). Tovophenones A (19) and B (20) were isolated from the roots of
678
T. mangle [29]. A lavandulyl chain (CioHn) was identified by the presence of two terminal methylene groups and verified by one-step cleavage of the double bonds with OSO4-HIO4. Tovophenone B showed to be a derivate of tovophenone A where a pyran moiety is generated by arrangement of a prenyl rest. Tovophenone B was not considered an artefact formed during the process of purification carried out because it was observed in the crude extract. However, many PBDs have showed to be unstable in solutions and the process of structural transformation can occur rapidly. A new study permitted to obtain the known tovophenones A and B and the new compound tovophenone C (21) [30] from T. brevistaminia. This compound showed a dihydrofuran ring with a hydroxyisopropyl group substituent and, like tovophenone B, can be also considered as a tovophenone A derivative. Grandone (22) was obtained from the floral resins of C. grandiflora as its dimethyl derivative. It contains three 3-methyl-2-butenyl groups and constitutes the first SBD isolated from floral resins of Clusia spp [19]. Machuone (23) was found in the fruits of C. sandiensis together with clusiaphenones A-B, and clusianone [27]. Structure was established on spectral basis and two tautomers were present in a c.a 4:1 ratio. Machuone is closely related to grandone. Initially the main PBDs isolated from floral resins of Clusia spp. showed to be bicyclo-[3.3.1]-nonane derivatives but a new study demonstrated the presence of several SBDs in these sources. Structures of weddellianone A (24) and B (25), lanceolatone (26) and hilarianone (27) were established on spectroscopic evidences of their methyl derivatives obtained after treatment of fresh floral resins with diazomethane [20]. All the structures were represented with a 1,3,5-triketone system because of the treatment before mentioned don't facilitate the determination of the exact structures. On the other hand, many natural SBDs have shown to have methoxyl groups in their structures and then in the methyl derivatives above mentioned is not possible to establish the origin of the methoxyl groups certainly. A study of V. guianensis led to isolation of new SBDs [12]. Vismiaguianones A-E (28-32) were identified as benzophenone derivatives characterized by the presence of an unsubstituted benzoyl group. Vismiaguianones A-C showed an additional ring suitable to cyclization between a hydroxyl and isoprenyl group at C-5. Vismiaguianone A has a hydroxy-dimethyl-dihydropyran ring, while vismiaguianones B and C have a dihydrofuran ring with a hydroxyisopropyl group substituent.
679
OH
OH OH (20) Tovophenone B
O
(21)Tovophenone C
O
OH
(23)Machuone
(24) Weddellianone A
(26) Lanceolatone Fig. (3). Simple benzophenone derivatives from (20) to (27).
(25) Weddellianone B
(27)Hilarianone
680
The position of ring C on the acetate derived ring was based on the presence of only one hydrogen-bonded proton in the H NMR spectrum of vismiaguianones A and C (§H 12.60 and 12.71, respectively) in respect to two weakly hydrogen-bonded hydroxyl protons in the H NMR spectrum of vismiaguianones B (5H 9.82 and 8.14). The hydroxyl groups were hydrogen-bonded to the carbonyl group of the benzophenone moiety Vismiaguianones D and E exhibited an additional aromatic ring which is associated with a phenylpropanoid rest. IR bands (1773 and 1774 cm"1) and 13 C NMR chemical shifts of the signals ascribed to esther groups (5 166.9 and 165.0), corresponding to each compound, were in agreement with the 5-lactone function observed for the first time in benzophenone derivatives. Pseudoguttiaphenone A (33), isolated from heartwood of G. pseudoguttifera together myrtiaphenone A-B and vismiaphenone C, is a 4methyl- vismiaguianone A derivative [31]. Pseudoguttiaphenone A could be biogenetically derived from vismiaphenone C via a cyclization between a hydroxyl and isoprenyl group and epoxidation in the 2,2dimethylchroman ring. A phytochemical study of fruits of C. nemorosa led to isolation of nemorosinic acid A (34) and B (35) which contained an oxidized lavandulyl chain characterized by the presence of a carboxyl group [32]. Both sets of NMR spectra indicated the presence of two components in a ca 3:2 and 1:1 ratio respectively which demonstrated the existence of a keto-enolic equilibrium in these compounds. However, nemorosinic acid A was identified as a PBD and nemorosinic acid B as an alkylarylketone. The structural relationship observed between these two compounds was firmly established on spectral evidences. Other SBDs found in family Clusiaceae were also included in Table 1. i.e. 3-geranyl-2,4,6-trihydroxybenzophenone (36) [6], 4,6,4'-trihydroxy2,3'-dimethoxy-3-prenylbenzophenone (37) [33], garciosaphenone (38), a digeranylbenzophenone isolated from trunk bark and stems of G. speciosa [34]. Four new SBDs, cudraphenones A-D (39-42), were isolated from the roots of Cudrania cochinchinensis (Moraceae) [13]. The ring B of these compounds showed a different substitution pattern with respect to PBDs isolated from Clusiaceae. Cudraphenones A-D presented a 3-hydroxy-2prenyl substitued ring B and only one isoprenyl chain on the ring A. The methyl protons of the 2,2-dimethylpyran ring of cudraphenone C appeared at a higher field position than those of cudraphenone A. The upfield shifts were caused by the anisotropic effect of the ring B.
681 OH
OH
HO
OH
0
(30)Vismiaguianone C
(29) Vismiaguianone B
(28)R=H Vismiaguianone A (33)R=CH3 Pseudoguttiaphenone A
OH
(32)Vismiaguianone E
(31) Vismiaguianone D
R:
(34) Nemorosinic acid A
CO,H (35) Nemorosinic acid B
HO
xo O
°" OCH3
(37) 4,6,4-trihydioxy-2,3-dimethoxy-3-prenyl benzophenone OH
O
0
R
R=Geranyl (38) Garciosaphenone
R—Geranyl (36)3-geranyl-2,4,6-trihydroxybenzophenone
(40) Cudraphenone B R,=R3=H, R2=C5H, (39) Cudraphenone A
(42) C
"d
Fig. (4). Simple benzophenone derivatives from (28) to (42)
(41) Cudraphenone C
682
Bicyclo-[3.3.1]-nonane derivatives Elucidation of the structures of xanthochymol (43) and isoxanthochymol (44), the first members of the bicyclo-[3.3.1]-nonane benzophenone derivatives was very important in the chemistry of these secondary metabolites. Their structures were established by chemical transformations and spectroscopic means which included X-ray analysis [35,36]. Initially, xanthochymol and isoxanthochymol were isolated from G. xanthochymus fruits by Karanjgoakar et al. [37]. Both structures were deduced on the basis of spectral evidences. The structure of isoxanthochymol was firmly established by an X-ray crystallographic analysis but, the structure of xanthochymol was suggested by comparison with the first one. Dreyer also reported the presence of xanthochymol in the mature fruit of C. rosea [38], the structural analysis was developed considering the structure mistakenly identified as xanthochymol in the previous report [37]. Blount and Williams revised the structure of xanthochymol employing spectroscopic methods that included an X-ray crystallographic method [35]. Finally, the structure of xanthochymol was deduced as (43). Significant differences in the 'H NMR spectra of xanthochymol and isoxanthochymol included the presence of signals due to two terminal methylene groups in xanthochymol and two methyl groups on a satured carbon (§ 0.93 and 1.24) in the isoxanthochymol spectrum. These findings are confirmed by 13C NMR spectra which showed two signals at S 109.8 and 113.5 for two terminal methylene carbons (-C(Me)=CH2) and two signals at 5 123.9 and 124.2 for methine carbons of two trisubstituted olefin groups (-CH=CMe2) [35]. Xanthochymol is converted in isoxanthochymol by acid-catalysed reactions and this method has been frequently used in order to define or compare these structures. These structures presented the same relative stereochemistry associated with the bicyclo moiety. New exhaustive chemical studies confirmed the above structures mentioned [7,36]. As xanthochymol, many PBDs isolated from Guttiferae show a structure based on the bicyclo-[3.3.1]-nonane-2,4,9-trione system (Table 2).
683 Table 2. Bicyclo-[3.3.1]-nonane derivatives. Compounds Xanthochymol (43)
Isoxanthochymol (44)
Cambogin=isogarcinol (45)
Camboginol=garcinol (46)
18-O-methyl isogarcinol * (47) 18-O-methyl garcinol * (48) Guttiferones A-D (49,53,50,51) Guttiferone A Guttiferone G (52) Guttiferone E (54)
Guttiferone F (55) Clusianone (56)
7-epiclusianone (57) Nemorosone (58)
Hydroxy nemorosone (59) 7-epinemorosone (60) Spiritone (61)
Insignone (62)
Sources C. rosea G. subelliptica G. xanthochymus G. pyrifera G. staudtii G. mannii G. ovalifolia G. purpurea G.ovalifolia G. subelliptica G. xanthochymus G. purpurea G.cambogia G. indica G. assigu G. purpurea G.cambogia G. indica G. assigu G. assigu G. assigu S. globulifera G. livingstonei G. macrophyl/a G. macrophylla G. ovalifolia C. rosea G. pyrifera A. stuhlmannii C. spiritu-santensis C. lanceolata C. pana-panri C. weddelliana C. fluminensis C. burchellii C. paralicola C. pernambucensis C.congestiflora C.sandinensis G. assigu H. sampsonii Rheedia gardneriana C. rosea C. grandiflora C. insignis C. nemorosa C. nemorosa C. nemorosa C. insignis C. renggerioides C. spiritu-sanctensis C. fluminensis C, burchellii C. pernambucensis C. weddelliana C. insignis
References 3 7 37 16 54 57 58 62 3,58 7 37 7,86 43 41,42 44 7,62 43 41,42 44 44 44 3 3 15 15 3 3 16 4 19,20 20 20 20 20 20 20 20 45 27 44 54 10 19 19 19 19 19 18 18 18 20 20 20 20 20 20
684 Table 2. Bicyclo-|3.3.1]-nonane derivatives. Compounds Scrobiculatones A and B (63,64) Plukenetiones D-E (65-66) Plukenetiones F-G (67-68) Chamone I and II (69,70) Aristophenones A-B (71) Propolone A (72) Sampsoniones K-M (73-75) Hyperibones A-I (76-84) Ochrocarpinones A-C (85-87) 15,16-dihydro-16-hydroperoxyplukenetione F (88) Garcinielliptone I (89) * Possible structural mistake
Sources C. scrobiculata C. plukenetii C. plukenetii C. havetiodes var. stenocarpa C.grandiflora
G.aristata propolis H. sampsonii H. scabrum O. punctatus C. havetiodes var. stenocarpa 0. punctatus G. subelliptica
References 20 47 47 52 9 49 11 51 8 14 52 14 53
We proposed to group PBDs that present a bicyclo-[3.3.1]-nonane2,4,9-trione system in accord with benzoyl moiety position: type A if it is on C-l, type B if it is on C-3 and finally type C if it is on C-5, Fig.(5) [40]. The structures previously reported as type C, nemorosone and 7epinemorosone, have been corrected and all polyisoprenylated benzophenones derivatives with a bicyclo-[3.3.1]-nonane-2,4,9-trione system isolated, therefore, are derivatives of type A or B [18,19]. In this work, we use this classification and a unique numbering system in order to facilitate the structural comparisons. Type A: R]= benzoyl group or derivative. R2,R3,R4: prenyl groups Type B: R2= benzoyl group or derivative. Ri,R3, R4:prenyl groups Type C :R3= benzoyl group or derivative. R], R2, R-t prenyl groups R5 = CH 3 or prenyl groups
*This type has not been observed
OH Fig. (5). Bicyclo-[3.3.1]-nonane derivatives. Type A-C
These PBDs isolated so far from 7 genera, presented several common NMR spectroscopic features which have been firmly established. 1. All benzophenone derivatives show an unconjugated carbonyl at C-9 (5 207-210) belonging to bicyclo moiety. 2. All compounds possess one or two aliphatic methyl singlets (5 1.0-1.6) that correlate in HMBC with an aliphatic quaternary carbon at C-8.(5 47-51)
685
3. All derivatives present a sole aliphatic methylene group (CH26) in the bicyclo and one of their protons is usually observed as double doublet (8 1.9-2.2) in *H NMR spectrum. 4. Most of them show an aromatic AMX or AA BBX system in the B ring. AA BB system has been also observed. 5. Gem-methyl group at C-8 shows two ranges of 13C chemical shifts and they seem to be associated with the configuration at C-7. If 3 JH6ax-H7=10-13 Hz, ranges will be 8 15-17 and 5 22-24. If 3JH6ax-H7= 78 Hz, ranges will be 8 22-25 and 8 26-28 (Table 3). The bicyclic ring system requires the group at C-l (benzoyl or 3methyl-2-butenyl), and the prenylated chain at C-5 to be in an equatorial orientation and then there are only two stereochemical possibilities associated with the last one. Prenyl rest at C-7 has been observed both axial and equatorial, thus 4 absolute configurations are the maxima number of possibilities for this bicyclo. Structures of xanthochymol (43), cambogin (45) and clusianone (56) have been firmly defined by X-ray analyses [35,41,42]. When the molecular models of isogarcinol and clusianone are compared they showed to have the same relative configuration in the bicyclic rest (considering only the bridgehead asymmetrical carbons). Xantochymol contains another stereochemical possibility that originates the fusion of rings. These results suggest that the arrangements are not stereospecifics and any absolute stereochemistry can be observed. From de latex of G. cambogia, cambogin and camboginol were isolated in large quantities (5.5 and 37% respectively) [43]. Their structures (45) and (46) were elucidated respectively by chemical and spectral means which included NMR spectra (no bidimentional techniques). These compounds are very closely related to xanthochymol (43) and isoxanthochymol (44) respectively. All NMR, physical and chemical data suggested they are optical antipodes. Camboginol (46) was converted to cambogin by refluxing with benzene solution containing traces of HC1 or CF3COOH. Thus the absolute configuration of cambogin was easily deduced by comparison with camboginol.
686
(+) form
(44) Isoxanthochymol (45) Cambogin=Isogarcinol (optical antipode)
(43) Xanthochymol
(47) 18-0 methyl isogarcinol
(46) Camboginol-Garcinol (48) 18-O-methyl garcinol (54)Guttiferone E (optical antipode) (55)Guttiferone F( C-23 epimer)
(52) Guttiferone G. Ri=R2= 3-methyl-2-butenyl (53) Guttiferone B. R,=geranyl R2=CH3
(49) Guttiferone A
\ ^ Y ^ ~ ^ ^
(50) Guttiferone C
^
(51) Guttiferone D
Fig.(6). Bicyclo-[3.3.1]-nonano derivatives from (43) to (55).
A study of the hexane extract of the fruit of G. indica indicated the presence of garcinol and isogarcinol and suggested a modification for the structure of camboginol on UV spectral evidences [41]. Initially both products were suggested as type A PBDs but, a new study about the structure of isogarcinol based on X-ray crystallographic analysis permitted to corrected them and garcinol and isogarcinol were included as type B PBDs. They represent the same structures of camboginol and cambogin respectively [42]. Fuller et al. have suggested precedence for the names
687
camboginol and cambogin based on chronological and structural accuracy standpoints. Besides, the name garcinol has been also attributed to an aryl benzofuran isolated from Garcinia spp. From G. assigu collected in Papua, New Guinea, the 18-O-methyl ethers of isogarcinol (47) and garcinol (48) were isolated. Compounds were identified on the basis of NMR data and by comparison with the known garcinol and isogarcinol and the authors suggested the same relative stereochemistry in all these compounds [44]. The same type of Cotton effect by CD analysis was observed both in isogarcinol and garcinol with respect to the above mentioned methylated derivatives. However, both 3 JH6ax-H7= 13.9 Hz (prenyl group at C-7 in eq. position) and the 13 C chemical shifts assigned to gem-methyls at C-8 in the O-methyl derivatives are not in agreement with the stereochemistry of isogarcinol determined by X-ray analysis. Considering the X-ray study, isogarcinol exhibits a prenyl group at C-7 in axial position (chair conformation) and the NMR data of 18-O-methyl ether of isogarcinol suggest a prenyl group at C-7 in eq. position (chair conformation). Probably, the PBDs founded in G. assigu are O-methyl derivatives of the C-7 epimers of isogarcinol and garcinol respectively. Guttiferones A, C and D (49-51) isolated, from the extract of ground S. globulifera roots, [3] and guttiferone G (52), isolated from G. macrophylla [15], are the unique bicyclo-[3.3.1]-nonane derivatives that exhibit only one aliphatic methyl singlet belonging to bicyclo moiety. In these cases one isopentenyl group was attached on one of the alyphatic methyl carbons. The same relative configuration was suggested for guttiferones A, C and D. Guttiferone B (53), obtained from the same source [3] showed an oppositive configuration at C-7. A 10.4 Hz coupling constant between H-6 and H-7 determined a diaxial orientation between them and thus the 2,2dimethyl allyl group at C-7 was equatorial. Guttiferone E (54), isolated from G. ovalifolia and C. rosea [3], showed 'H and 13C NMR spectra identical to those of camboginol. However, its optical rotation [a]o = + 101°, was opposite in sign to that reported of camboginol [OC]D = - 125°. It was converted to isoxanthochymol by acid-catalyzed cyclization. Guttiferone E is thus the optical antipode of camboginol and a double bond isomer of xanthochymol. Guttiferone F (55) constitutes the unique PBD isolated from genus Allanblackia so far [4]. Its structure is very closely related to camboginol (46) and guttiferone E (54 (+) camboginol), two PBDs isolated from genus Garcinia and Clusia. Physical and spectroscopic data suggested that guttiferone F is the C-23 epimer of camboginol or
688
guttiferone E. Chemical evidences obtained by acid-catalyzed conversion of guttiferone F to 23-epi-cambogin permitted to the authors the verification of the epimeric configuration at C-23. De Oliveira et al. have published three main studies about chemical composition of Clusia's floral resins [18-20]. They have treated the floral resins with diazomethane in order to facilitate the separation of the major components. The first two works led to postulate the existence of the following PBDs in Clusia spp: grandone (22), clusianone, nemorosone, hydroxynemorosone, 7-epinemorosone and nemorosone II. Except grandone all of them showed to be bicyclo - [3.3.1]-nonane derivatives. Clusianone had been isolated both from the roots of C. congestiflora [44] and from fruits of C. sandinensis [27], later McCandlish et al. reported an X-ray diffraction analysis of clusianone [45] that defined the structure as (56), in which a type B nucleus and the equatorial 3-methyl-2butenyl group at C-7 were firmly established. The C-7 epimer (57) of clusianone (7-epiclusianone) has been successively isolated from Rheedia gardneriana [10]. Clusianone reported in two different works, [27 and 19] showed differences of C chemical shifts that suggest an epimer relationship between these compounds i.e. gew-methyl group at C-8 (Table 3). NMR data of structures proposed for nemorosone [19] and nemorosone II [18] isolated by De Oliveira et al. were identical with those of O-methyl derivatives obtained employing nemorosone isolated from Clusia rosea in a new study [40]. Nemorosone as it is in the nature was fully characterized by NMR spectroscopy techniques that included nOe difference spectroscopy experiments on the natural product. After comparison of the H and C NMR chemical shifts of the natural product nemorosone and its methyl derivatives with O-methyl nemosorone and O-methyl nemorosone II previously described, we proposed that only there exist one nemorosone. The O-methyl nemorosone (type C) and O-methyl nemorosone II (type A) previously isolated from Clusia spp. are the O-methyl tautomers of the same natural product that is named nemorosone (58). A structure type A for nemorosone was confirmed on the basis of nOe difference spectroscopy experiments. Saturation at the frequency of the Me-33 axial gave positive increments at the aromatic protons signals (H-12 and H-16) of the benzoyl group. In the same way, irradiation of the H-6 eq. showed an interaction with methylene protons at C-22 and when the latter protons were irradiated, interactions with methylene protons at C-6 and Me-25 were observed. These results prompted us to define that
689
nemorosone is a type A PBD. An analogous situation was proposed for hydroxy nemorosone (59) but it has been isolated only by De Oliveira et al. up to date. Nemorosone was identified as the major component from the floral resin of C. rosea (48%), C. grandiflora (69 %), C.insignis (43 %) and C. nemorosa (38 %) [19]. Table 3. "C chemical shifts of gem-methyls at C-8 in bicyclo-[3.3.1|-nonane derivatives. Compounds h 13C (gem-methyls at C-8) Me ax.: Me eq. Methyl Clusianone 15.9 :22.4 Hidroxynemorosone 16.1 :24.4 Clusianone* 22.5:27.0 Methyl-O-7-epinemorosone 23.9:27.2 Methyl-O-nemorosone 15.7:23.4 Guttiferone F 23.2 :27.3 Guttiferone B 16.5:23.8 Guttiferone E 23.2 : 27.3 Isoxanthochymol 22.7:27.1 O-methyl Chamone I 16.4 :24.7 Chamone II 16.6:24.7 Propolone A 15.9:23.7 Aristophenone * 15.8:23.7 Nemorosone* 15.6:23.2 Plukenetione D (acetate) 23.3 :27.1 Plukenetione E (acetate) 22.0 :26.0 Plukenetione F 23.5 :27.3 Plukenetione G 22.5 :26.8 18-O-methyl isogarcinol 16.3 :23.9 18-O-methyl garcinol 16.3 :22.6 15,16-dihydro-16-hydroperoxyplukenetione F 23.6:26.8 Sampsonione K 22.2 :26.8 Sampsonione L 22.2:26.8 Sampsonione M 24.0 :26.5 Xanthochymol 23.2:27.5 Insignone 23.9:27.2 Spiritone 15.8:24.1 Hyperibone A 16.1.: 23.9 Hyperibone B 16.0:23.9 Hyperibone C 16.5 :24.7 Hyperibone D 16.5 : 23.3 Hyperibone E 16.9:23.9 Hyperibone F 17.0:23.8 Hyperibone G 16.5 : 23.3 Hyperibone H 25.1:26.8 Hyperibone I 23.9:27.3 Ochrocarpinone A 15.9:23.8 Ochrocarpinone B 16.5:23.2 * Only one tautomer has been considered
References 19 19 27 18 18 4 3 3 35 9 9 11 49 40 47 47 47 47 44 44 52 51 51 51 35 20 20 8 8 8 8 8 8 8 8 8 14 14
A similar misassignment was suggested for 7-epinemorosone (60) but due to the lack of authentic sample the observation was only a hypothesis [40]. Bittrich et al. reinvestigated the structure of 7-epinemorosone
690
employing the O-methyl derivative isolated from Tovomitopsis saldanhae. Finally the structure was corrected and considered as type A BPD [46]. From Clusia spp. spiritone, insignone and scrobiculatones A and B were isolated [20]. Their structures (61-64) were established on spectral evidences which included 2D NMR techniques. Comparison of 13C chemical shifts assigned to gem-methyl group at C-8 of spiritone and 7epiclusianone [19] suggested that the stereochemistry between them is very closely related. When the same comparison was done between insignone and 7-epinemorosone, similar 13C chemical shifts were observed between above mentioned groups and then insignone and 7-epinemorosone show a similar stereochemistry respect to bicyclo moiety. On the other hand, scrobiculatones A and B seem to be very closely related to nemorosone (58) on the basis of the same analysis. Henry et al. as part of their phytochemical studies of Caribbean Guttiferae examined the extract of C. plukenetii [47]. The investigation permitted to isolate plukenetiones D and E as acetyl derivatives and plukenetiones F and G. Plukenetiones D and E (65,66) are a tautomeric pair of PBDs. The NMR data of regioisomeric pair plukenetiones F and G (67,68) provided evidence for a 2,2-dimethyl-2H-pyran moiety. The unusually high field signal observed for one methyl of pyran ring of plukenetione F may be due to shielding effects from the phenyl group. This effect is absent in plukenetione G. Although, the absolute configurations of plukenetiones D and E have not been determined Henry et al. suggested that plukenetiones D/E and 7epinemorosone are the same product. This consideration is logic but, these products could be enatiomers in like manner guttiferone E and camboginol. Grossman and Jacobs [48] developed a comparative study among PBDs isolated from Clusiaceae in order to clarify some structural aspects but, structures mistankely identified until that moment were included, i.e: Omethyl nemorosone [19] and O-methyl-7-epinemorosone [18]. This situation conditioned comparisons between compounds that nowadays are considered to have the same structure (i.e. O-methyl nemorosone [19] and O-methyl nemorosone II [18] are the methylated tautomers of the same compound named nemorosone (58). Therefore, the conclusions obtained should be considered carefully. Particularly, the conformations associated (chair or twist-boat) with the more saturated ring in bicyclo-[3.3.1]-nonane derivatives. Some authors have suggested that the conformation of the mentioned ring depends of the configuration at C-7 [18,40,47,48]. When prenyl group at C-7 is equatorial predominates the chair conformation and
691
when the isopentenyl group is in axial position (chair conformation) the twist-boat conformation has been suggested as predominat on the basis of the existence of two 1,3-diaxial interactions between the isopentenyl group at C-7 and C-2 and C-4 if the chair conformation is conserved. These observations are logics but, it is also possible to consider that both conformations are represented at the equilibrium in similar quantities. We developed a semiempirical computational procedure for conformational search and energy minimization (AMI, MOPAC v6) in order to clarify this point [49]. The results assessed the chair as the predominant conformation for stereoisomer R (equatorial isoprenyl group at C-7). However, the twist boat and the chair conformers of S stereoisomer (axial isopentenyl group at C-7 in chair conformation) showed to have an energy difference < 0.7 Kcal/mol, suggesting that both conformations are represented at the equilibrium. Keto-enolic equilibrium observed in PBDs is associated to the process of conversion between tautomers which has been evidenced by two sets of NMR signals in compounds as nemorosone (58), clusianone (56), aristophenones (71) and xerophenones A and B (95,96). Equilibriums showed in Fig. (8) are suggested on the basis of that the velocity of conversion between conformers is faster than that between tautomers. On the other hand, all PBDs analysed by X-ray diffraction methods present a chair conformation both when isopentenyl groups occupies an axial or equatorial position [27,40,49,50]. Examination of the reported values of vicinal coupling constant for a series of PBDs gave two ranges. In the case of nemorosone (58) (as it is in nature or in O-methyl derivatives) and guttiferone B (53), the coupling constant between H-6 axial and H-7 has a value of 10-13 Hz, suggesting that the 3-methyl-2-butenyl substituent occupies an equatorial position in the predominant chair conformation [3,18,40]. On the other hand, there are other examples where the coupling constant between H-6 axial and H-7 reaches a value of 7-7.5 Hz. That is the case for the compounds guttiferone A and F (49,55), plukenetione E acetate (66) and plukenetione G (68) [3,47]. All of them possess one isopentenyl group in axial position (if the chair conformation is considered) but their 3J values are not characteristic of the chair conformation of cyclohexane derivatives (Jaa=10-12 Hz, Jae=Jee=2-5 Hz). This result can be justified if both chair and twist-boat conformations contribute to the vicinal coupling constant observed.
692
(64) Scrobiculatone B. R=C 5 H 9 eq*.
(56)Clusianone R=C 5 H 9 eq*
(67) Plukenetione F. R= C 5 H 9 ax •
(57) 7-epiclusianone R=CsH 9 ax*
(58) Nemorosone R(= R 3 = H, R 2 =eq*. (59) Hydroxy nemorosone R,= H., R 2 = eq*., R 3 = OH (60) 7-epinemorosone R ( - R 3 - H, R 2 - ax*. (65) a Plukenetione D R,= CH 3 CO, R 2 =ax*., R 3 =H (66) b PUkenetione E R,= CH 3 CO, R 2 =ax*., R 3 =H R 2 - C5H9
(63) Scrobiculatone A. R=C 5 H 9 eq*. (68) Plukenetione G. R=C 5 H, ax*.
(70) Chamone II R=C 5 H 9 eq*
(69) Chamone I R=C 5 H 9 eq* *axial or equatorial in chair conformation
(62) Insignone
Fig. (7). Bicyclo-[3.3.1]-nonane derivatives from (56) to (70).
(61)Spiritone
693
Fig. (8). Tautomers and conformers in bicyclo-(3.3.1)-nonane drivatives
Lokvan et al. studied the chemical composition of the trunk latex of C. grandiflora [9]. After treatment with diazomethane three PBDs were obtained: the known nemorosone and two new compounds closely related named chamones I and II (69,70). Comparison with the spectral data of nemorosone established that chamone I contained an additional prenyl group and a terminal methylene carbon. The relative configuration of chamone I and nemorosone was determined using ID NOESY pulse sequences. Selective irradiation of the methoxyl group at C-2 permitted to suggest the chair conformation in the B ring and the same relative configuration for the mentioned compounds. The NMR data and the lack of reactivity with diazomethane suggest that the C-3 isopentenyl group has ring closure at the C-2 enolic hydroxyl in chamone II. A study of the fresh fruits of G. aristata led to isolation of tautomeric pair of PBDs, aristophenones A and B [49]. Aristophenone (71) was presented in CDCI3 as a tautomeric pair in a ratio 1:1, as evidenced by two sets of NMR signals, the consequence of the presence of the enolizable 1,3-diketone system. *H and 13C chemical shifts of the 12 olefinic methyl groups were assigned. Ideally, the protons of each methyl group could be irradiated and the nOes recorded of those are cis to the corresponding methylene groups (13, 25 and 30). However, selective saturation and NOE detection is difficult when both target and enhanced signals are situated as close together as they are in a tautomeric pair in a ratio 1:1. Another approach to this problem involves C chemical shifts. The shifts of methylene carbons in conjugated unsaturated fatty acids are sensitive to whether they are situated in CM (Z) or trans {E) configurations. The former
694
are shielded (ca. 5 27.5) relative to the latter (ca. 8 32.5) due to stericcompression effects. Methyl carbons exhibit the same type of geometrical characteristics in corresponding systems. If we examine the three pairs of olefinic methyl groups (13, 14; 25, 26; and 30, 31), we see that methyls 13, 25, and 30 are cis to methylene 10, 22, and 27, respectively, while methyls 14, 26, and 31 are trans to these groups. From these discussions the chemical shifts of the cis methyl carbons were shielded relative to those of trans methyls. The chemical shifts of the directly attached methyl protons could then be determined by means of the HSQC experiments. The *H and 13C chemical shifts of the 12 olefinic methyl groups [49] were reported in Table 4. Table 4 . ' H a n d Position 13 14 25 26 30 31
1
C chemical shifts of olefinic methyl groups la 8 13 C 5'H 17.7 1.58 25.3 1.73 18.0 1.75 26.1 1.59 17.7 1.53 26.0 1.81
lb 8 13 C 17.5 25.6 17.9 25.7 17.1 26.2
I 'H .62 .71 .68 .65 .46 .82
The tautomeric mixture was acetylated employing Ac2O/pyridine in order to facilitate the structural analyses and two tri-acetylated compounds were obtained. Aristophenone is similar to clusianone the main difference was associated with the different substitution of phenolic ring. Propolone A (72) was isolated in large quantities from an ethanol extract of Cuban propolis [11]. This finding and the isolation of nemorosone from C. rosea has permitted to suggest the role of this plant in the chemical composition of Cuban propolis. However, propolone A has not been reported as a component of Clusia rosea's floral resin, the main Clusia sp. distributed in Cuba. Thus, propolone A can be a derivative of nemorosone and the conversion would occur whereas the process of conservation and treatment of propolis samples and extracts are carried out. Some authors have reported that natural PBDs are unstable mainly in solutions and our experiences are in agreement with those observations. On the other hand, Porto et al. noted the structural similarities between the PBD isolated from floral resins of C. scrobiculata scrobiculatone A (63) [20] and propolone A. However, the first one has not been isolated from floral resins of C. rosea so far.
695
30
31
O
OH <"•
y
HO.
O
26
(71) Aristophenone A 25
O
Aristophenone B
O / O
OH (72) Propolone A
(73)SampsonioneK R= (74)SampsonioneL R=CH3
(75) Sampsonione M R=geranyl Fig. (9). Bicyclo-[3.3.1]-nonane derivatives from (71) to (75).
Sampsoniones K-M (73-75), isolated from H. sampsonii [51] are very closely related to PBDs isolated mainly from genera Garcinia and Clusia and they seem to be derivatives of superior homologues to plukenetiones D and E or 7-epinemorosone. Molecular models of sampsoniones K-L disclosed that, by its formation the tricyclic system itself sets up the relative configuration at the C-l and C-8. NOE data indicated that the cyclohexanone ring adopts the chair conformation and the isoprenyl group at C-7 has an axial position.
696 A phytochemical study of H. scabrum, a medicinal plant used in Uzbekistan, led to isolation of hyperibones A-I [8]. Hyperibones A-G (7682) showed structures very closely related and all of them seem to be derivatives of nemorosone (type A PBDs). The structural differences among them were associated with modifications or arrangements in the 3methyl-2-butenyl rests. C chemical shifts exhibited by gem-dimethyl at C-8 indicated an equatorial orientation of isoprenyl chain at C-7. On the other hand, hyperibone H-I (83,84) can be considered derivatives of type B PBDs. In all of them a dihydrofuran ring was firmly established on the basis of spectral evidences. It is very interesting to note that the dried aerial parts of H. scabrum were extracted employing hot methanol (60° C) which could facilitate arrangements of the prenyl groups at C-3 and C-5. Chaturvedula et al. identified other derivatives of nemorosone named ochrocarpinones A-C (85-87) which constitute the first PBDs isolated from genus Ochrocarpus [14]. The structures of ochrocarpinones B-C showed NMR data very similar to those observed in hyperibones A-G. The presence of a 2,2-dimethyl-3-hydroperoxy-2-H-dihydropyran ring in ochrocarpinone A was indicate by a positive peroxide test with FeSCN. Ochrocarpinone A was a derivative of plukenetione G, but, the 13C chemical shifts were most similar to scrobiculatone A (C-7 epimer of plukenetione G) a derivative of nemorosone isolated from floral resins of Clusia sp. Ochrocarpinone C (87) seems to have the same structure of hyperibones A or B, two epimers isolated from H. scabrum, stereochemistry of ochrocarpinones was not reported. 15,16-dihydro-16-hydroperoxyplukenetione F (88) isolated from fruits of C. havetiodes [52], is a regioisomer of ochrocarpinone A. The proton and carbon shifts at position 18 (5c 93.5, 8H 4.66) were deshielded relatively to those of a secondary alcohol suggesting that a hydroperoxy group was present at this position. The presence of hydroperoxy group was confirmed by FeSCN test. Garcinielliptone I (89) was isolated together other two phloroglucinol derivatives and terpenoids from the seeds of G. subelliptica [53]. NMR data were very similar to hyperibone B but the authors observed differences between the optical rotations of these compounds and suggested an enantiomer relationship between them. Ethylenic functions in the side chains of PBDs seem to be very sensitive to acid solutions. In organic or inorganic acids converted xanthochymol, guttiferone E, guttiferone F and cambogin to the corresponding pyran ether
697
derivatives [6,15,40]. Knowledge of this chemical behaviour is very important, as some PBDs described as natural products might well be artefacts formed as result of the methods used for the isolation or the treatment associated to the conservation process of the natural source. HO
(76) Hyperibone A
(77) Hyperibone B (89) Garcinielliptone I (optical antipode)
OH (78) Hyperibone C
HO-
OH (79) Hyperibone D
(82) Hyperibone G
(80) Hyperibone E
OH (81) Hyperibone F
(83) Hyperibone H
HOO
(85) Ochrocarpinone A
(86)Ochrocarpinone B
Fig. (10). Bicyclo-[3.3.1]-nonane derivatives from (76) to (89).
(88) 15,16-dihydro-16-hydroperoxyplukenetione F
698
Other PBDs Fractionation of the hexane extract of the fruit of C. plukenetii permitted to obtain plukenetione A (90), the first PBD with an adamantyl nucleus [54]. Its tri-oxigenated ring is non-aromatic and contains three carbonyl groups (8 201-203). The structure was determined by tracing the connectivities shown in the HMBC spectra and nOe experiments. The stereochemistry at H-6 was deduced by the W-coupling to H-10, and nOe interaction with C-26 methyl protons and HMBC observations. It is very interesting to note that the individual protons of the methylene pairs both at C-ll and C-16 (attached to asymmetric carbons) showed magnetic equivalence. Plukenetiones B and C, two colourless oils, were isolated from the same source of plukenetione A, the ground fruits of C. plukenetii [47]. Plukenetione B (91) contains a tetracyclo [5.3.3.19>12O1>5] tetradecane10,11,14-trione moiety and plukenetione C (92) showed a 4,5 dioxatetracyclo [7.3.3.111>14O1>7] hexadecane -12,13,16 trione moiety. Although the numbering between both compounds differs they are closely related. Plukenetione C exhibited an uncommon peroxide group among natural PBD. All of them were characterized by the presence of three unconjugated carbonyls included in a six member ring where the ketoenolic equilibrium can not be established. Their molecular formulas and spectroscopic evidences permitted to identify the presence of a benzoyl group and four five-carbons units. Policyclic structures were determined on the basis of NMR data essentially. New derivatives of plukenetiones A and C were isolated from C. havetiodes var. stenocarpa [52]. The new compounds 28,29-epoxy plukenetione A (93) and 33-hydroperoxy isoplukenetione C (94) showed to be very closely related with above mentioned compounds respectively. All of them were observed in minor quantities and the structural differences respect to above mentioned compounds were associated with modifications of prenyl rests. Essentially, oxygenated functions for example epoxy and hydroperoxy groups were identified by chemical and spectroscopic means. Xerophenones A and B (95,96) were obtained from the hexane extract of the leaves and twigs of C. portlandiana [50]. The H NMR spectrum showed a 4:1 mixture of tautomers in CDCI3. These compounds are derivatives of 11-oxatricyclo [4.3.1.14'10] undecane-7,9-dione and their structures were deduced on the basis of NMR data. Considering the existence of the keto-enolic equilibrium in xerophenones was possible to
699
suggest some structural possibilities very closely related among them. In a new study xerophenone B was corrected on the basis of careful analysis of the nOe interactions and HMBC connectivities. Structures of xerophenones deserve comment because some structural details result very interesting. In xerophenones A and B no differences were observed between chemical shifts of the same carbon in each tautomer (i.e: C-6 and C-9). Other PBDs that have been analysed in their keto-enolic equilibrium showed differences up to 6 ppm. For example: nemorosone, aristophenones, clusianone and machuone [40,49,27].
(91) Plukenetione B
(90) Plukenetione A: R,=,
(93) 28,29-Epoxyplukenetione A: R,=
(92) Plukenetione C:R=
OOH (94)33-hydroperoxyisoplukenetione C: R=
o-H'"o
(96) Xerophenone B (95) Xerophenone A Fig. (11). Other polyisoprenylated benzophenone derivatives from (90) to (96).
700 Table 5. Other PBDs isolated from Clusiaceae Compound Plukenetiones A-C (90-92) 28,29-epoxiplukenetione A (93) 33-hydroperoxy isoplukenetione C (94) Xerophenones A and B (95,96) Sampsoniones A-J (97-106) Nemorosonol (107)
Sources C. plukenetii C. havetiodes var. stenocarpa C. havetiodes var. stenocarpa C. havetiodes var. stenocarpa C. portlandiana H. sampsonii C. havetiodes var. stenocarpa C. nemorosa
References
47,58 52 52 52 50 51 52 43
Extensive phytochemical studies developed with H. sampsonii led to the isolation of a family of caged PBDs named sampsoniones A-J (97-106) [51]. Many of them exhibited complex arrangements that were firmly established on the basis of spectroscopic evidences. Authors have suggested a very interesting biosynthetic via in which the same precursor for all these compounds, a bicyclo-[3.3.1]-nonane derivative, has been considered. Sampsoniones C-J showed three unconjugated carbonyls (5 203-207) similar to plukenetiones A and B isolated from C. plukenetii. Sampsoniones K-M have been also isolated but they were mentioned when PBDs with a bicyclo-[3.3.1]-nonane were considered. The stereochemistry of plukenetione B (91) was not determinated in the publication describing this compound but the isolation of sampsoniones from H. sampsonii, permitted to suggest the stereochemistry by comparison with sampsoniones C and G [48]. Both plukenetione B and sampsoniones C and G seem to have the same policyclic system. The main differences among these compounds are associated with R group and configurations at H-a and H-b (a,jS or ftcc). Nemorosonol was obtained from the benzene extract of ground fresh fruits of C. nemorosa. Spectral data indicated that (107) is a PBD incorporating four five-carbons units. Structural details were determined on the basis of NMR data. NMR spectral parameters were recorded in C&D6 solutions because of the predominance of one tautomer was observed. Nemorosonol represents the unique PBD with a tricyclo[4.3.1.O ' ]-decane skeleton isolated so far [39]. The structure and stereochemistry at C-5 were revised in a new study employing an X-ray crystallographic method. This study also permitted to identify the presence of two cristallographically independent molecules for nemorosonol in the unit cell [55]. In plukenetiones A-C and sampsoniones A-J the benzoyl moiety was vicinal to the aliphatic quaternary carbon that supports gem-methyls. This structural characteristic could be considered in order to extend the
701
classification proposed for PBDs with a byciclo [3.3.1] nonane system. All PBDs isolated so far in which a prenyl group is arrangement by attach on the acetate derived ring to generate a new C-C bond could be classified as type A or B PBDs. Only those PBDs considered by us as simple derivatives are excluded. Xerophenones A and B can be classified as type B PBDs considering the vicinal position of benzoyl group to the two oxygenated carbons included in the keto-enolic equilibrium.
o R= geranyl
(97) Sampsonione A R= Geranyl
(99)Sampsonione C R,=
(98) Sampsoni one B R=^
(100) Sampsonione D Ri=< (101) Sampsonione E R, = 0 (ketone)
H
H
(102) Sampsonione F R= geranyl R]= O (103) Sampsonione GR= 3-methyl-2-butenyl R,=^-OH (104) Sampsonione H R= geranyl R|= H ;
(105) Sampsonione I R= Geranyl
H O
O O
OH
O
(106) Sampsonione J R= Geranyl (107) Nemorosonol Ri=R2=R3= 3-methyl-2-butenyl Fig. (12). Other polyisoprenylated benzophenone derivatives from (97) to (107)
702
BIOLOGICAL ACTIVITY Natural products of plant origin offer a wide variety of active compounds that could meet the demand for base compounds of drugs. PBDs have shown different biological properties but, probably the three most important are the cytoprotection against HIV-1 in vitro of guttiferones [3-4], antimicrobial properties [5-11] and cytotoxic activity found in diverse nucleus [12-16]. Interesting bioactivity such as antibacterial activity against methicillin-resistant Straphylococcus aureus [6-8], antioxidant activity [44,59,60] and cytotoxic activity due to induction of apoptosis in human leukemia cells have been reported [61,62]. Recently garcinol (46), also called camboginol, has attracted considerable interest because of its associated beneficial health properties, including antiulcer activity [59], anti-glycation activity [60], cancer chemopreventive activity against colonic aberrant crypt foci (ACF) in an animal model [63], induction of apoptosis through cytochrome c release and activation of caspases in human leukemia cells [61]. It also showed strong antioxidant activity [59,60]. Anti-HIV-1 activity There is current widespread interest in the compounds from Guttiferae (Clusiaceae) because the plants of this family has proved to be a valuable source of leads to HIV-1 inhibitory natural products. The chromeno-coumarin calanolide A, isolated from Calophyllum langiferum var. Austrocoriaceum, is nowadays in clinical trials as a nonnucleoside specific inhibitor of HIV-1 reverse transcriptase [64,65]. Boyd et al. in a bio-assay oriented study reported that the anti-HIV activity exhibited by extracts of some Guttiferae species was attributed to a series of prenylated benzophenone derivates. Bioassay-guided fractionation of the HIV-inhibitory activity extracts of three different genera of Guttiferae {Garcinia, Clusia and Symphonia) led to isolation of prenylated benzophenones, guttiferones A-E, as the principal active constituents [3]. Guttiferones A-E were tested in the NCI's primary anti-HIV screen and all showed a similar level of activity. Their inhibited the cytopathic effects of in vitro HIV infection in human Tlymphoblastoid CEM-SS cells, with EC50 values of 1-10 \ig mL"1, while cytotoxicity occurred at concentrations greater than 50 jag mL" .
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Isoxanthochymol (44), a cyclized derivate of guttiferone E (54), was inactive [3] suggesting that the presence of an enol functionality is necessary for anti-HIV activity. The HIV-inhibitory activity in extracts of Allanblackia stuhlmannii was tracked, via bioassay-guided fractionation, to guttiferone F [4]. As other guttiferones, guttiferone F (55) exhibited partial (not achieving 100%) cytoprotection against HIV-1 in vitro (EC50 23 jug mL"'), as well as direct cytotoxicity (IC50 of 82 |ig mL"1) to the host cells. The investigation of the extract of Marila laxiflora, which showed activity in the NCI's anti-HIV primary screen, led to the isolation of laxifloranone a new prenylated phloroglucinol, laxifloranone having a a cinnamic acid residue. [66]. As guttiferones, this compounds exhibited only partial cytoprotection (80%) and moderate inhibition of the cytopathic effects of HIV-1 infection in CEM-SS cell line with an EC50 value of 0.62 |4.g mL"1 and an IC50 = 6.6 \ig mL"1. Four SBDs (7-10), were isolated from leaves of Vismia cayennensis [25]. Only vismiaphenone D exhibited a significative activity (EC50 ca 11 (j,g mL" ) and as guttipherones complete cytoprotection was not achieved. Vismiaphenones might be viewed as precursors of guttipherones, additional prenylation and subsequent carbocyclization woukd lead to guttipherone analogues. Garciosaphenone A (38) showed interesting anti-HIV-1 activity [34]. It was very active in HIV-1 reverse transcriptase assay (IC50 = 23.9 fig mL"1). Its activity was comparable to those of fagaronine hydrochloride, but 7-13 times less sensitive than nevirapine. Nevertheless, garciosaphenone A was toxic in the syncytium test. Antimicrobial activity The PBDs showed antimicrobial properties. The most interesting is the the antibacterial activity against methicillin-resistant Straphylococcus aureus (MRSA). Kolanone (18) ( 1 % EtOH), isolated from the fruit pulp of G. kola, exhibited antimicrobial activity against a range of organisms (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumoniae and Candida albicans) [5]. 3-geranyl-2,4,6- trihydroxybenzophenone (36) showed inhibitory effects against Candida albicans secreted aspartic proteases (SAP) with
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IC50 values of 40 \xg mL"1 [6]. The SAP of C. albicans have been shown to be a major virulence factor in Candida infections [67]. Inhibition of SAP has been proposed as a new approach in the treatment of candidosis [68]. 3-geranyl-2,4,6-trihydroxybenzophenone was also found to have antimicrobial activity against C. albicans, C. neoformans, S. aureus and MRSA with IC50/MIC of 4.0/25 ng mL'1, 1.5/3.13 ng mL"1, 1.5/3.13 \xg mL" and 2.0/3.13 p.g mL" , respectively (positive control for C. albicans and C. neoformans is amphotericin B, IC50/MIC of 0.04/0.078 (ig mL"1 and 0.3/0.625 ng mL"1 respectively; tetracycline for S. aureus and MRSA, IC50/MIC of 0.15/0.3125 ng mL"1 for both organisms) [6]. Also some bicyclo-[3.3.1]-nonane derivatives have shown antibacterial activity against MRSA. Bioassay-guided fractionation of pericarp extracts of two Garcinia species (G. purpurea and G. subelliptica) resulted in the isolation of five benzophenone derivates, garcinol(46), isogarcinol (45), xanthochymol (43) and isoxanthochymol (44). These compounds were evaluated for their antibacterial activity against MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) [7]. Garcinol and xanthochymol showed strong anti-MRSA activity. The MIC value of xanthochymol ranged from 3.13 to 12.5 |j.g/mL against MRSA and is nearly equal to that of the antibiotic, vancomycin (6.25 (j.g/mL) which is currently used to treat MRSA infections. Isogarcinol and isoxanthochymol were less active than garcinol and xanthochymol, which suggests that a chelated hydroxyl group at C-l is involved in the inhibitory activity. Also hyperibones A-D (76-79) were screened for antibacterial activity by the disc diffusion test against MSSA and MRSA [8]. Hyperibones A, B and D showed mild activity (zone diameter: 9.5, 9.2, 6.0, 9.3 mm, for MRSA and 9.0, 9.1, 6.0, 9.0 mm, for MSSA, respectively; tetracycline 34 mm, for MRSA and MSSA; quercetin 8 mm, for MRSA and MSSA) Nemorosone (58), and chamone I (69), isolated from floral resins and trunk latex of Clusia grandiflora respectively, showed pronounced antibacterial activity on honeybee pathogens Paenibacillus larvae and Paenibacillus alvei in TLC bioassay [9]. These compounds are structurally similar, differing only the degree of prenylation. Chamone II and methyl ethers of nemorosone and chamone I were inactive and this suggest that the presence of an enol functionality is necessary for antibacterial activity in these molecules.
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7-Epiclusianone (57) was active against the phytobacteria Clavibacter michiganense (MIC = 4 (ig mL"1) and the enterobacteria Listeria monocytogenes and Straphylococcus aureus (ICioo = 80 jag mL"1), and in vitro against the trypomastigote forms of Trypanosoma cruzi (LC50 = 260 |wg mL* , 5 1 8 jxM). From this in vitro activity, 7-epiclusianone was also tested in vivo in experimentally infected mice, but it was resulted inactive [10]. Propolone A was tested for its antimicrobial and fungicidal activities against several Actinomyces, Gram positive and Gram negative bacteria and yeast [11]. Major activity was observed against two Gram positive bacteria, Streptomyces chartrensis and Streptomyces violochromogenes with a MBC (minimal concentration required to eliminate 99% of the microorganism inoculated) = 50 for both bacteria, MBC = 10.5 for gentamycin. The antimicrobial action of nemorosone isolated from floral resins of Clusia rosea against 44 micro organisms was tested employing the method of agar plate double radial diffusion, using 6.8 x 10 colony forming units per mL in each case. Both Gram positive and Gram negative bacteria were evaluated and the best results were observed against two Gram positive bacteria, Bacillus subtilis (MIC = 200 fig mL"1) and Staphylococcus aureus. (MIC = 500 \xg mL*1). Only the 20.6 % of the Gram negative bacteria studied were inhibited by the presence of nemorosone. The product was more active against Gram positive bacteria [69]. Antioxidant activity Free radicals and reactive oxygen species play an important physiological role, they are essential for production of energy, synthesis of biologically essential compounds, in signal transduction and phagocytosis, a critical process of immune system [70]. On the other hand, there is increasing evidence that these reactive oxygen species may play a causative role in a variety of disease including heart disease, atherosclerosis, aging and cancer [71,72]. Consequently, the role of antioxidants has received increased attention during the past decade. Krishnamurthy [73] observed that garcinol had no antioxidative activity in lipid peroxidation. Yamaguchi et al. evaluated the chelating, antioxidative and radical scavenging activities of garcinol, demonstrating that it has a strong antioxidant activity [59,60].
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Their confirmed these results in the mycellar system [60]. Garcinol exhibited weak antioxidative activity in measurement of lipid peroxidation in mycellar system, being at most one-half as strong as a-tocopherol and weaker than catechin. In the Fe2+ chelating activity assay garcinol interfered with the formation of ferrous ion and bipyridyl complex (chelating agent), suggesting that it has chelating activity [60]. The rate was the same of citric acid and lower than EDTA and DTPA. Garcinol was the only waterinsoluble reagent used in this assay, and it was added to the reaction mixture with SDS. This condition might be disadvantageous for this substance. In the DPPH (l,l-diphenyl-2-picrylhydrazyl) assay, garcinol exhibited potent radical scavenging activity at an extent almost 3 times higher than a-tocopherol and comparable to 85% of the activity of ascorbic acid [84]. Also in the superoxide anion scavenging assay in the phenazine methosulfate/NADH-nitroblue tetrazolium system, garcinol exhibited potent superoxide anion scavenging activity almost comparable to that of gallic acid and stronger than that of (+)-catechin [60]. The radical scavenging activity of garcinol have been confirmed using the electron spin resonance (ESR) spin trapping method which observe the reaction between superoxide anion and radical scavenger more directly. In the hypoxanthine/xanthine oxidase system, emulsied garcinol suppressed the signals of the superoxide anion-DMPO adduct in the ESR method, in a dose dependent manner [59]. The superoxide anion scavenging rate of garcinol was almost the same as that of a-tocopherol and less than those of ascorbic acid and catechin at the same weight/volume concentration. The activity of garcinol and a-tocopherol might depend on their solubility in an aqueous system. In the Fenton reaction system, garcinol suppressed hydroxyl radical formation, dose-dependently, more strongly than a-tocopherol and catechin [59]. The suppression of hydroxyl radical in this system may be caused by direct scavenge but also by inhibition of the Fenton reaction by Fe + chelation for chelating activity of garcinol. In the H2O2/NaOH/DMSO system, designed to evaluate both watersoluble and oil-soluble free radical scavengers, garcinol also suppressed the formation of free radicals such as methyl radical, hydroxyl radical and superoxide anion [59]. Garcinol suppressed hydroxyl radical in this nonFenton type ROS generating system, in this case the reaction mechanism
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was the direct radical scavenge and not the inhibition of Fenton reaction by chelation. The specificity against each radical species was compared with that of a-tocopherol, a typical lipophilic natural antioxidant. In methyl radical scavenging activity, garcinol was weaker than a-tocopherol, but against hydroxyl radical scavenging activity, it was stronger. The superoxide anion scavenging activity of garcinol showed almost the same level of activity as a-tocopherol. These tendencies were consistent with the results in the hypoxanthine/xanthine oxidase and Fenton systems. This results confirmed that scavenging activity of garcinol against hydroxyl radical was stronger than that a-tocopherol and its other scavenging activities were weaker. Clinical applications of garcinol was performed by Yamaguchi et al. that investigated the anti-glycation and antiulcer activities of this compound [59,60]. Many reports showed a significant role for glycation (nonenzymatic reaction of protein with reducing sugar) in diabetic complications, Alzheimer's disease and aging processes. Oxidative reactions are known to be included in the later process of glycation. The chelating, antioxidative and radical scavenging activities observed for garcinol were presumed to contribute to the mechanism of glycation inhibition. Garcinol exhibited potent glycation-suppressing activity in a bovine serum albumin/fructose system [60]. It suppressed fluorescence and protein cross-link formation in the reaction system. The mechanism of the activity of garcinol was inferred to be the chelation of metal ions which catalyzed the glycation. The importance of metal ion-catalyzed oxidation was mentioned by Hayase [74] and by Sajithlal [75] and to confirm this finding, cupric ion was added to the assay system and the activity of garcinol was then reduced. Reactive oxygen species (ROS) have been shown to play a critical role also in gastric ulcer [76]. Radical scavenging activity of garcinol is expected to be useful for preventing diseases caused by ROS, such as stress-induced gastric ulcer. The antiulcer activity of garcinol was examined in vivo using both indomethacin-induced and water immersion stress-induced models [59]. In the water immersion stress model, garcinol suppressed gastric injury formation at the same extent of cetraxate-HCl used as a positive control, and reduced the indomethacin-induced gastric injury. These results suggested that garcinol may have potential as an antiulcer drug. Although the mechanism of its antiulcer activity is not yet
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understood, garcinol may scavenge reactive oxygen species on the surface of gastric mucosa, thus protecting cells from injury. To evaluate the antioxidant mechanism of garcinol Sang et al. developed studies with the objective of characterize the reaction products of garcinol with the stable radical DPPH and with peroxyl radicals generated by thermolysis of the azo initiator azo-bis-isobutyrylnitrile (AIBN) [77,78]. The antioxidant process of reaction among DPPH and antioxidants is thought to be divided into two stages: 1. DPPH' + AH -»• DPPHH + A" 2. A" + X' -» nonradical products AH is the antioxidant, A' is the antioxidant radical and X is another radical species or the same species as A . Although the first stage is a reversible process, the second stage is irreversible and must produce stable radical terminated compounds. The reaction of garcinol with the stable radical DPPH generated two major reaction products, GDPPH-1 and GDPPH-2. The antioxidant process of reaction between garcinol and peroxyl radicals generated by thermolysis of AIBN is thought to divided into three stages: 1.
RN=NR-* 2R'+N 2 2R" + 2O2 -• 2ROO'
2.
2ROO" + AH -* ROOH + A"
3.
A' + X' -» nonradical products
AIBN decomposed thermally to yield alkyl radicals (R) which react with oxygen rapidly to generate peroxyl radicals (ROO). Although the second stage is a reversible process, the third stage is irreversible and produces stable radical termination compounds. Four major reaction products, GDPPH-1, GDPPH-2, GAIBN-1 and GAIBN-2 (isogarcinol or cambogin), were formed by reaction of garcinol with peroxyl radicals derived from AIBN in a homogeneous solution.
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Structural information about these nonradical products would afford important contributions to the knowledge of the mechanism of oxidative reactions. Based on the chemical structures of reaction products, generated by reaction of garcinol with DPPH and peroxyl radicals derived from AIBN, Sang et al. proposed the antioxidant mechanism of garcinol as illustrate in figure (13). Garcinol has been proposed to react with stable radical DPPH or peroxyl radical by a single electron transfer followed by deprotonation from the hydroxyl group of the enolized 1,3-diketone system to form a resonance pair. If reaction was initiated at the hydroxyl group of C-3 GDPPH-1 and GAIBN-1 would be formed, and GDPPH-2 and GAIBN-2 would be formed if reaction was initiated at the hydroxyl group of C-l. The identification of GDPPH-1 and GDPPH-2 as reaction products provided the first unambiguous evidence that the principal oxidation sites of garcinol are the 1,3-diketone and the phenolic ring part. The formation of GAIBN-1 and GAIBN-2 showed that the double bond of the isoprenyl group was also a principal site of the antioxidant reaction of garcinol. Other PBDs assayed for their antioxidant activity were 18-O-methyl isogarcinol (47), 18-O-methyl garcinol (48), isogarginol (45) and clusianone (56) [44]. In the DPPH assay, only isogarcinol, with a cathecol group in its structure, caused rapid decolorization of the DPPH solution, indicating marked radical-scavenging activity (IC50 = 13.3 |J,M; vitamin E IC50 = 22.8 \\M), whereas isogarcinol 13-O-methyl ether, garcinol 13-0methyl ether, and clusianone had almost no effect (IC50 > 100 pM). The potential antioxidant activity of nemorosone (58) was assessed on the basis of the scavenging effect on the stable free radical of 1,1-diphenylpicrylhydrazyl (DPPH). Nemorosone was active to scavenge the DPPH free radical in the same order of magnitude that the reference a-tocopherol (IC50 values of 44.1 |j,M and 24.4 \\M respectively). The methyl ethers of nemorosone were also evaluated as mixture employing the same method. The methylation of nemorosone abolished practically the antioxidant properties of this natural product because of the IC50 value observed was > 200 \iM.
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OOH
OH
GDDPH-2
Fig. (13). Proposed mechanism for the formation of oxidation products of garcinol.
Cytotoxicity Some PBDs have shown cytotoxic activity. The SBDs vismiaguianones B, D and E, cudraphenones A-D and the bicyclo-[3.3.1]-nonane derivatives ochrocarpinones A-C, guttiferones A, E and G, xanthochymol possess this biological activity. arcinol showed in vivo cancer chemopreventive activity, and the isogarcinol-xanthochymol mixture displayed a strong apoptosis-inducing effect against human leukemia cells.
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Vismiaguianones A-E (28-32) were tested for DNA strand-scission activity and were evaluated for cytotoxicity against the KB (human oral epidermoid carcinoma) cancer cell line [12]. Vismiaguianone B exhibited moderate DNA strand-scission activity (43 ± 12% nicked at 2.5 |Lig mL*1). Vismiaguianones D and E were found to be moderately cytotoxic when tested against the KB cell line, with EC50 values of 2.4 and 3.3 jj,g mL"1, respectively. Vismiaguianones A-C have similar structure, except for variations in the substitution pattern and functionality of ring C. Only vismiaguianone B, which has ring C attached at C-4 and C-5, showed moderate DNA strand-scission activity, whereas vismiaguianones A and C, which have ring C affixed to C-5 and C-6, did not show any activity. Vismiaguianones D and E are regioisomers varying in the position of ring D. Both compounds showed moderate cytotoxic activity for KB cells, indicating that their differential substitution of ring D did not affect such activity. Tovophenones A-C (19-21), were found to be inactive in the KB cell line (EC50 > 50 ^ig ml/ 1 ) [30]. Hou et al. have investigated the cytotoxic activities against the human oral squamous cell carcinoma cell line HSC-2 and normal human gingival fibroblasts (HGF) of cudraphenones A-D (39-42) and of prenylated xanthones and flavonoids isolated from the roots of Cudrania cochinchinensis [13]. Cudraphenones A-D showed higher cytotoxic activities against HSC-2 [CC50=0.17, 0.036, 0.092, 0.052 mM, respectively] than other compounds. Moreover, cudraphenones A-D showed higher cytotoxic activities against HSC-2 cells than against HGF (tumor specificity: CC50 HGF/CC50 HSC-2 = 2.5, 2.5, 2.1, 3.7 for cudraphenones A-D, respectively). Previously, Hou et al. reported that substitution of a hydrophobic group (isoprenoid unit) in polyhydroxylated flavones, flavonols, isoflavones modified their cytotoxic activities against HSC-2 cells [79,80]. Furthermore, flavones and flavonols with two sets of hydrophobic and hydrophilic (hydroxyl) groups showed higher cytotoxic activities against HSC-2 cells than monoprenylated flavonoids and a high tumor specificity (B/A = 1.5-2.6). The presence of two sets of hydrophobic and hydrophilic groups in separate domains might play a role in the mediation of tumor-specific action. Ochrocarpinones A-C (85-87), three bicyclo-[3.3.1]-nonane derivatives isolated from the bark of Ochrocarpos punctatus, were tested for cytotoxicity against A2780 ovarian cancer cells [14]. All compounds were
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found to be weakly cytotoxic, with IC50 of 6.9, 7.4, 8.2 |j.g mL"1 for Ochrocarpinones A-C, respectively. Also guttiferones A (49) and G (52) were weakly cytotoxic in the A2780 human ovarian cell line, with IC50 values of 6.8 and 8.0 fig mL"1, respectively [15]. Actinomycin D was used as a positive control with an IC50 = 0.003 (igmL"1. In the course of search for anticancer agents in the plant kingdom, Roux et al. [16] found that an EtOAc extract of the fruits of G. pyrifera exhibited significant inhibitory activity on the disassembly of microtubules into tubulin (63% inhibition at 6.67 |ug mL"1). This activity did not correlate with a positive cytotoxicity in KB cells (12% inhibition at 10 (j.g mL"1). Substances able to interact with tubulin/microtubule system represent a potential inhibitor of cell replication. Bioassay-guided fractionation led to the isolation of xanthochymol (43) and guttiferone E (54) as compounds with microtubule disassembly inhibitory properties. These two compounds showed a strong inhibitory activity of the disassembly of microtubules into tubulin (IC50 of 2 and 1.5 |aM, respectively) similar to that exhibited by paclitaxel (IC50 = 0.5 |iM). Moreover, observation by electron microscopy of the microtubules assembled in the presence of xanthochymol and guttiferone E and cooled to 0°C showed a classic pattern for microtubules. In contrast to paclitaxel, xanthochymol and guttiferone E did not promote the assembly of tubulin at 0°C or in the absence of GTP. A Structure-Activity relationship study was undertake in order to determine some structural features necessary for microtubule stabilizing activity among this class of PBDs. Etherification of the enol by methylation or cyclization led to a complete loss of inhibitory activity of microtubule disassembly. The same is true if both phenolic groups are methylated or oxidized. However, some activity is preserved if only one of the two phenolic groups is methylated, ethylated or glycosylated. Hydrogenation of the double bonds also led to a total loss of activity. These results show that the cathecol and enol portion of the molecule are not the entire pharmacophore responsible for the biological activity; the lipophilic domain having the unsaturated prenyl chains is also essential because the octahydro derivate is not active, although the catechol and enol parts are not modified. As for the cytotoxicity on KB cells, the IC50 values for the cell growth inhibition were similar for all the compounds. It appears
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that cytotoxicity is probably not related to the interaction of the products with tubulin inside the cell [16]. Cancer chemopreventive activities During the past decade, a large number of natural products and dietary components have been evaluated as potential chemiopreventive agents [81]. Dietary factors play an important role in human health and in the development of certain chronic diseases including cancer. Recent research has focused on the presence of minor constituents or non-nutrients, which possess antimutagenic and anticarcinogenic properties, in diets [82]. Such compounds are candidates for chemiopreventive agents against cancer development in humans. Garcinol is a PBD isolated from G. indica and other species. The dried rind of G. indica (Kokum) which is used as a garnish for curry and in traditional medicine in India contains 2-3% of garcinol by weight. Pan et al. have studied the induction of apoptosis signaling pathway by garcinol in human leukemia cell line, HL-60. Their results clearly demonstrate that garcinol strongly induced apoptosis in a dose-dependent manner in HL-60 cells [61]. Apoptosis, a morphologically distinct form of programmed cell death, is an evolutionary highly conserved phenomenon that plays an important role in the regulation of cellular activities in eukaryotes. The caspase family of proteases plays key roles in promoting the cell death associated with apoptosis. Recently, several in vivo and in vitro studies have indicated that some potent chemopreventive agents, such as sulindac and other nonsteroidal anti-inflammatory drugs, induce apoptosis in colonic tumor, leading to the prevention of colon cancer [83,84]. The induction of apoptosis by garcinol occurred within several hours, consistent with the view that garcinol induces apoptosis by activating the pre-existing apoptosis machinery. Indeed, treatment with garcinol caused an induction of caspase-3 activity and degradation of poly(ADP-ribose) polymerase (PARP), which precedes the onset of apoptosis. Pretreatment with the caspase-3 inhibitor repressed garcinol-induced caspase-3 activation and DNA fragmentation, suggesting that apoptosis induced by garcinol involves a caspase-3 mediated mechanism. In vitro studies have identified Apafl, cytochrome c, and caspase-9 as participants in a complex important for caspase-3 activation [85]. These data suggest a linear and specific activation cascade between caspase-9 and
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caspase-3 in response to cytochrome c release from mitochondria. Release of cytochrome c from the mitochondria has been shown to be an almost universal phenomenon during apoptosis. In their study, Pan et al. found that garcinol strongly induced the release of cytochrome c, and the cleavage of caspase-9. The cleavage of D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. Of these, Bcl-2, Bad, and Bax were also studied by Pan et al. The level of expression of Bcl-2 slightly decreased, while the levels of Bad and Bax were dramatically increased in cells treated with garcinol. These results indicate that garcinol allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. It is suggested that garcinol induced-apoptosis is triggered by release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3 and caspase-2, degradation of PARP, and DNA fragmentation caused by the caspaseactivated deoxyribonuclease through the digestion of DFF-45. This study suggested that garcinol induced apoptosis through a caspase-dependent mechanism, which may contribute to the cancer chemopreventive actions. Carcinogens usually cause genomic damage in exposed cells. As a consequence, the damaged cells may be triggered either to undergo apoptosis or to proliferate with genomic damage, leading to the formation of cancerous cells that usually exhibit cell cycle abnormalities and which are more susceptible to various apoptosis-inducing agents [86,87]. Thus, treatment with garcinol may preferentially cause apoptosis in those abnormal cells, ultimately leading to the prevention of cancer. Furthermore, the ability of garcinol alone to induce apoptosis suggests its potential use as a chemotherapeutic agent because many anticancer drugs are known to achieve their antitumor function by inducing apoptosis in the target cells [88]. Also the effects of isogarcinol (45) and xanthochymol (43) on growth inhibition in four human leukemia cell lines (NB4, HL-60, U937 and K562) were examined by Matsunoto [62]. This two compounds displayed more potent growth inhibition than garcinol. The potency of the growth inhibitory effect was isogarcinol> xanthochymol> garcinol. Matsumoto further investigated whether the cytotoxic effect of the compounds was mediated by apoptotic mechanism in leukemia cell lines. Isogarcinol,
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xanthochymol and garcinol induced apoptosis in all four leukemia cell lines. Cancer chemopreventive activity of garcinol have been also demonstrated in animal model: dietary administration of garcinol significantly inhibited azoxymethane(AOM)-induced aberrant crypt foci (ACF) formation in male F344 rats without causing any adverse effects [63]. ACF are precursor lesions for colon carcinoma. In this study several explanations for the inhibitory effects of garcinol on ACF formation by AOM are considered. Several natural compounds could induce detoxification enzymes [89]. Dietary administration of garcinol significantly elevated glutathione S-transferase (GST) and quinone reductase (QR) activities in liver. This biological action may contribute to its 'blocking' effect on AOM-induced ACF formation. Activated leukocyte-derived O2" and NO generation have both been reported to be involved in colon carcinogenesis [90,91]. Increased iNOS and COX-2 expression also involves colon carcinogenesis [92,93]. Garcinol inhibited TPA-induced O2" generation in human promyelocytic HL-60 and LPS- anf IFN-y-induced NO generation in mouse macrophage cells. It showed also suppressing effects on LPS- anf IFN-y-induced iNOS and COX-2 expression in Western blot analysis. These findings might suggest possible chemopreventive ability of garcinol, through induction of liver GST and QR, inhibition of O2" and NO generation and/or suppression of iNOS and COX-2 expression, on colon tumorigenesis. Cancer preventive effects have often been attributed to antioxidant actions. In order to understand whether oxidation products of garcinol play a functional role in biological system, the antitumor activities of four oxidation products of garcinol (GDPPH-1-2 and GAIBN-1-2) were individually tested. The induction of apoptosis in human leukemia HL-60 cells, the inhibition of NO generation and the inhibition of LPS-induced iNOS gene expression by Western blotting analysis by garcinol and its reaction products were investigated by Sang et al. [77]. Garcinol, GDPPH-1, GDPPH-2 and GAIBN-2 appeared to be more potent and dose-dependent on the induction of cell apoptosis. When the HL-60 cells were treated with the same concentration (20 pM) of these compounds, the apoptotic potency was the same. The effects of garcinol, GDPPH-1, GDPPH-2, GAIBN-1 and GAIBN-2 on the inhibition of NO generation in macrophages LPS-stimulated showed that all markedly reduced NO generation in a concentration-dependent
716
manner (inhibitory potency at 10 yM: garcinol> GDPPH-1> GDPPH-2> GAIBN-2>GAIBN-1). Garcinol, GDPPH-1, GDPPH-2, GAIBN-1 and GAIBN-2 were examined also to determine whether they affect iNOS protein in macrophages activated with LPS. These compounds significantly inhibited expression of iNOS protein (inhibitory potency at 10 |J,M: garcinol>GDPPH-2>GAIBN-2>GDPPH-1 >GAIBN-1). These tests indicated that like garcinol, GDPPH-1, GDPPH-2 and GAIBN-2 showed strong inhibitory effects on these assay. GAIBN-1 showed very weak activity. The potency of these compounds in apoptosisinduction may vary with the different cell lines. These findings might suggest possible chemopreventive ability of garcinol and its oxidation products. Analysis of these products could provide a unique tool for assessing the contribution of antioxidant reactions to the disease preventive effects of garcinol. Nemorosone and its methyl derivatives as mixture were evaluated against the human cervix carcinoma (HeLa), the human larynx carcinoma (HEp-2), prostate carcinoma (PC-3) and central nervous system carcinoma (U-251) cell lines [94]. Cuesta-Rubio et al. found that the natural product nemorosone was active against the four cell lines (IC50 values of 3.3, 3.1, 7.2 and 3.9 JJM, respectively). When the mixture of methyl derivatives was used, the required concentration to reach IC50, in the cellular lines, was between ten and thirty times more concentrated. These results suggest that the presence of the keto-enolic equilibrium in nemorosone plays an important in its cytotoxicity. REFERENCES [1] [2] [3] [4] [5] [6]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
721
THE BENZOPHENONES: ISOLATION, STRUCTURAL ELUCIDATION AND BIOLOGICAL ACTIVITIES SCOTT BAGGETT,1 EUGENE P. MAZZOLA,2 AND EDWARD J. KENNELLY1* 'Department of Biological Sciences, Lehman College and The Graduate Center, The City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468 University of Maryland-FDA Joint Institute for Food Safety & Applied Nutrition, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 ABSTRACT: The benzophenones are a group of ca. 146 compounds comprising a 13carbon core that can be prenylated and/or further cyclized producing numerous structurally unique compounds. Benzophenones have a limited natural distribution and are concentrated in the Clusiaceae and a few other plant families such as the Moraceae. Benzophenones display many biological activities including antioxidant, antimicrobial, antifungal, cytotoxic, and anti-HIV activities. This chapter reviews the biosynthesis, sources, isolation, structural determination, and biological activities of benzophenones. Our experiences with the activity-guided isolation and structural elucidation of xanthochymol by ID and 2D NMR from Garcinia xanthochymus fruits are discussed in detail.
INTRODUCTION The purpose of this chapter is to review the benzophenones, a class of compounds consisting of ca. 146 compounds. Given the structural diversity, bioactivities, and paucity of previous reviews, the benzophenones are examined comprehensively. Benzophenones have been isolated from the leaves, roots, fruits, bark, and floral resin of plants belonging to at least 13 families including: Asteraceae, Clusiaceae, ("HO")
Davalliaceae, Fabaceae, Gentianaceae, Iridaceae, Lauraceae, Magnoliaceae, Moraceae, Rosaceae, and Thymelaeaceae. The majority of b e n z o p h e n o n e s (96 Of 146, 6 6 % ) are found in t h r e e C l u s i a c e a e genera:
0 Fig. (I).
OH
Basic chemical structure of benzophenones
722
Clusia, Garcinia, and Hypericum. Benzophenones have a range of structures but share a common 13-carbon skeleton, Fig. (1). There is no standard numbering systems for benzophenones. The shikimate-derived A-ring is a benzene ring usually containing 0, 1, or 2 substituents. The acetate-malonate derived B-ring undergoes prenylation and cyclization producing numerous structurally unique compounds containing bi-, tri-, and/or tetracyclic ring systems. Isolated benzophenones exhibit a range of biological activities including antifungal, antioxidant, antimicrobial, antiviral, and anticancer. Historically, reviews of benzophenones were included as small sections with the biogenetically related xanthones. Previous reviews include Bennett and Lee [1] who discussed the distribution and isolation of 20 benzophenones included in a review focused on xanthones. They also discussed the biosynthetic relationships between xanthones and benzophenones. Kumar and Baslas [2] summarized the chemical composition, medical importance, and biologically active benzophenones, biflavonoids, and xanthones isolated from Garcinia species. Two reviews by Waterman focused on the chemotaxonomic significance of benzophenones, along with biflavonoids and xanthones, often African Garcinia species [3,4]. Locksley and Murray [5] discussed the distribution of thirteen benzophenones in higher plants and Sultanbawa [6] included five benzophenones in a review on xanthonoids from tropical plants. Sultanbawa also discussed the biosynthesis of benzophenones and xanthones. Erdelmeier et al. [7] discussed 20 polyprenylated benzophenones in a chapter on the various properties of St. John's Wort (Hypericumperforatum). This chapter briefly discusses benzophenone biosynthesis, lists known benzophenones and their sources, and discusses their isolation, structural determination, and biological activities. BENZOPHENONE SYNTHESIS Previous researchers have suggested [6,8] that benzophenones are biosynthesized by condensation of metabolites from the shikimate pathway, forming the A-ring, and the acetate-malonate pathway, creating the B-ring. This produces the basic 13-carbon benzophenone skeleton, Fig. (1). Support for this biosynthetic pathway includes the isolation of benzophenone synthase from Centaurium erythraea [9] and research by Atkinson et al. [10] who examined benzophenones as intermediates in the synthesis of xanthones. (Xanthone biosynthesis is reviewed by
723
Bennett et al. [1]). Using 14C and 3H labeled precursors, Atkinson et al. discovered that the shikimate-derived phenylalanine was incorporated into the A-ring and three acetates were condensed to form the B-ring. OH COO" I H2C-Co-SCoA 3 Malonyl-CoA
SACo O m-Hydrozybenzoyl-CoA (Shikimate)
3CO 2 4 CoASH
(HO). \
(OH) I Hn ^ ^ nu,
6
^ s>^
OH
^ "
prenylation ~^^- ~^^*and cyclization
Examples include the guttiferones, plukenetione a nd sampsonione
OH
13-carbon base benzophenone Fig. (2). General pathway for the synthesis of benzophenones. Adapted from [6,9]
During these experiments 14C phloroglucinol was also added and was not incorporated into either the A- or B-rings, thus supporting the synthesis scheme shown in Fig. (2). Addition of 14C labeled phloroglucinol is significant because some researchers have suggested that benzophenones are not derived from the basic 13-carbon skeleton, shown in Fig. (1), but from phloroglucinol (1,3,5-trihydozybenzene). Examples include the sampsoniones, isolated from Hypericum sampsonii, by Hu et al. The sampsoniones were initially described as "polyprenylated benzophenones" [11,12] and later called "polyprenylated phloroglucinol" derivatives [13,14]. Hu et al. also proposed a biosynthetic pathway for the sampsonione-type "benzophenones" from the basic 13-carbon benzophenone skeleton shown in Fig. (1). In their proposed synthesis scheme, it is unclear if the basic benzophenone skeleton is derived from the shikimate and the acetate-malonate pathways or from phloroglucinol. Additionally, Fuller et al. [15] suggested that the vismiaphenones (35, 44, 45, 53) are related to and possible precursors of the guttiferones, a
724
class of polyprenylated benzophenones represented by xanthochymol (138). It remains unclear whether the basic 13-carbon benzophenone is constructed (from the shikimate and acetate pathways) and then prenylated and cyclized or the B-ring is derived from phloroglucinol and later the 7-membered carbon substructure (A-ring) is added. Given the structural diversity of benzophenones, their biosynthesis is suggestive of multiple pathways. Further research is needed to fully understand the biosynthesis of benzophenones. ISOLATED BENZOPHENONES Information on 146 isolated benzophenones is summarized in Table 1, sorted by molecular weight from lowest to highest. Each entry contains the common name(s), chemical structure, biological source(s), and reference(s). For convenience benzophenones are divided into two types: basic benzophenones and polyprenylated benzophenones (PPBs). Basic benzophenones have the 13-membered skeleton, Fig. (1), with various numbers of -OH and -OMe groups attached, and at most one prenyl group. Examples include compounds 1-26. Fuller eta I. [15] suggested that the vismiaphenones (35, 44, 53) are possible intermediates in the biosynthesis of PPBs, the second type of benzophenone. This group has additional prenyl or geranyl groups attached, bi-, tri-, or tetracyclic rings systems, or oxo bridges, peroxide or epoxide groups. Examples are the guttiferones, plukenetiones, and sampsoniones, represented by xanthochymol (138), plukenetione A (76), and sampsonione A (126), respectively. The discussion below highlights features of the basic and polyprenylated benzophenones.
725
Table 1: Chemical Structures, Molecular Weights, and Sources of Isolated Benzophenones Compound
MW
Structure
Species
Ref
OH
4-Hydroxybenzophenone
198
2,4,6-Trihyroxybenzophenone
Talauma mexicana [16]
230 Hypericum sampsonii [17]
O
OH OMe
Cearoin
O
OH
Dalbergia cearensis, Dalbergia coromandeliana, [18244 Dalbergia odorifera, 23] Dalbergia parviflora, Dalbergia sissoides, Dalbergia volubilis
OMe
244
Cotoin O MeO
[24, 25]
OH
Isocotoin O
244
Helichrysum spp.
246
Dalbergia melanoxylon
[26, 27]
OH
2,4,4',5-Tetrahydroxy benzophenone O
[28]
OH
2,4,6,3'-Tetrahydroxy benzopheone
246
OH
Aniba duckei
Garcinia multiflora, [10, Gentiana lutea 29]
O
246
Iriflophenone O
OH
Irisflorenlina, Iris germanica. Iris pouminii, Moms alba
[30-
33]
726 #
Compound
Structure
|MW
Species
| Ref
258
Allanblackia floribunda
[5]
260
Aniba duckei, Anemarrhena asphodeloides
260
Dalbergia melanoxylon
OMe
Hydrocotoin 0
OH OMe
10
2,6,4'-Trihydroxy-4methoxybenzophenone 0
[34,
OH OMe
11
12
Melannoin
ii
i
O
OH
2,4,6,3',5'Pentahydroxybenzophenone
[28]
262
arcinia pedunculata [36]
262
Garcinia assugu, Garcinia multiflora
[29, 37]
272
Aniba pseudocoto, Cascara sagrada
[5]
274
Allanblackia floribunda, Garcinia multiflora
[5, 29]
274
Machaerium scleroxylon
[38]
276
Garcinia multiflora
[29]
1 II OH 13
0
Macurin O
OH OMe
14
Methlyhydrocotoin II 0
15
OMe OMe
2,3'-Dihydroxy-4,6dimethoxybenzophenone
II
1
0
OH OH OMe
16
Scleroin "OMe 0
17
4,6,3',4',Tetrahydroxy-2methoxybenzophenone
OH
HO 0
OMe
727 Compound
Structure
MW
Species
Ref
276
Hypericum annulalum
[39]
MeO 18
Annulatophenone
OH
O HCK
19
_OMe
2',3',6-Trihydroxy2,4-dimethoxybenzophenone OH
0
O MeO.
[5]
Aniba pseudocoto
OH OMe
304 Garcinia subelliptica
[41]
MeO
O MeO 22
302
OMe
Protocotoin
6,4'-Dihydroxy-2,3',421 trimethoxybenzophenone
[40]
OMe
MeO
20
290 Garcinia subelliptica
OH OMe
Methyl protocotoin O
316
Aniba pseudocoto
[5]
328
Cudrania cohochinchinensis var. genontogera
[42]
OMe OMe
23
Cudranone Prenyl O
OH OMe
24
2,4,6,3',4'Pentamethoxybenzophenone
MeO
OMe
332 Garcinia pedunculata
[43]
332 Garcinia pedunculata
[36]
OMe
OMe O MeO
25
2,4,6>3',5'Pentamethoxybenzophenone
MeO
OMe
MeO
O
OMe
HO 26
4,6,4'-Trihydroxy2,3'-dimethoxy-3prenylbenzophenone
358 MeO
Prenyl O
OMe
Garcinia mulliflora
[29]
728 Compound
27
Clusiaphenone A
362
O Prenyl
28
Clusiachromene C
32
[46]
364
Clusia multiflora
[46]
364
Clusia multiflora
[46]
364
Clusia multiflora
[46]
364
Clusia mulliflora
[46]
OH
Clusiacyclol B
OH
0
Clusia multiflora
OH
Clusiacyclol A
0
364
OH
Clusiacitran B
O
31
Clusia ellipticifolia, [44, Clusia sandiensis 45]
OH
Clusiacitran A
O
30
Ref
OH
O
29
Species
Structure
OH
729 U I
33
Compound
Structure
Cudraphenone A
Prenyl O
MW
Species
364
Cudrania cochinchinensis
[47]
364
Clusia eliipticifolia, Vismia decipiens
[44, 48]
364
Clusia eliipticifolia, Vismia decipiens
[48]
366
Clusia sandiensis
[45]
366
Cudrania cochinchinensis
[47]
378
Garcinia myrtifolia, Garcinia pseudogultifera
[49, 50]
380
Helichrysum triplinerve
[26]
OH
OH
34
| Ref
Isovismiaphenone B Prenyl O
35
OH
Vismiaphenone B Prenyl 0
36
OH Prenyl
Clusiaphenone B Prenyl O
OH Prenyl OH
37
Cudraphenone B HO
Prenyl O
OH
OMe 38
Myrtiaphenone B Prenyl 0
OH
MeO
0—Geranyl
4-Geranyloxy-239 hydroxy-6-dimethoxy' benzophenone O
OH
730 U
Compound
40
Clusiaphenone C
Structure OH
0
41
Species
Ref
380
Clusia ellipticifolia [44]
380
Clusia ellipticifolia [44]
OH
Clusiaphenone D
O
42
MW
OH
Cudraphenone C
380
Cudrania cochinchinensis
[47]
Prenyl 0 MeO 43
Marupone
380
0
44
OH Prenyl 380
Vismiaphenone A O
Moronobea pulchra [51]
Vismia decipiens
[48]
OMe
Prenyl OMe 45
Vismiaphenone C
380
Garcinia [49, pseudoguttifera, 50] Garcinia myrtifolia
380
Vismia cayennensis
[15]
38
Cudrania cochinchinensis
[47]
Prenyl II 0
46
OH
Vismiaphenone E
OH
47
Cudraphenone D
Prenyl Prenyl 0
731 Compound
48
Structure
MW
Vismiaguianone A
Species
Ref
382
Vistnia guianesis
[52]
382
Vismia guianesis
[52]
382
Vismia guianesis
[52]
Prenyl 0
49
OH
Vismiaguianone B Prenyl O
50
OH
Vismiaguianone C Prenyl 0
OH OGlc
51
Garcimangosone D
392 Garcinia mangostana [53]
II 0
52
OH Prenyl
Myrtiaphenone A
394
Garcinia [49, pseudoguttifera, 50] Garcinia myrtifolia
394
Vistnia cayennensis [15]
Prenyl 1! 0
53
OH
Vismiaphenone F Prenyl O
54
OH
Pseudoguttiaphenone A
396 Prenyl 0
OH
Garcinia pseudoguttifera
[50]
732
Compound
Structure
MW
Species
Ref
396
Vismia cayennensis
[15]
408
Hypericum annulatum
[54]
408
Coleogyne ramosissima
[55]
408
Davallia solida
[56]
412
Vismia cayennensis
[15]
422
Gnidia involucrata
[57]
424
Gnidia involucrata
[57]
424
Hypericum annulatum
[39]
Prenyl OMe 55
Vismiaphenone D Prenyl O
OH
MeO 56
Annulatophenonoside OAra O HO
57
Iriflophenone-2-O-|3D-glucopyranoside O
OGIc OGIc
58
Iriflophenone-4-O-pD-glucopyranoside O
OH Prenyl OMe
59
Vismiaphenone G
60
2,4',6-Trihydroxy-4methoxybenzophenone-2-Oglucoside
O
OH .OMe
O
61
OH
2,3,4',5,6Pentahydroxybenzophenone-4-Cglucoside
0
OH OGIc
HO 62
Hypericophenonoside
OH
0
733
Compound
63
Structure Prenyl
Vismiaguianone D
MW
Species
Ref
428
Vismia guianensis
[52]
Prenyl
64
Vismiaguianone E
428
Vismia guianensis
[52]
65
Machuone
432
Clusia sandiensis
[45]
434
Clusia grandiflora
[58, 59]
436
Tripterospermum japonicum
[60]
450
Hypericum annulatum
[54]
464
Tovomita mangle, Tovomita brevistaminea
[61, 62]
OH Prenyl
66
Grandone
0 MeO. 67
OH ,OMe
Triptephenoside OGlc
OH
0
MeO 68
Acetylannulatophenonoside
OH
Prenyl OMe
69
Tovophenone A O
OH
734
Compound
Structure
MW
Species
464
Tovomita mangle, Tovomita brevistaminea
[61,
466
Cassia angustifoUa
[63]
480 Cassia angustifoUa
[63]
| Ref
OH 70
Tovophenone B
71
Cassiaphenone, a-2glucoside
OH
COOH CH 2 OH
OGlc 0 OH
OH COOH COOH
72
Cassiaphenone, p-2glucoside OGlc O HO.
73
Tovophenone C
O
74
OH
Tovomita brevistaminea
[62]
486
Garcinia dulcis
[27, 64]
486
Garcinia dulcis
[27]
500
Clusia plukenetii
[65]
OH
Garciduol A
OH
480
OH
0
OH
OH
75
Garciduol C
OH
OH
76
Plukenetione A
0
735
U \
77
Compound
Plukenetione F
Structure
YX
r
Species
Ref
500
Clusia havetioides var. stenocarpa. Clusia plukenetii
[DO,
500
Clusia havetiodes var. stenocarpa. Clusia plukenetii
[66, 67]
500
Clusia scrobicuiata
[68]
500
Clusia scrobicuiata
[68]
/
—Prenyl
y
MW
I
67]
Prenyl
78
Plukenetione G
r/ ^
n—^ J\
0
1 ~~~~
—Prenyl 11 Prenyl
X 79
Scrobiculatone A
—Prenyl
II
X Prenyl
^Y 80
Scrobiculatone B
[
—Prenyl
II
II Prenyl
Prenyl 81
7-Epi-clusianone
^>\>
°\
1T 0
Prenyl 502 Rheedia gardneriana
^Y\ T OH
Prenyl
[6971]
736 Compound
Structure Prenyl HO
MW|
Species
Ref
502
Clusia insignis, Clusia nemorosa, Clusia renggerioides
[59]
502
Clusia spiritusanctensis, Clusia sandiensis, Garcinia assugu
[37, 45, 58]
502
Carcinia dulcis
[27, 64]
502
Clusia hilariana
[68]
502
Garcinia kola
[72]
502
Clusia lanceolata
[68]
1
82
—Prenyl
7-Epi-nemorsone
Prenyl Prenyl 83
Prenyl
V
Clusianone
Prenyl OH
84
Garciduol B
Prenyl
85
Hilarianone
fl fl 0
0 Prenyl
86
Kolanone
0
87
Lanceolatone
OH Prenyl
737
u
Compound
Structure Pren yi
MW
Species
Ref
Y
502
Clusia grandiflora, Clusia insignis, Clusia nemorosa, Clusia rosea
[58, 73]
//.o
88
Nemorosone
[ 0
-^—Prenyl
\
T
Prenyl Prenyl
89
Nemorosone II
I
,1
S 0
^—Prenyl
Y
Clusia grandiflora, 502 Clusia renggerioides, Clusia rosea
[59, 74]
Prenyl Prenyl Preny 90
Nemorosonol
{
Plukenetione D
Clusia mulliflora, Clusia nemorosa
[46. 75 76]
502
Clusia plukenelii
[67]
502
Clusia plukenetii
[67]
502
Propolis from Cuba
[77]
II
OH
91
502
\J>—Prenyl ff 0 Prenyl
I
-^—Prenyl
V \ o[5 Pren yi
rV 0H Prenyl
92
Plukenetione E
l
Y
j—Prenyl
II
0
0 yi
/} '\Jj 93
Propolone A
Pren
">C/Prenyl
— o=r^ |
0
738 Compound
Structure Prenyl
MW
Species
Ref
94
Weddellianone A
502
Clusia weddelliana
[68]
95
28,29-Epoxyplukenetione A
516
Clusia havetioides var. stenocarpa
[66]
518
Clusia nemorosa
[58, 59]
518
Ochrocarpos punctatus
[78]
518
Ochrocarpos punctatus
[78]
518
Clusia plukenetii
[67]
Prenyl Prenyl
96
Prenyl
Hydroxynemorosone
Prenyl
97
Ochrocarpinone B
Prenyl
98
Ochrocarpinone C Prenyl
Prenyl
99
Plukenetione B / 0
Prenyl
739 Compound
100
MW
Structure
- -Prenyl
Sampsonione B
Species
518 Hypericum sampsonii
Ref
[12]
OH Prenyl
101
Sampsonione G
518
Clusia havelioides var sienocarpa, Hypericum sampsonii
[II. 66]
Prenyl
0
Prenyl Prenyl 102
Sampsonione L
518 Hypericum sampsonii
[13]
Pren1
103
Xerophenone A
Clusia plukenetii, Clusia portlandiana
[67, 79]
518 Clusia portlandiana
[79]
528 Hypericum sampsonii
[11]
518 Prenyl OH
104
O
Xerophenone B
O
105
OH
Sampsonione H Geranyl
106 Nemorosinic acid A
530 O
OH
Clusia nemorosa
[80]
740 Compound
Structure
107 Nemorosinic acid B 0
MW
Species
Ref
530
Clusia nemorosa
[80]
534
Clusia havetioides var. stenocarpa, Ochrocarpos punctatus
[66, 78]
534
Garcinia aristata
[81]
534
Garcinia aristata
[81]
534
Ochrocarpos punctatus
[78]
O
OOH
108
15,16-Dihydro 16hydroperoxyplukenetione F
OH
109
Prenyl
Aristophenone A Prenyl O
OH Prenyl
110
Aristophenone B Prenyl O
111
O
Ochrocarpinone A
Prenyl
Prenyl
112
542 Hypericum sampsonii
Sampsonione E
O (/
Geranyl
[II]
741 Compound
113
Plukenetione C
114
Spiritone
Structure
MW
Species
Ref
550
Clusia havetioides var. stenocarpa, Clusia plukenetii
[66, 67]
556
Clusia spiritusanctensis
[68]
iPrenyl
Prenyl Prenyl
Prenyl
115
Insignone
560
Clusia insignis
[68]
116
Chamone II
568
Clusia grandiflora
[74]
Prenyl
742
Compound
117
Structure
MW
Hypersampsone F
Species
Ref
568 Hypericum sampsonii
[IV]
568 Hypericum sampsonii
[11. 17]
570
[74]
Prenyl Prenyl
118
Sampsonione D
Geranyl Prenyl Prenyl 119
Chamone I
Clusia grandiflora
Prenyl
120
Hypersampsone D
570 Hypericum sampsonii
[17]
570 Hypericum sampsonii
[IV]
570
[68]
0
0 0 Geranyl
121
Hypersampsone E O O
Geranyl
Prenyl
122
Weddellianone B
Prenyl Prenyl
Clusia weddelliana
743
Compound
123
Structure
33-Hydroperoxyisoplukenetione C
MW
Species
Ref
582
Clusia havetioides var. stenocarpa
[66]
OOH
124
Sampsonione I
125
Sampsonione J 0
o
584 Hypericum sampsonii
[14]
584 Hypericum sampsonii
[14]
586 Hypericum sampsonii
[12]
586 Hypericum sampsonii
[II]
586 Hypericum sampsonii
[11]
586 Hypericum sampsonii
[13]
Geranyi Prenyl
126
Sampsonione A 0
127
Sampsonione C
°
128
OH Geranyi
0
Geranyi
Sampsonione F Geranyi Prenyl Prenyl
129
Sampsonione K
Prenyl
744 Compound
Structure Prenyl
MW
Species
Ref
Geranyl 130
Sampsonione M
OH
131
Prenyl
N < '
OH
OH
[8284]
602
Garcinia indica, Garcinia ovalifolia, Garcinia xishuanbannanansis
602
Garcinia pyrifera, Garcinia subelliptica
90]
602
Garcinia cambogia
[91]
[85-
Prenyl
Garcinol (camboginol) 11
135
Garcinia assugu, larcinia pedunculala
Prenyl
133 Cycloxanthochymol
134
602
Prenyl
132 (+)-Isoxanthochymol
OH
[13]
.Prenyl
(-)-Isoxanthochymol (Isogarcinol, cambogin)
1
586 Hypericum sampsonii
iPrenyl
Guttiferone A Prenyl O
OH
Calophyllum brasiliense, Garcinia intermedia, Garcinia livingstonei, 92602 Garcinia 94] macrophyUa, Symphonia globulifera
745
Compound
Structure OH
136
Preny[
MW
Guttiferone E Prenyl
OH
137
Prenyl
Species
Ref
Prenyl Clusict rosea, Garcinia assugu, 602 Garcinia huillensis, Garcinia ovalifolia, jarcinia pedunculata
[37, 83, 88, 95]
,\ Prenyl
Guttiferone F
602
Allanblackia sluhlmannii
[96]
Prenyl
OH
138
Prenyl
602
Xanthochymol
OH
139
Prenyl.
Prenyl
Clusia rosea, Garcinia indica, Garcinia mannii, Garcinia ovalifolia, Garcinia staudtii, Garcinia xanthochymus, Garcinia xishuanbannanansis, Rheedia madrunno
97100]
%v \Prenyl
Pedunculol
604 Garcinia pedunculata
[83]
616
Garcinia assugu
[37]
616
Garcinia assugu
[37]
Prenyl
OMe
140
Prenyl
Garcinol, 13-Omethyl ether Prenyl
OMe
141
Prenyl_ O,
^Prenyl
Isogarcinol, 13-Omethyl ether I Prenyl
746 Compound
142
Bronianone
Structure Geranyl
Geranyl .0
HO
MW
Species
670
Garcinia hombronlana
670
Symphonia globulifera
[88]
670
Symphonia globulifera
[88]
670
Symphonia globulifera
670
Garcinia macrophylla
Ref
101]
Preny!
0 OH 143
0
Preny[
.Geranyl
Guttiferone B Geranyl O
OH Prenyl
144
Guttiferone C
Prenyl OH
145
Prenyl.
Guttiferone D
Prenyl
OH 146
Prenyl,
''^tf
^Geranyl
Guttiferone G
[93]
''Prenyl O
OH
Benzophenones and xanthones are known to co-occur in the Clusiaceae [1], and the garciduols (74, 75, 84) are three benzophenonexanthone dimers [27,64]. The cudraphenones (33, 37, 42, 37), a group of benzophenones isolated from the Moraceae family, are prenylated on the A- and B-rings [47]. These are the only four compounds that are prenylated on both the A- and B-rings. Ten benzophenone glycosides, nine O-glycosides (56-58, 60, 62, 6768, 71-72) and one C-glycoside (61) have been isolated from five different plant species. None of these are prenylated, and the glycoside substituent is attached to either the A- or B-ring [39,54-57,60,63].
747
The most common polyprenylated benzophenones have a bicyclo[3.3.1]nonane B/C-ring system. Approximately 38 bicyclo[3.3.1]nonane benzophenones have been isolated. A typical bicyclo[3.3.1]nonane benzophenone is xanthochymol (138), which is discussed in detail below. The floral resin of twelve Clusia species has yielded at least fourteen (66, 79-80, 82, 85, 87-89, 93-94, 96, 114-115, 122) bioactive bicyclo[3.3.1]nonane polyprenylated benzophenones. These compounds have the isoprenyl and benzoyl residues attached at different positions on the bicyclo[3.3.1]nonane ring [58,59,68,73,102]. In addition, the floral resin is used by pollinating bees for nest construction [77]. Four isolated PPBs, with the bicyclo[3.3.1]nonane system, contain peroxide bonds: 15,16-dihydro-16-hydroperoxyisoplukenetione F (108), ochrocarpinone A (111), plukenetione C (113), and 33hydroperoxyisoplukenetione C (123) [66,78]. The tautomeric benzophenone pair xerophenone A (103) and B (104) present an interesting variation in benzophenone chemistry by having a 7-membered C-ring and an oxo bridge between carbons 4 (C-ring) and 10 (B-ring). These compounds feature an oxatricyclo[4.3.1.1]undecane-7,9dione system [79]. A number of PPBs occur as tautomeric pairs; examples include aristophenone A and B (109, 110) [81] and plukenetione D (91) and E (92), which were isolated after acetylation [67]. Plukenetione A (76) was the first PPB isolated with a adamantyl skeleton and an methylpropenyl group [65]. Sampsonione D (118) and I (124) each have one isopentenyl side chain replaced by an isopropenyl moiety [11,13,14]. Grossman et al. details the relationships of the plukenetiones, nemorosone II, and sampsoniones [103]. They showed that plukenetiones B, D, and E are diastereomeric to nemorosone II and sampsonione G. Nemorosonol (90), isolated from Clusia nemorosa fruits, has a novel tricyclo[4.3.3.0]decane acetate-derived B-ring [75]. This compound's structure was determined by X-ray crystallography [76], and is the only isolated benzophenone with a tricyclo[4.3.3.0]decane system. There has been considerable confusion in the nomenclature and structural elucidation of garcinol/caboginol (134) and isoxanthochymol/isogarcinol (131). Their naming history is discussed by Bennett et al. [1] and by Fuller et al. [96]. The synonyms for these compounds are listed in their respective entries in Table 2. As noted by Fuller et al., the common name garcinol refers to more then one
748
compound. In addition, from a structural and chronological standpoint the common name caboginol has precedence for 134. Details on the structural elucidation of these compounds has been described [104-110]. ISOLATION OF BENZOPHENONES Multiple chromatographies are needed to purify benzophenones, and a variety of normal and reversed-phase solvent systems and solid phases including column chromatography over silica gel, reversed-phase, and Sephadex LH-20, as well as preparative TLC and HPLC have been employed. Isolation methods for selected benzophenones are given, and our experiences with the activity-guided isolation of xanthochymol (138) from G. xanthochymus fruits are discussed. Typically, the roots, leaves, fruits, wood, or floral resin are extracted with a single solvent, or solvents, of increasing polarity including CeH6 [75], hexane [67,81], CH2C12 [96], acetone [45], petroleum ether [111], MeOH [96], and/or EtOH [13,47]. After in vacuo concentration, the residue is resuspended in water and sequentially partitioned with solvents of increasing polarity, including w-hexane, CeH6, CH2CI2, EtOAc, and BuOH. After partitioning, extracts are passed over silica gel, either open column [67,83] or vacuum-liquid chromatography (VLC) [27], usually with mixtures of hexane-EtOAc [49,50] or CHCh-MeOH. Separation via Sephadex LH-20 using isocratic systems of MeOH [27], CH 2 Cl 2 -Me0H [88], CHCh-MeOH [29,37], or a gradient solvent system of CH2C12 -> CH2Cl2-MeOH, [93] has also been employed. After initial separation benzophenone-enriched fractions are rechromatographed (using preparative TLC, column chromatography, or HPLC) over a variety of stationary phases (in order of decreasing times employed) including silica gel, C,8, Sephadex LH-20, C8 [88], diol [88], MCI gel CHP-20P [55], and Toyopearl HW-40 [55]. Usually, multiple chromatographies over the same stationary phase or a combination of stationary phases are used to purify benzophenones. Recrystallization has been described in a few publications [54,81,83]. Isolation methods for selected benzophenones are given below. The anti-HIV guttiferones A-D (135, 143-145) were isolated from Symphonia globulifera by extracting with CH 2 Cl 2 -Me0H and then MeOH. The combined organic extracts were partitioned with EtOAc and, after in vacuo concentration, were passed over a diol column, eluted with CH 2 Cl 2 -Et0Ac-Me0H. The HIV-active fractions were combined and rechromatographed over a diol column, eluted with CH2C12. Next, the
749
HIV-active fractions were combined and rechromatographed over a column, eluted with 9:1 MeOH-H2O and 100% MeOH. Final purification was achieved by C8 HPLC using M e O H ^ % H 2 O-0.01% TFA [88]. Sampsoniones A-M (100-102, 105, 112, 118, 124-130) were isolated from whole air-dried Hypericum sampsonii which was extracted with 95% EtOH. The EtOH extract was concentrated under reduced pressure and partitioned between CH2CI2 and H2O. The CH2CI2 phase was chromatographed over silica gel, eluted with hexane-EtOAc mixtures. The sampsonione enriched fraction(s) were rechromatographed over silica gel, eluted with hexane-CHCb-acetone mixtures. Individual sampsoniones were isolated by preparative TLC or VLC over Cis [1114]. The vismiaphenones (35, 44, 45, 53) were isolated using Sephadex LH-20 with 1:1 CH 2 Cl 2 -Me0H, followed by normal-phase HPLC (17:3 hexane-'PrOH) with a cyano column [15]. Percolation with hexane [67] or extraction with CI-bCh-MeOH, then MeOH [67] were used in isolating plukenetiones B-G (77, 78, 91, 92, 99, 113). After extraction, Henry et al. subjected the hexane extract to repeated chromatography over silica gel using Me2CO-hexanes mixtures to yield plukenetiones B-G [67]. Chaturvedula et al. isolated the related benzophenone 15,16-dihydro-16-hydroperoxyisoplukenetione F (108) from Ochrocarpos punctatus by fractionation over Sephadex LH-20 with n-hexane-EtOAc, followed by preparative reversed-phase TLC and reversed-phase HPLC Chaturvedula et al. used a similar procedure to isolate the ochrocarpinones A-C (97, 98, 111) from O. punctatus [78]. The benzophenone glycoside iriflophenone-4-OP-D-glucopyranoside (58) was isolated from Davallia solida by chromatography of the nBuOH layer over Sephadex LH-20, eluted with MeOH [56]. The first fraction was further purified by preparative cellulose TLC and then reversed-phase HPLC with MeOH-H2O to yield 58. Porto et al. methylated a Clusia floral resin extract, chromatographed the derived compounds over silica gel, rechromatographed using preparative argentation TLC (5% silver nitrate), and thereby isolated seven polyisoprenylated benzophenones [68]. Lokvam et al. [74] and de Oliveira et al. [59] also methylated a crude extract before isolating chamones I, II (119, 116), and nemorosone II (89) from Clusia species. The isolation of xanthochymol (138), shown in Fig. (3), illustrates typical methods used to purify benzophenones. Two partitioning methods were developed in the course of our laboratory work with Garcinia
750
xanthochymus. The first method dissolved the MeOH extract in 9:1 H2OMeOH and partitioned sequentially with hexane and EtOAc. This was a less-than-optimum system because the benzophenones, biflavonoids, and xanthones were found in both organic phases. An optimized method resuspended the dried MeOH extract in 100% water and partitioned sequentially with CHCI3 and EtOAc concentrating the benzophenone and xanthones in the CHCI3 partition and the biflavonoids into the EtOAc layer, Fig (3). After partitioning, the CHCI3 layer was separated over Sephadex LH20 and eluted with MeOH. The benzophenone-enriched fraction was chromatographed repeatedly over re versed-phase (2:8-0:1 H2O-MeCN, 5% steps) to yield two novel benzophenones, the known benzophenone aristophenone A (109) and fractions A and B, each a mixture of benzophenone double-bond isomers [112]. Fraction A, was a mixture of 136 and 138, Fig. (3C), and fraction B was a mixture of 131 and 133. Repeated attempts to separate these fractions using normal-phase and reversed-phase preparative TLC; column chromatography over Sephadex LH-20, silica gel, C\%, polyamide, and cyano columns; and HPLC over Ci8, C8, cyano, phenyl, and silica columns were unsuccessful. Other researchers have encountered difficulties in separating benzophenone double-bond mixtures consisting of 136 and 138 and related compounds [88,90]. After a protracted method development using various types of argentation (silver) chromatography systems, compounds 136 and 138 were isolated with a quaternary solvent system (40:10:1.25:0.2 hexaneEtOAc-95% EtOH-TFA) over normal-phase TLC impregnated with a 10% solution of AgNO3, Fig. (3D and 3E). This procedure was also used to separate 131 from 133 [112]. The separation of G. xanthochymus was monitored by HPLC (described below) and TLC. Two d 8 TLC systems, 1:1 and 15:85 10 mM ammonium acetate-MeCN, were used to combine collected fractions. After development, compounds were visualized with 1% vanillin in acidified EtOH. After heating, benzophenones turned greenyellow. These two TLC systems proved very useful in monitoring the separation of benzophenones in our studies on G. xanthochymus and for the dereplication of a number benzophenones in other Clusiaceae species. Table 2 lists analytical and preparative HPLC methods developed for the isolation and quantification of benzophenones. Most methods have utilized reversed-phase Cig columns with mixtures of MeCN or MeOH and H2O, with or without an acid modifier. Exceptions include
OUh
C
138 and 136
U.H" Ei.tr
^
D
O 10-
PP5"
138
u.w
*
mmJ
QCrJft-
|
I
E
frfljfr
136
• 1
OI>HV
4t.U(V
Fig. (3). Isolation of xanthochymol (138). A: EtOAc partition, B: CHCI3 partition; HPLC system, gradient: 9:1 10 mM ammonium acetate—MeCN to 100% MeCN (see text for details), PDA extracted at 254 nm C Fraction A, before Ag-TLC D. Isolated xanthochymol (138) E. Isolated guttiterone E (136); HPLC system, gradient 1 1 1 0 mM ammonium acetate—MeCN to 100% MeCN in 26 mm at 1 mL/min
752
Nucleodex p-PM [90], C8 [88], and cyano [15] columns used in three different methods. During our isolation work with G. xanthochymus two HPLC methods were developed. The HPLC methods used a Phenomenex Luna Cig (5 jum, 250 x 4.6 mm) column and a solvent system of A = 10 mM ammonium acetate and B = MeCN. In the first method the initial conditions were 9:1 A-B, and a linear gradient was initiated until minute 45. The final solvent mixture was 0:1 A-B. The column was held at 100% B until minute 55, and then the initial conditions (9:1 A-B) were reinitiated at minute 56. The column was equilibrated for 10 minutes before the next injection. In the second system the initial conditions were 1:1 A-B, and a linear gradient was initiated at minute 4 until minute 26. The final solvent mixture was 0:1 A-B. Sample chromatograms are shown in Fig. (3). Both HPLC systems were used to track compounds isolated from G. xanthochymus fruits and to dereplicate benzophenones in a number of other Clusiaceae species. Table 2: Published HPLC Methods Used for the Isolation and Quantification of Benzophenones Compound(s) Analyzed 48-50 69 93 70,71 97,98 6, 53, 55, 59 109,110 108, 111 136,138 136', 138" 117,120,121 131,135,136 135,144,145 132,133, 136,138 Compound(s) Analyzed 66, 88, 96 88 79,80,85,87,94,112, 114,115 "isolated as a mixture
Isolation Methods Column; Solvent System MetaChem, Intersil ODS-3 (8 urn, Cm, 250 x 20 mm) 10 mL/min; isocratic 4:1 MeOH-H 2 O; gradient 70:30 to 90:10 MeOH-H 2 O in 30 minutes Column not reported; 9:1 MeOH-H 2 O Waters ^Bondapack C, 8 2 mL/min; 9:1 MeOH-H 2 O Column not reported; 88:12 MeOH-H 2 O Shimadzu ODS C, 8 (250 x 10 mm); 70:30 MeCN-H 2 O Dynamax-cyano (4.1 x 30 cm), 80 mL/min; 17:3 hexane-'PrOH Waters .uBondapack C l g 2 mL/min; 9:1 MeOH-H 2 O Shimadzu ODS C 18 (250 x 10 mm); 75:25 MeCN-H 2 O Nucleodex P-PM (5 fim, 250 x 10 mm) at 0 °C; 52.5:47.5:0.1% MeCN-H 2 O-TFA Rainin Dynamax (1.0 x 25 cm); 97:3 MeCN-H 2 O Cosmosil 75 C, 8 Prep; 9:1 MeOH-H 2 O and 1:0 MeOH-H 2 O Rainin Dynamax (1.0 x 25 cm); 24:1 MeCN-H 2 O Rainin Dynamax (1.0 x 25 cm); MeOH^t% H 2 O-0.01% TFA Phenomenex Luna C, 8 (5 fim, 250 x 4.6 mm) 1 mL/min; gradient 9:1 10 mM ammonium acetate-MeCN to 100% MeCN in 45 minutes Quantification Methods (column; Solvent System) Waters Novapak C18 (4 ^m, 3.9 x 150 mm) 1 mL/min; gradient 60:40 to 100:0 MeCN-H 2 O in 60 minutes (quantified as Me esters) Column not reported; gradient 50:50 to 100:0 MeOH-AcOH 2% in 15 min Waters Novapak Cu (4 fim, 3.9 x 150 mm) 1 mL/min; gradient 60:40 to 100:0 MeCN-II 2 O in 60 minutes (quantified as Me esters)
Ref.
[52] [621 [77] [62] [78] [15] [811
US] [90] [88] [17] [881 [88] [112]
[59] [102] [68]
753
STRUCTURAL ELUCIDATION OF BENZOPHENONES The structures of benzophenones have been established by UV, IR, MS, and most extensively, by ID and 2D NMR. The structures of a few benzophenones have been determined by X-ray crystallography including: nemorosonol (88) [76], 7-epi-clusianone (81) [71], xanthochymol (138) [113], and (-)-isoxanthochymol (131) [104,114]. Chemical tests with FeCU [47] or Gibbs reagent [27] and acetylation [27] or methylation [42] are used to show the phenolic nature of benzophenones. IR has been useful in showing that benzophenones contain hydroxyl groups, both conjugated and nonconjugated ketone groups, and aromatic C=C bonds. Xanthochymol (138), to our knowledge, is the only benzophenone analyzed, in detail, for its electron impact MS fragmentation behavior [110]. Reported MS losses for benzophenones include an mlz 105 (C6H5CO+) for a unsubstituted phenyl ketone A-ring [44,65], mlz 137 (C6H5 O2-CO"1") for a 3,4-dihydroxybenzophenone moiety, and mlz 68 (CsHg) for a prenyl-type group. The positive electrospray ionization (ESI) mass spectrum, Fig. (4), of xanthochymol (138) showed a base peak at [M + H] + = mlz 603 and ,M ao 75: JO
s.l
ut.11 3>lm
2»£5_"3°z a , * , 300
150
UO
ISO
»"• «0
+i»«r| J t f »
i i
<SO
150
SOJ
Fig. (4). Positive ESI mass spectrum of xanthochymol (138)
754
losses at mlz 467, loss of the 10 carbon side chain attached at position 8 or the loss of the 3,4-dihydroxybenzophenone moiety. The mlz 411 ion likely represents an additional prenyl group loss. Additional fragments were observed at mlz 343, 286, and 233 likely corresponding to additional losses of 1, 2, or 3 prenyl, or parts of prenyl, groups. The negative ESI mass spectrum (not shown) only displayed a base peak at [ M - H T = ;»/z601. The structures of most benzophenones have been determined by 1D ('H, 13C, and DEPT) and 2D (COSY, HSQC, HMBC, and NOESY) NMR experiments. The majority of benzophenone NMR spectra have been recorded in CDCI3 and CD3OD. Benzene-c/6, mixtures of benzened(, and CDCI3 [75], or pyridine-ds have also been used [88,93]. The aforementioned solvents were used to resolve overlapping signals of studied compounds. Deuterated TFA (0.1%) has also been used to increase the rate of keto-enol interconversion in benzophenones. We now turn our attention to the structural elucidation of xanthochymol (138). The 500-MHz *H spectrum of xanthochymol (138) is displayed in Fig. (5). The chemical shifts, coupling data, multiplicity, COSY, and HMBC correlations are shown in Table 4. Because of the tautomeric nature of benzophenones, NMR spectra are sometimes acquired after the addition of deuterated TFA (0.1%). We found this to be essential when using CDCI3, but not with CD3OD. However, carbon spectra recorded with CD3OD-TFA displayed sharper signals for C-l, 3, 4, 8, and 10. To aid in the structural determination of xanthochymol (138), we divided the proton spectrum into three regions. Region one, from 5 0-3.0, contains numerous overlapping signals, but yields some valuable information, specifically the number of methyl groups. Due to overlapping signals, the exact number of methyl groups is not conclusive, but 7 or 8 is a good approximation. Furthermore, the methyl signals appear as two groupings, two upfield signals at 8 1.01 and 1.17 and 5-6 signals between 5 1.5-1.8. The two upfield signals are characteristic of geminal-dimethyl protons on a sp3 carbon. The number of methyl groups from 5 1.5-1.8, along with data from the next region (5 4.0-5.2) indicates the number and type (isopent-2-enyl versus isopent-3-enyl) of prenyl groups in xanthochymol (138). The 'H spectrum of xanthochymol (138) displays signals for a least one olefinic proton at ca. 5 5.00 (indicating two isopent-3-enyl groups) and two signals for terminal methylene protons, indicating the number of isopent3-enyl groups. Due to overlapping 'H signals, DEPT-135 and HSQC experiments are needed to confirm the number and type of prenyl groups.
Methyl groups
Methine and methylene groups
AMX spin system
Terminal methylenes
Olefinic protons
uw I
• • • • I • • • • I ' ' ' ' I • • • • I • ' • . I • ' ' • I ' • • • I • • ' ' I ' • ' ' I ' • ' • I ' • • ' I • • • • I ' ' ' ' I ' ' ' ' I
7 . 5
7 . 0
6 . 5
6 . 0
5 . 5
5 . 0
4 . 5
4 . 0
3 . 5
3 . 0
Fig. (5). 'H spectrum of xanthochymol (138) recorded at 500 MHz in CD,OD
2 . 5
2 . 0
1 . 5
756
The last region (8 6.0-8.0) reveals one structural fragment. In polyprenylated benzophenones, the aromatic A-ring typically has either a 3- or 5-spin pattern. Xanthochymol (138) clearly displays an aromatic 3spin system consisting of protons at 5 7.22 d (J = 2.1 Hz), 7.00 dd (2.1, 8.1 Hz), and 6.72 d (8.1 Hz). Even though the ] H spectrum is crowded, three important structure features are obtained: i) the type of aromatic Aring; ii) the approximate number and types of prenyl groups; and iii) the presence of an aliphatic geminal-dimethyl group. The 13C, DEPT-135, and DEPT-90 experiments showed a nonconjugated ketone at 5 209.8, flanked by two quaternary bridgehead carbons (8 59.5 and 68.9), an enolized 1,3-diketone [8 117.9 and 194.5 (2x)], a methylene carbon at 8 42.7, and a methine carbon at 8 47.0 which, together with a quaternary carbon at 8 50.4, established the bicyclo[3.3.1]nonane system in xanthochymol (138). The DEPT-135 showed five sp2 methines and six aliphatic methylenes. The number of methine carbons, five sp2 and two aliphatic, was confirmed by a DEPT90 experiment. The carbon and DEPT experiments further indirectly confirmed the AMX 3-spin system with 13C quaternary aromatic resonances at 8 128.7, 145.3, and 151.4, and the number of prenyl groups as four (two isopent-2-enyl and two isopent-3-enyl groups). The HSQC spectrum, displayed in Fig. (6), unequivocally establishes the number and type of protonated carbons in xanthochymol (138). Even though the DEPT-135 experiment displays the number of CH3, CH2, and CH carbons, the HSQC spectrum resolves the following: i) an olefinic CH proton is under the H2O signal, thus 138 contains two isopent-2-enyl groups; ii) xanthochymol (138) has two terminal methylenes, and, therefore, two isopent-3-enyl groups; and iii) the exact number of CH3, CH2, and CH carbons. Therefore, the HSQC spectrum establishes that 138 contains eight methyl, eight methylene, and seven methine carbons. Resonances for C-H pairs are listed in Table 3. Table 3. Protonated Carbons of Xanthochymol Based on the HSQC Spectrum CH
CH,
CH3
"H .01 .17 .52 .63 .67 .71 .71 .75
5 C 26.4 22.2 17.2 16.8 25.0 17.3 21.8 25.4
S
H 1.9 2.0
1.3,1.5 2.05, 2.25 2.08,2.28 4.52 4.65 2.55,2.73
S
C
35.8 36.7 32.1 42.7 29.8 112.5 109.4 26.0
S
H
1.52 2.6
4.90 5.05 6.72 7.00 7.22
"C 47.0 43.7 124.1 120.4 114.0 124.2 116.2
-J
Cn
-
u> -
•I
O
I
o o
I
I • " 1X1
CO
o
o
o
I "
o
o
Fig. (6). HSQC spectrum of xanthochymol (138) recorded at 500-MHz in CD,OD
l in
758
The COSY spectrum connects 3 multi-spin networks in xanthochymol (138). Analysis of the COSY spectrum shows one olefinic proton coupled to a methylene group. The second olefinic proton is also coupled with a second methylene group, and an additional methylene and methine group. Also, a CH2-CH-CH2-CH2 coupled spin system, possibly connecting two prenyl groups, is observed. The three observed proton spin systems are: i) 5 5.05 (18), 2.73 (17A), and 2.55 (17B); ii) 8 4.92 (25), 2.28 (24A), 2.08 (24B), 1.52 (6), 2.25 (7A), and 2.05 (7B); and iii) 5 2.0 (29AB), 2.6 (30), 1.5 (34A), 1.3 (34B), and 1.9 (35 AB). At this point, using the 'H, 13C, DEPT, COSY, and HSQC spectra, we have tentatively identified five structural fragments shown in Fig. (7). We now use the HMBC spectrum, Fig. (8), to confirm these structural fragments and to assemble the complete structure of xanthochymol, Fig. (9). The HMBC spectrum, Fig. (8), confirms the following substructures: i) HMBC correlations from H-12 (5 7.22) to C-13 (5 145.3) and C-14 (5
X
Fig. (7). Structural fragments of 138 deduced by ] H,
I3
\
C , DEPT, COSY, and HSQC spectra
151.4) and from H-15 (5 6.72) to C-13 (5 145.3) and C-14 (5 151.4) confirms the 3,4-dihydroxybenzophenone moiety, and a HMBC cross peak from H-12 (5 7.22) to C-10 (5 194.8) connects the AMX system to the carbonyl carbon at C-10 (5 194.8), Fig. (7A); ii) HMBC cross peaks from H-7B (5 2.05) to C-l (5 194.5) and from H-6 (5 1.52) to C-7 (5 42.7), C-5 (5 50.4) confirmed the bicyclo[3.3.1]nonane system, Fig. (7B); and iii) the geminal-dimethyl protons which displayed HMBC cross peaks to each other, Fig. (7C); iv) HMBC cross peaks from the methyl groups to the olefinic carbons and correlations from the CH2 groups to their respective olefinic carbons confirmed the two isopent-2-enyl groups, Fig. (7D); and v) HMBC cross peaks from H-29 (5 2.0) to C-30 (5 43.7) and C-31 (5 147.9), from H-35 (5 1.9) to C-34 (5 32.1), C-30 (5 43.7), and C-37 (5 109.4), and from H-33 (5 1.63) to C-30 (5 43.7) confirmed the two isopent-3-enyl groups, Fig. (7E), are connected at C30.
Fig. (8). HMBC spectrum of xanthochymol (138) recorded at 500 MHz in CD,OD
760
Finally, the HMBC spectrum is used to connect the structural fragments to the bicyclo[3.3.1]nonane' system and complete the structure of xanthochymol, Fig. (9). Observed HMBC cross peaks from H-17B (5 2.55) to C-3 (5 194.5) and from H-7B (5 2.05) to C-24 (5 29.8) placed the isopent-2-enyl groups at C-4 (5 68.9) and C-6 (5 47.0). HMBC cross peaks from Me-22 and Me-23 to C-4, 5, and 6 (5 68.9, 50.4, 47.0, respectively) placed the geminal-dimethyl group at C-5. An HMBC correlation was observed from H-29 to C-l, thus the two isopent-3-enyl groups, Fig. (7E) were placed at C-8. Therefore, using ID and 2D NMR experiments we assigned the structure in Fig. (9) to xanthochymol. 27
Fig. (9). Arrows denote key HMBC cross peaks and bold lines indicate key COSY correlations for xanthochymol (138)
1 For clarity the stereochemistry of the bicyclo[3.3.1]nonane is not shown, as not to confuse it with the bolded COSY cross peaks.
761 Table 4. ] H, 13C, DEPT, COSY, and HMBC NMR Data for Xanthochymol (138) in CD3OD H" DEPT" C COSY" HMBC"
(5, mult. J= Hz) (8) 1 194.5 2 117.9 3 194.5 68.9 4 S 50.4 1.52, m 47.0 6 7 2.05,2.25, m (12.8) 42.7 8 59.5 9 209.8 10 194.8 11 128.7 12 7.22, d, (2.1) 116.2 13 145.3 14 151.4 15 6.72, d, (8.1) 114.0 16 7.00, dd, 2.1,(8.1) 124.2 17 2.55, 2.73, m 26.0 18 5.05, t, (5.5) 120.4 19 134.7 20 1.75 25.4 21 1.71 17.3 22 1.17 22.2 23 1.01 26.4 24 2.08,2.28 29.8 4.92, 124.7 25 26 132.6 1.67 25.0 27 1.52 17.2 28 2.0, m 36.7 29 2.6, m 43.7 30 147.9 31 4.52, brs 32 112.5 1.63 16.8 33 34 1.3, 1.5, m 32.1 1.9, m 35.8 35 145.9 36 4.65, brs 109.4 37 1.71 21.8 38 •Recorded at 500 MHz; "Recorded at 300 MHz
C C
c c c
CH CH2 C C C C CH C C CH CH CH2 CH C CH, CH, CH, CH, CH2 CH C CH, CH, CH2 CH C CH2 CH, CH2 CH2 C CH2 CH,
7,24 6
5, 7, 23, 24 1,24
16
10, 13, 14, 16
16 12, 15 18 17
11, 13, 14 12, 14 3,18,19
18,19,21 18, 19,20 5, 6, 23 4, 5, 6, 22 6,25 24
30 29,34
30,35 34
25, 26, 28 25, 26, 27 1,30,31
32 30,31,32 30 30, 34, 36, 37 35,38 35,36,37
BIOLOGICAL ACTIVITIES Isolated benzophenones display a number of biological activities, Table 5. Reported biological activities are grouped into eight categories: antioxidant, antibacterial, antifungal, antiviral, cytotoxic, molluscicide, trypanocidal, and additional. Of the 146 isolated benzophenones only 49, (33%) have been evaluated for their biological activity. This section discusses the various biological activities of isolated benzophenones.
762 Table 5. Reported Biological Activities for Benzophenones Antioxidant Compound 7 13 17 83 88 131
Assay
DPPH (1,1 -diphenyl-2-picrylhydrazyl)
Result ICso = 66.3/iM IC5O = 5.3^M IC 50 = 7.8/jM IC 5 0 >100//M IC50 = 44.1/iM ICso=13.3//M
134
ICsu = 10.2/iM
139 140 131 134
IC a , ICso Weak Weak
134
Compound
86
89 119 93
Peroxide determination Hypoxanthine/xanthine oxidase system Fenton reaction system H 2 O 2 /NaOH/DMSO system Antibacterial Assay/organism Bacillus subtilis Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumoniae Candida albicans Paenibacillus larvae Paenibacillus alvei Paenibacillus larvae Paenibacillus alvei Streptomyces chartrensis, Streptomyces violochromogenes
131 132 133 134 138
Methicillin-resistant Staphylococcus aureus (MRSA)
134
Helicobacter pylori
138
Compound 81 86 93
Compound 13 83 131 134 140
Staphylococcus aureus Escherichia coli Streptococcus faecalis Klebsiella pneumonia Antifungal Assay/organism Cladosporium sphaerospermum A range of organisms Candida albicans, Candida tropicalis, Pseudomonas aeuruginosa Antiviral Assay
Epstein-Barr virus early antigen
> 100 > 100 activity activity
Ref. [29] [371 [1021 [371 [37. 115,116] [37] [117]
Suppressed the superoxide, hydroxyl, and methyl radicals
[115, 118]
Result 14.2 ± 0.2 mmh 15.0±0.2mm b 14.4 ±0.1 mmb 14.0 ± 0.2 mmb 14.1 ±0.1 mm' 19.5 mm b 2.3 mm' 19.7 mm" 10.0 mm"
Ref.
50"
[77]
12-25/jg/mL 25/ig/mL* 25/jg/mL" 6.25 - 25/jg/mL 3.1 -12.5/ig/mL
[89]
Complete inhibition at 31.5 /jg/mL at 6h and 3.9 fig/mL at 12h Active Inactive 0.78 fig/mL* 1.56 ^g/mL11
[72]
[74] [74]
[37,119] [87] [120]
Result Inactive Activite
Ref. [1211 [72]
16.2"
[77]
Result
52.7±2.3® 52.7±2.3® 56.0*1.9® 47.0 ±2.5® 54.9 ±2.6®
16nmol 16nmol 16 nmol 16nmol 16nmol
Ref.
[37]
763 Antiviral (cont.) Compound 141 46 53 55 59 132 135 136 137 141 142 143 117 120 121
Compound 33 37 42 47 48 49 50 63 64 69 70 73
Assay
Anti-HIV
Anti-HBeAg secretion on MS-G2 hepatoma cell line Cytotoxic Assay HSC-2 human oral squamous cell HGF normal human gingival fibroblasts HSC-2 human oral squamous cell HGF normal human gingival fibroblasts HSC-2 human oral squamous cell HGF normal human gingival fibroblasts HSC-2 human oral squamous cell HGF normal human gingival fibroblasts
KB cell line
131/133" 136/138" 97 98 108 111 135 144 124 125 88
A2780 human ovarian cells
P388 cell line HeLa human cervix carcinoma Hep-2 human larynx carcinoma PC-3 prostate cancer U251- central nervous system HL-60 leukemia cells
134
Compound 81
K562 leukemia cells NB4 leukemia cells U937 leukemia cells Molluscicide Assay/organism Biomphalaria glabrata
Result 60.1 ±2.1 @16nmol Inactive Inactive EC51, ~ 11 /jg/mL Inactive Inactive ECso = 1 -10/jg/mL EC 50 = 1 -10,ug/mL ECso = 23 ^g/mL EC 5 o=l-lO/ig/mL EC5() = 1 -10^g/mL EC SO =1 -10^g/mL 10,ug/mL 10/ig/mL 10/ig/mL
Ref.
Result CC50 = 0.17mM CCSI, = 0.43 mM CC50 = 0.036 mM CCa, = 0.090 mM CC50 = 0.092 mM CC50 = 0.19mM CC50 = 0.052 mM CC5l, = 0.19mM ECso > 20 ^g/mL EC5IJ > 20 /jg/mL ECso > 20 //g/mL ECso = 2.4 ± 0.9/ig/mL EC51> = 3.3 ± 1.5/ig/mL ECso = 10.0 /ig/mL (inactive) ECso = 9.0 ^g/mL (inactive) EC50 = 8.2 fig/mL (inactive) IC50 = 5.8 fiM IC50 = 10/iM ICso = 7.4±O.2/jg/mL ICso = 8.2 ± 0.2 /ig/mL ICs,, = 8.4±0.6/(g/mL lCso = 6.9±0.3^g/mL ICso = 6.8 ,ug/mL ICso = 8.0 ^g/mL EDso = 6.9/(g/mL ED50 > 30 /jg/mL (inactive) ICso = 3.3 MM \C5a = 3A/iM
Ref.
ICso = 7.2 /iM ICso = 3.9 juM ICso = 9.42 ftM ICso = 5-20 MM ICso = 5-20/;M ICso = 5-20 MM ICso = 5-20 / J M
Result Inactive
[15]
[88]
[17]
[47]
[52]
[62] [90] [78]
[93] [14]
[102] [1221 [82]
Ref. [121]
764 Trypanocidal Compound
Assay/organism
81
Trypanosoma cruzi
135
Epimastigotes Trypomastigotes
Result LC5o = 518 fiM active in vitro, (not in vivo) in mice 100/iM 83 fiM
Ref. [121] [92]
Additional Compound 7 13 17 81 10 49 131/133' 136 138 131 134 138 138
Assay Brine shrimp lethality
Testosterone 5a-reductase DNA strand-scission activity Microtubule disassembly inhibition Apoptosis detected by Western blot analysis
Result LD 5U > 100/JM LD5,, = 43.1 fiM LD 5U > 100,uM LC 50 = 49.7 MM IC 50 > 1.00 mM 43 ± 12% nicked at 2.5 /ig/mL Inactive IC S 0 =1.5//M IC50 = 2 ^ M Apoptosis activation of caspase-3
Male F344 rats using a aberrant crypt foci (ACF) bioassay w/ azoxymethane (AOM)
No CNS effect at 1/5 LD 50 (LD 5 ,,= lOOOmg/kgi.p.) Significantly inhibited AOM induced ACF formation
Apoptosis induction in HL-60 cells
EC5,, = 8.4^M
Cardiovascular effects in cats
Ref. [29] [1211 [35] [52] [90]
[82] [120] [123]
[116] Reduced NO production 49, 87, and 92% at 2.5, 5, and 1 0 ^ M , respectively Ulceration induction in rats by Significantly prevented adverse [118] affects indomethacin "tested as a mixture; bzone of inhibition; ctotal inhibition, dminimum inhibitory concentration, "concentration required to eliminate 99% of organism 134
Griess reaction
A number of benzophenones have been assayed for their cytotoxicity towards ovarian [78,93], leukemia [14,82], and CNS [102] cancer cell lines. In addition, the guttiferones (132 and 135-142) have displayed potent cytotoxicity toward leukemia [82] and ovarian cancer cell lines [37,93]. Garcinol (134), also a guttiferone, has been evaluated for a number of biological activities. Garcinol was found to neutralize the superoxide anion, methyl, and hydroxyl radicals in a variety of antioxidant assays [118] and displayed anti-glycation activity in a fructose-BSA assay [115]. Sang et al. [116] studied the reaction mechanism between the stable free radical l,l-diphenyl-2-picrylhydrazyl (DPPH) and 134. They also isolated the DPPH/garcinol oxidation products [116] and tested 134 in a NO generation, apoptosis, and H2O2 antioxidant assays. Two animal studies have been conducted on garcinol (134). The first study used F344 male rats and administered 134 at 0.01% or 0.05%. Garcinol provided significant in vivo protection against the development
765
of aberrant crypt foci (ACF) [123]. Another study, using male Wistar rats, evaluated a G. cambogia extract against indomethacin induced gastric ulcers [124]. They concluded that G. cambogia extract prevented gastric ulcer formation and maintained the rats at a near normal state, but the bioactive constituents were not identified. However, benzophenones 131 and 134 have been isolated from G. cambogia fruits [91]. An extract of G. cambogia, sold in the US as a dietary supplement, has also been evaluated as a weight loss aide [125]. Hydroxycitric acid, rather then a benzophenone, is believed to be the compound responsible for the weight loss properties of G. cambogia. Other reported activities for 134 include a bactericidal effect on Helicobacter pylori [119], strong cytotoxicity [82,122], and additional antibacterial activities [89]. The guttiferones (132 and 135-142) displayed partial cytoprotection toward HIV-1 infection in human lymphoblastoid CEM-SS cells; however, no decrease in viral replication was observed [88,96]. The vismiaphenones were also assayed for their anti-HIV activity in the NCI primary HIV screen; only 55 was active [15]. Guttiferone A (135) and 7-epi-clusianone (81) were assayed against Trypanosoma cruzi, the etiologic agent of Chagas' disease, and both were found to be active [92,121]. Xanthochymol (138) and guttiferone E (136) displayed outstanding activity in a in vitro microtubule disassembly assay [90]. Kolanone (86), nemorosone II (89), propolone A (93), chamone I (119), and xanthochymol (138) displayed significant antimicrobial and antifungal activity against a variety of pathogenic yeasts and bacteria [72,74,77,120]. Compounds 131-134 and 138 displayed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) at 3.1 - 12.5 ,ug/mL, nearly equal to the antibiotic vancomycin [89]. CONCLUSION Benzophenones are an interesting class of compounds displaying much structural diversity and numerous bioactivities. By far, the richest sources of benzophenones have been Clusiaceae species. However, of the approximately 37 genera and 1610 Clusiaceae species [126], only a few genera/species have been exhaustively analyzed. It is likely that a number of novel bioactive benzophenones are still undiscovered.
766
ABBREVIATIONS ACF CH2C12 CHCb COSY DEPT-135 DPPH ESI EtOH geranyl group HMBC HPLC HSQC IR Me2CO MeCN MeOH MS NMR PPB prenyl group TLC VLC
Aberrant crypt foci Methylene chloride Chloroform Correlation spectroscopy Distortionless enhancement by polarization transfer 1,1 -diphenyl-2-picrylhydrazyl Electrospray ionization Ethanol -CH2-CH=C(Me)-CH2-CH2-CH=C(Me)2 Heteronuclear multiple-quantum correlation High-performance liquid chromatography Heteronuclear single-quantum correlation Infrared spectroscopy Acetone Acetonitrile Methanol Mass spectroscopy Nuclear magnetic resonance Polyprenylated benzophenone -CH2-CH=C(Me)2 Thin-layer chromatography Vacuum-liquid chromatography
ACKNOWLEDGEMENTS Scott Baggett was supported by NIH-NCCAM National Research Service Award #F31-AT00062. This research was supported by funds from the NIH-National Institute of General Medical Sciences SCORE award S06GM08225 and the Professional Staff Congress of The City University of New York (PSC-CUNY) award 669662. Kurt Reynertson is thanked for his careful review of this manuscript. REFERENCES
[1] [2]
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BIOACTIVE COMPOUNDS FROM TRIPTERYGWM WILFORDII RENSHENG XU, JOHN M. FIDLER AND JOHN H. MUSSER Pharmagenesis Inc., Palo Alto, CA 94304, USA ABSTRACT: Tripterygium wilfordii (Celastraceae) is a wild shrub distributed in southern China. It was used in Traditional Chinese Medicine as an insecticide for killing fly larvae 'maggots and Oncomelania snails, a vector of Schistosoma japonicum. In 1969, the decoction of the radix of T. wilfordii was first used in China to treat patients with rheumatoid arthritis (RA); although efficacious, side effects were observed. Subsequently, a partially purified extract called "multiglycoside of the radix" (TWG) was used to treat autoimmune diseases including RA, lupus erythematosus, chronic nephritis and hepatitis. TWG appeared to have fewer side effects compared to the T. wilfordii decoction. In China, TWG is also used to treat various skin disorders, such as psoriasis and leprosy. In an unrelated area, TWG was found to have male spermicidal antifertility activity. Thus far, more than 46 diterpenoids, 20 new triterpenoids, 26 alkaloids and other small molecules have been isolated and identified from the plant. Among them the most potent are triptolide type compounds, which show immunosuppressive, anticancer and antifertility activities. The derivative of triptolide, PG490-88, is being evaluated in phase I clinical trials as an anticancer agent. The natural products structural chemistry of T. wilfordii has been well defined with several total syntheses of triptolide reported.
INTRODUCTION Traditional Chinese Medicine (TCM) is one of the most developed time-honored medicines in the world. It has a long history of use in practice and has been carefully recorded in ancient books. Shennong-Bencao (Shennong herbs) is a compendium book published back to A.D.200 [1]. The characteristics and medical applications of 356 medicines have been described in the book. Now TCMs have been developed to include 10,000 herbs and are popular in China for treating different kinds of diseases. The development of TCM with modern science and technology has allowed the exploration of many new drugs; some of them have
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been acknowledged worldwide. The famous antimalarial artemisinin (qinghausu)[2] and anti-dementia huperzine A [3] are examples. Recently the high efficiency of immunosuppressive activities of Tripterigyum wilfordii (Celastraceae) has attracted the attention of scientists. The herb was recorded early in "ShengnonBencao" as a toxic agent and used as an insecticide for killing fly larvae 'maggots' and oncomelania snails, a vector of Schistosoma japonicum [1,4]. According to TCM, TW radix activates blood circulation and relieves blood stasis, counters inflammation and relieves edema, purges internal heat and eliminates toxins. In 1969, the decoction of the radix was first used in a hospital in Fujian to treat rheumatoid arthritis (RA) patients and showed a high level of efficacy [16], but side effects were also observed. Later, the purified extract of xylem from its roots, the so-called "Reigongten-doudai" (multiglycoside of the herb, TWG), was developed as an over-the-counter drug popularly used in China to treat various immunosuppressive diseases (e.g., RA, lupus erythematosus, chronic nephritis and hepatitis) [5]. Studies on the plant further characterized its biological activities and chemistry. The following is a summary of these studies, with a focus on the chemistry of compounds extracted from Tripterygium wilfordii.
EXTRACTS OF THE TW PLANT AND THEIR BIOACTIVITIES There are several forms of purified extract reported in the literature: TWG/T2, EA and PG27. For TWG/T2, the xylem of the roots was extracted by water, then the chloroform extraction of the water extract was passed through silica gel columns, eluted with chloroform and subsequently with chloroform with 10% alcohol, and the later elution was concentrated, dried and used as TWG [6]. EA was the ethyl acetate extract of the xylem[7]. For PG27, the roots were extracted with alcohol, and the concentrated dichloromethane extract of the evaporated alcohol extract was passed through the silica gel column and eluted with CH2CI2 and CH2CI2: MeOH 95:5; the concentrated and dried late elution
775
fractions were used as PG27 for bioassays [8]. All of these extracts show immunosuppressive activity in vitro and in vivo. TWG inhibited Concanavalin A-induced proliferation of mouse spleen cells and thymocytes in a dose-related manner. The expression of interleukin-2 (IL-2) receptors by Concanavalin Aactivated spleen cells was completely inhibited by adding TWG, but IL-2 production was not completely suppressed [9]. T2 at 0.1-1 |ig/ml inhibited antigen- and mitogen-stimulated proliferation of T cells and B cells, IL-2 production by T cells, and immunoglobulin production by B cells. T2 did not affect IL-2 receptor expression by T cells, IL-1 production by monocytes, or the capacity of monocytes to present antigen. Inhibition could not be accounted for by nonspecific toxicity. These results supported the conclusion that T2 exerts a powerful suppressive effect on human immune responses, and this action might account for its therapeutic effectiveness in RA [10]. The immunosuppressive activity of PG27 was demonstrated by prolonging rat heart and kidney allograft survival. PG27 administered intraperitonially to Lewis recipients for 16 days at 1030 mg/kg/day significantly increased the median survival time of Brown Norway heart allografts from 7 to 22 days. Oral administration was also effective, in prolonging the cardiac allograft survival time to > 100 days when PG27 treatment was given for 52 days. At a dosage of 20-30 mg/kg/day, PG27 significantly extended the median survival time of Brown Norway kidney allograft recipients from 9 up to 77 days, and 30 mg/kg/day of PG27 for 52 days extended survival beyond 200 days. PG27 combined with cyclosporin (CsA) significantly enhanced heart and kidney allograft survival, even at doses of CsA that were ineffective when administered alone, providing evidence of synergy between PG27 and CsA. PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, again showing synergy, as well as completely inhibiting xenoantibody production. PG27 also suppressed graft-versus-host disease in murine allogeneic bone marrow transplantation [6,11,12]. TWG exhibited a significant anti-inflammatory effect on acute agar-induced edema of the rat paw and suppressed carrageenaninduced inflammation in vivo. The number of exudate cells and the concentration of Prostaglandin E2 (PGE2), nitrite and TNF-oc in the
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exudates obtained from TWG treated animals were significantly reduced (by 69%, 78%, 57% and 77%, respectively), compared to that from vehicle-treated animals. Cyclooxygenase-2 mRNA was markedly suppressed in the air pouch lining tissue of TWG treated rats (p > 0.001). In contrast, PGE2 content of the kidney and stomach, and the production of PGE2, nitrite and TNF-a by spleen cells, were not affected by treatment with TWG [5, 13]. TWG has an anti-spermatogenesis action similar to that of gossypol. The fertility of Wistar rats given 10 mg/kg of TWG daily was reduced after four weeks of treatment and completely lost after treatment for eight weeks. The spermatocyte density and activity was remarkably reduced (P < 0.01), but neither the organs nor sexual activity changed during treatment. There were degenerative changes in the seminiferous tubules and sperm of male rats when they were treated with TWG at 30mg/kg for 35 and 80 days. The number of spermatocytes was also subsequently reduced as well as its susceptibility [14,15]. In 1983, Yu reviewed 144 cases of clinical use of TWG to treat rheumatoid arthritis. The total effective rate was 93.3% (17.6% remission, 37.5% effective and 38.2% improvement). The course of treatment was three months with an oral dose of 1-1.5 mg/kg, and the maximum daily dose was 90 mg given in three doses. Side effects included gastrointestinal disturbances, irregular menstruation and amenorrhea in female patients and gynecomastia in males. Side effects subsided after withdrawal of the drugs [5]. Another report showed various degrees of improvement by TWG treatment in 24 of 26 cases of lupus erythematosus (92.3%)[16]. TWG 1 mg/kg/day orally was used to replace azathioprine in tripledrug therapy (CsA, prednisone and azathioprine) for 10 renal transplant recipients; all of the recipients have been examined up to 3-10 months with good renal function. Side effects including mild gastrointestinal reaction were acceptable. It was concluded that TWG was more effective than azathioprine [17]. Recently similar clinical reports appeared in the literature [18 -20]. CHEMICAL COMPONENTS OF THE TW PLANT Kupchan et. al., were first to study the chemical components of the plant. Three new diterpenoids, triptolide 1, tripdiolide 2 and
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triptonide 4 were separated from the extract of the roots, collected from Taiwan. All of them have three specific epoxides and an a,punsaturated y-lactone ring in their structures. The 14-P-hydroxyl group in triptolide and tripdiolide is hydrogen bonded to the C-9,11 epoxide oxygen atom. The authors cited this relationship to explain the antileukaemic biological activity of these compounds[21 - 23]. Since then 46 diterpenoids have reportedly been separated from the plant or its variety, growing in Japan, Tripterigium wilfordii Hook fil. var. regelii Makino. Among them 12 (compounds 1-12) belong to the triptolide type, 21 (16-36) are of the triptophenolide type and 10 (37-46) belong to the triptoquinone type (cf. table 1 & fig.l)[2144]. Table 1.
New Diterpenoids from TW plant. Name
Formulae
M.P. ("C)
(<X]D (solvent)
References
1
Triptolide
C 2 0 H24O 6
226-227
-154°(CH 2 C1 2 )
21
2
Tripdiolide
C20H24O7
210-211
-138°(CH 2 CI 2 )
21
3
Tripterolide
C 2 oH 24 0 7
225-228
4
Triptonide
C 20 H 22 O 6
251-252
5
Triptolidenol
C20H24O7
193-194
25
6
16-OH-triptolide
C20H24O7
230-232
27
7
Tripchloride
C20H25O6CI
230-231
26
24 -175°(CH 2 C1 2 )
21
8
Triptotetraolide
C2oH26Og
258-260
28
9
Isotriptetraolide
C 2 oH 2 fi08
250-252
29
10
Triptriolide
CMH26O7
260-262
30
11
Tripdioltonide
C 2 oH 2 40(i
222-224
26
12
13,14-epoxide-9, 11,12-trihydroxytriptolide
C 20 H 26 O 7
268-270
26
13
Tripterifordin
C20H30O3
255-256
-46.6°(CHC13)
31
14
Tripterinin
CMH30O4
244-245
1.86°(CHCI 3 )
32
15
13-t77/'-manoyl oxide18-oic acid
C20H32O3
33
16
Triptonolide
C 20 H 22 O 4
34
778 able 11. Contd....)
Name
Formulae
M.P. (°C)
17
Triptophenolide (Hypolide)
C 2 oH 24 03
232-234
35
18
Triptophenolide methyl ether
C 2 ,H 2 6 O 3
152-154
35
[<X1D (solvent)
References
19
Neotriptophenolide
C2,H26O4
189-191
35
20
Isoneotriptophenolide
C2IH26O4
185-187
36
21
Triptonoterpene
C20H28O2
153-155
22
Triptonoterpene methyl ether
C21H30O3
209-211
189.5 (MeOH)
25
23
Neotriptonoterpene
C 2 ,H 30 O 3
205-207
95.1° (MeOH)
38
24
Triptobenzene A*
C 2 oH 2 80 3
amorphous
89.3° (CHCI3)
39
25
Triptobenzene B*
C 2 oH 3 o0 2
amorphous
18.3° (MeOH)
39
26
Triptobenzene C*
C20H26O4
amorphous
-7.4° (CHCI3)
39
27
Triptobenzene D*
C20H26O2
174-177
48.1°(CHC13)
39
28
Triptobenzene E*
C 2 oH 24 04
amorphous
29
Triptobenzene F*
C20H24O4
amorphous
39.0°(CHCl3)
39
30
Triptobenzene G*
C20H24O4
amorphous
31.7°(CHC13)
39
31
Triptobenzene H
C 2 iH 2 8 O 4
amorphous
171°(CHCI3)
40
32
Triptobenzene 1
C20H24O4
amorphous
33
Triptobenzene L
C20H30O2
amorphous
34
Triptobenzene M
C20H28O3
35
Triptobenzene N
C 2 oH 2 60 3
36
Triptonoterpenol (Triptonodiol)
C 2 iH 3 o0 4
210-212
37
Triptoquinone A*
C20H24O4
179-182
128°(CHC13)
42
38
Triptoquinone B*
C20H26O4
amorphous
167"(CHO13)
42
39
Triptoquinone C*
C20H28O4
amorphous
-63" (CHC13)
42
40
Triptoquinone D*
C 2 oH 2 g0 3
amorphous
26.1°(MeOH)
42
41
Triptoquinone E*
C 2 oH 2 60 3
121-123
30.3° (MeOH)
42
42
Triptoquinone F*
C 2 oH 2 60 4
207-209
80.5° (CHCh)
42
43
Triptoquinone G*
C 2 oH 2 60 5
amorphous
35.0° (CHC13)
42
25
39
40 30.4° (MeOH)
41
amorphous
42.4° (MeOH)
41
amorphous
-42.3° (MeOH)
41 38,41
779 (Table 1. Contd....) Name
Formulae
M.P. (°C)
[<X|D (solvent)
References
44
Triptoquinone
C 20 H 22 O 4
55-58
121.6°(CHCI3)
43
45
Triptotin A
C 20 H 22 O 6
187-188
68.7°(CHC13)
44
46
Triptotin B
C20H26O6
amorphous
91.8°(CHC13)
44
* Isolated from Tripterygium wilfordii var. regelii Mankino R2
14
1 2 3 4 5 6
H H H H OH H
CH3 CH3 CH3 CH3 CH3 OH
P-OH H P-OH p-OH P-OH a-OH =0 H p-OH H p_OH H
Ri
R2
21
H
OH
22
H
OCH3
23 OH
0CH3
OH
24 Ri=O; R2 = CH2OH; R3=2H OH
28 R, = OH; R2 = CH3
25 R i = < n . R2 = CH3; R 3 = 0
29 R, = H; R2-CH2OH
26 R t - 0 ; R2-CH2OH;
30 epimerof29
R3-0
780 (Fig. l.Contd....) HO,
OH
HOOC
H OH
\
1
1=*6
R, R; R3 R4 R5 R
32
33 P-OH H H 34 0 H OH 35 0 H H 36 0 OH H
H H2 OH H H2 OH H 0 OH OMcH, OH
HOOC
Fig. 1. Structures of new diterpenoids from TW plant (1).
There are about 20 alkaloids separated from the herb. Most of them are three-ring sesqueterpenes linked with a pyridine through two ester groups. Many of these alkaloids have immunosuppressive activities {c.f. table 2 & fig. 2) [45-55].
781 Table 2.
Alkaloids from TW plant Name
Formulae
M.P. (°C)
[<x]o (Solvent)
References
47
Wilfordine
C43H4,O,8N
175-176
5° (CHC13)
45
48
Deacetylwilfordine
C 4 ,H4,Oi 8 N
182-184
49
Wilforine
C 43 H 4 ,O, 8 N
170-171
30°(Me 2 CO)
45
50
Deacetylwilforin e(Wilforzine)
C 4 ,H 47 O 17 N
177-178
6° (Me 2 CO)
47
51
Wilfornine (2Nicotinylwilforine)
C 42 H 4g Oi 8 N 2
187-189
52
Wilforgine
C 4 iH, 7 Oi,N
211-212
53
Wilformine (Euonine)
C 38 H 47 O 18 N
155-157
45
54
Wilforjine (Deacetyl wi 1 formine)
C36H45O17N
156-158
22
55
Wilfortrine
C 4 ,H 47 O 20 N
235-237
56
Deacetylwilfortrine
C 39 H 45 O,,N
179-182
48
57
Wilforidine
CWR.SO.KN
176-178
49
58
Wilfordlongine
Cj(,H45Ol8N
179-181
50
59
Isowilfortrine
C 4l H4 7 O 20 N
329-331
51
60
Wilfordconine
C 4 ,H 47 0 2 oN
192-193
52
61
Neowilforine
C 43 H,9O| 7 N
156-157
62
Celafiirine
C 2 ,H 27 O 3 N 3
154-155
-1P(CHC1 3 )
54
63
Celacinine
C 25 H,,O 2 N 3
203-204
-19°(CHC1 3 )
54
64
Celabenzene
C 23 H 2 ,O 3 N 3
156-158
0° (CHC13)
54
65
Wilforcidine
C 36 H 38 O 8 N 2
188-189
55
66
Triptofordinine A-1 *
C41H43O12N
193-194
56
67
Triptofordinine A-2*
C 4 ,H 43 O, 2 N
94-95
56
48
45,48
25° (Me 2 CO)
10°(Me 2 CO)
45,48
46,47
53
* isolated from Tripterygium wilfordii var. regclii Mankino
Approximately 20 new triterpenoids {cf Fig. 3) have been separated from the TW plant or its variety {cf 5-7) [56-65]. Among them, tripterine or celastrol type compounds can be separated from other celastrus species of the
782
R4
AcO Benzoyl Benzoyl Benzoyl Benzoyl Nicotinyl Furanoyl Ac H Furanoyl Furanoyl H
R,0
Ac Ac Ac H Ac Ac Ac Ac Ac Ac Ac
OH OH H H H H H H OH
OH OH
.OCOQH;
58 R, = Ac; R2 = H 59 R| = Ac R2 = Furanoyl 60 R, = Fuanoyl R 2 =H
64 R =
67
R=-
Fig. 2. Structures of alkaloids from TW plant.
same family of plants. Celastrol type compounds show immunosuppressive and anti-inflammatory activity. In addition, sesquiterpenoids (cf. Fig. 4) are reported to be separated from the TW plant and its variety [66-68] (c.f. fig. 8). Among all of the components isolated from the TW plant, the most potent compounds, with immunosuppressive, anti-
783
HO :::|-|<-:;.:y
WilforiidcA WiiforiidtB
i '
2,3-DihydKBty-friedel-6H9(l t)-eci-29-oic acid
• I ; , . . ! . . • ! :•
Demelhylzdastrol
3,24-Dk.w-fridclan-W-oic acid
Friedel-11,13-dien-3-one
R = p-OH R-0
TriptolriECipoioit acid A
R^n-OH R, =HRj=CH 3
TripotrilcrpnuBCacid B R.ip-OH
Rn'HRj'CH,
Tripnurilsipcnoicacid C R, =a-OH R ] = C H J R J = H Triplodihydroxy acid
R, = R; =H Rj= CH,OH
m e t h y l '•!.;•
Polpunome acid R = H
Tripterigic add
R,=a-OH R, = C
1-Hydroxypiilpanic acid R = OH
1-Epikaonic add
Cangorcmm R = CHO .?••:.= • ; . i i ' . - . . - : - : i i -
WilfOriC acid A
Orthosphenic Acid
K = OH
.:! i-:I:I:. .i.nl
R=H
..• i i
Wilforic acid B
Fig. 3. Structures of triterpenoids from TW plant.
WilforicacidC
784
OR,
PhOCO
OAC
OCOPh
P ^ O C H = CHPh
PhOCO
OH
o Triptofordin A R, = H, R2 = COCH=CHPh Triptofordin C-l
R=O
Trtptofordin D-l Ri - A c , .1*2 = 0 OAc
Triptofordin B R, = OH. R2 = COPh
R =*i
Triptofordin D-2 Ri = Ac, R2
Triptofordin C-2
H
~"H
Triptofordin F-3 Ri = Ac, R2 PhOCO
OAc / OR
Triptofordin F-4 Ri = H, R2 -O"H OAC
\|
^l
\ , 0
1
ORil
Triptofordin E
R = COPh
Wilfomide
Neotriptonolide
Triptofordin F-1 R = Ac Triptofordin F-2 R = COCH = CHPh
Fig. 4. Structures of new sesqueterpenoids from TW plant.
inflammatory, anti-cancer and anti-spermatogenic activities, are triptolide type diterpenoids. ISOLATION, CHARACTERIZATION AND STRUCTURAL MODIFICATION OF TRIPTOLIDE Triptolide can be found in all parts of the plant: roots, stems and leaves. The content of 1, 2 and 4 depends upon the growth area, age and size of the plant. The yield of 1 from the roots or from the leaves is usually around 10 ppm [21, 27]. One isolation method begins with alcohol extraction, followed by a chloroform extraction and finally use of repeated silica gel column separation to obtain compound 1 and other diterpenoids [26]. The basic physical constants of triptolide are shown in the Table 1. Triptolide has a molecular formula C20H24O6, MW of 360; UVmax (EtOH) of 218 nm (e 14,000) and IR absorption at 3460 (hydroxyl group), 1773, 1686, and 1173 (oc,|3-unsaturated y-lactone ring) cm'1. Triptolide *H- and 13C-NMR data is shown in Table 3 [21,27].
785 Table 3.
' H- and I3C-NMR data of triptolide 1
Carbon
1
2
Carbon
29.76 t
17.04 t
§H
1.21
10
35.76 s
1.55
11
56.74 d
3.91
2.11
12
54.51 d
3.51
2.32
13
66.62 s
14
73.41 d
3
125.52 s
4
129.91 s
15
28.16d
5
40.42 d
2.67
16
16.84 q
0.87
6
23.64 t
1.96
17
17.72 q
1.01
2.17
18
173.17s
19
69.93 t
4.67
20
13.59 q
1.11
7
60.05 d
8
60.73 s
9
65.73 s
3.37
3.41 2.24
Triptolide 1 is a stable compound, but in basic conditions (pH 7.4, such as treatment with HNMe2) it will isomerize to isotriptolide 68, with loss of bioactivity. Among the three epoxide rings in the molecule, the a-epoxide at C-12-C-13 is the most reactive to nucleophillic attack. In acid conditions this epoxide ring will open to give tripchlorolide 7, which retains the bioactivity. Tripchlorolide 7 is a prodrug of triptolide in human plasma [69]. In vitro and in vivo tripchlorolide reverses to triptolide 1 by loosing a chlorine ion. When triptolide 1 was reacted with nucleophillic reagents: HBr, CH3CH2CH2SH, HO Ac, MeOH, ammonium thiocyanide or 4methoxybenzenesulfonyl chloride, the same C12-C13 epoxide reacted to form compounds 69-73 [70-72]. Refluxing 1 for 96 hrs in phosphate buffer solution at pH 4 generated triptriolide 10 in 43% yield [72]. In contrast, when 1 was stirred with sodium cyanoborohydride followed by dropwise addition of neat boron trifluoride diethyl etherate at room temperature for 16 hrs, the epoxide ring at C-7-C-8 opened and the compound 74 was obtained. Subsequently, reaction of compound 74 with HC1 at room
786
temperature produced the 12-chlorocompound 75 and when heated to 85°C for 26 hrs a new compound 76 was formed [73]. Interestingly, 1 stirred in concentrated HC1 at room temperature, provided compound 77. Its structure was confirmed by X-ray refractometric analysis. The mechanism was proposed as shown in Fig. 5 [70].
= C17;Br69;SPPr70; OCH371;SCN72; OSO,-C6H4-CH,73; OH 10
COOH
Fig. 5. Schema of structure modification of triptolide.
To obtain water-soluble prodrugs, triptolide was reacted with succinic anhydride or similar reagents and DMAP in dried
787
dichloromethane. The resulting free acids were reacted with sodium bicarbonate, which provided sodium triptolide succinate 78 and similar compounds in high (up to 96%) yields. Compound 78 exhibited the same bioactivity as triptolide 1 due to conversion to the original compound by metabolic processes in vivo [74]. SYNTHESIS OF COMPOUNDS
TRIPTOLIDE
AND
RELATED
Total Synthesis of Racemic Triptolide Since the triptolide-type diterpenoids are the most potent chemical class isolated from TW, scientists have focused on total syntheses of this class of compounds. Berchtold's group first accomplished the total synthesis of racemic triptolide and related compounds [75]. Ketone 79 was used as the starting material. It was reacted with 2(P-iodoethyl) butyrolactone 80 in a dispersion of 50% NaH in oil to give diastereomeric lactone 81 that in turn reacted with dimethyl amine to afford a 1:1 mixture of diastereomeric amide 82. Oxidation of 82 with Collins reagent provided aldehyde 83. Aldol condensation of 83 yielded 84 and 85. When the mixture of 84 and 85 was heated with p-toluenesulfonic acid in benzene to effect quantitative dehydration of 84 and afford 1:2 mixture of 85 and 86, which could be separated by fractional crystallization. Reduction of the equilibrium mixture of 85 and 86 with sodium borohydride and subsequent treatment with 2N HC1 gave lactone 87. Base-catalyzed isomerization of 87 to 88 was effected in a methanol solution of sodium methoxide. Benzylic oxidation (CrOs/HOAc) of 88 gave 89. Ether cleavage (BBr3/CH2Cl2) of 89 gave phenol 90, and subsequent borohydride reduction of the ketone afforded 91 in an 80% yield from 89. The Alder periodate reaction converted 91 to epoxy dienone 92. Reaction of 92 with a large excess of w-CPBA gave racemic 4. Reduction of 4 with sodium borohydride gave racemic triptolide 1 (cf. fig. 6). Synthesis of /-triptolide via /-dehydroabietic Acid The first natural optically active triptolide was synthesized starting with the natural /-dehydroabietic acid 93 [76], which is isolated from natural gum rosin [77]. Natural product 93 was oxidized by
788
NMe? 85 a-C-3 amide
& 86 3 C-3 amide
Fig. 6. Schema of total synthesis of racemic triptolide.
AICI3 to phenol compound 94. Curtius degradation of trifluoroacetate 95 provided isocynate 96, which was converted to tertiary amine 97 by UAIH4 in refluxing THF followed by refluxing AcOH-aqueous HCHO. After oxidation to the N-oxide of 97 by mCPBA, 30 min reflux in CHCI3 effected a Cope elimination giving olefin 98. Oxidative cleavage by OsO4/NaIO4, AcOH-dioxane-H2O
789
afforded ketone 99. Compound 99 was transformed to p-hydroxy ketone 100 by generation of the enolate with (/-Pr)2NLi followed by reaction with gaseous HCHO in THF at -78°C. After blocking the alcohol blocked as the 2-methoxypropyl ether [MeOC(CH3)=CH2AcOH ], a second protected methylol unit was appended, giving triol monobenzyl ether 101 by a successive treatment with 2 equiv of PHCH2OCH2Li in THF, then pHl HC1-THF. After the formation of phenolic monoacetate of 102 through successive exposure to MeOC(CH3)=CH2-AcOH, Ac2O-pyridine and pHl HCl-MeOH, oxidation with CrO3-pyridine/HCl yielded aldehyde 103. Dehydration to the oc,p-unsaturated aldehyde 103 was managed by treatment with 0-C6H4(NH2)2-PhCOOH, followed by hydrolysis (pHl, hydrochloric acid, EtOH, 20°C) of the presumed intermediary o-phenylene diamine imine of 92. Oxidation of 103 to the carboxylic acid level (NaC102-H0S02NH2) followed by hydrogenolysis of the benzyloxy group (H2-Pd-C, EtOH) resulted in spontaneous lactonization, affording butenolide 104. Betenolide 104 was initially oxidized by C1O3 to the ketone 105, then saponified to the unisolated free phenol and finally reduced to the benzyl alcohol 106 by NaBH4. Benzyl alcohol 106 was converted to epoxy dienone 107 and then treatment with H2O2-KOH gave a mixture of bisoxide 108 and the 12,13-P-epoxy isomer. The mixture was immediately further oxidized with 3,5(NO2)2C6H3COOH-Na2HPO4 to form /-triptonide 4. NaBH4 easily reduced compound 4 to /-triptolide 1 and its epimer l-14-epitriptolide (cf. fig. 7). Enantioselective Total Synthesis of Triptolide Yang and her co-workers successfully accomplished enantioselective synthesis of the natural triptolide [78,79]. 2Isopropyl phenol was used as the starting material. The hydroxyl group of the phenol starting material was protected reacting with MOMC1. Then the resulting MOM ether 109 was treated by nBuLi, cooled to -78°C and treated with iodomethane to give 110. It was further lithiated by s-BuLi/THF and treated with 3,3-dimethyl bromide to give 111. The MOM ether group of 111 was deprotected by TMSCl/ LiBF4 and methylated by Me2SO4 to give 112. Allylic oxidation of 112 with SeO2/TBHP provided 113.
790
Fig. 7. Schema of total synthesis of l-triptolide through 1-dehydroabietic.
Compound 113 was brominated to give 114 and then dianion displacement furnished the acyclic precursor 115. (+)-8Phenylmenthol 116 was used as a chiral auxiliary for the synthesis of (-)-triptolide. Ester exchange of 115 with 116 in the presence of DMAP provided the acyclic precursor 117. Cyclization of 117 afforded the major diasteriomer 118. Vinyl triflate 119 was reduced
791
by 2.2 equiv of DIBAL-H in dichloromethane at -78 to -30°C for 20 h, affording allylic alcohol (+) -120. The carbonylation of 120 under standard conditions gave the key product, (+)-triptophenolide methyl ether 18. Benzylic oxidation of 18 by CrO3/HOAc-H2O and subsequent demethylation and reduction provided p -alcohol 121. Compound 121 was oxidized first by NaIO4 to give 122 and then by oxone in acetonitrile with aqueous Na2 (EDTA) solution followed by addition of CF3COCH3 via precooled syringe to give 123. Compound 123 was oxidized by hydrogen peroxide in alkaline solution to get (-)-triptonide 4, which gave (-)-triptolide 1 and its epimer by reduction with NaBH.4. The key step is cyclization of 117 to 118. The authors found that lanthanide triflate could catalyze the Mn(III)-based radical cyclization reaction through strong chelation to the (3-keto ester group. In the presence of Yb(OTf)3; the cyclization reaction was accelerated and the trans/cis ratios were improved. Catalyzed asymmetric radical cyclization of 117 by Yb(OTf>3 in the presence of CF3CH2OH at -5°C afforded 118 and its diastereomer in a significantly higher ratio 38:1 as well as higher yield (ll%){cf. fig. 8). BIOACTIVITY OF CHEMICAL COMPONENTS FROM TW PLANT Tao et al. [80] have described the immunosuppressive components in TWG or T2 and EA as triptolide 1 and tripdiolide 2. The concentration of each compound in the extract was 0.36 (ig of 1 and 0.68 |lg of 2 in 1 mg of T2 and 1.08 ng of 1 and 0.31 |ig of 2 in 1 mg of EA. Fidler et al. [11,12] indicated that 1 (PG490 and its semisynthetic succinate ester prodrug of 1, PG490-88), as well as PG27, was efficacious as an immunosuppressive agent and prevented allograft rejection after cardiac transplantation. PG27 contained 0.36 % of 1. PG490-88 at 0.335 mg/kg/d significantly increased cardiac allograft mean survival time from 7 to 47 days, and significantly increased cardiac allograft survival when used in combination with CsA. Yang et al. [81] demonstrated 1 is the major active component in the plant extract. It induced caspasemediated apoptotic death of T cell hybridomas and peripheral T cells, but not thymocytes. In a comparison of several epoxide
792
Fig. 8. Schema of enantioselective total synthesis of triptolide.
diterpenoids extracted from the herb, the immunosuppressive activity order according to the therapeutic index (TI) was: triptolidenol 5 (30.7)> tripchlorolide 7 (16.7) > 16-hydroxytriptolide 6 (15.8)> triptolide 1 (13.7) > tripdiolide 2 (8.8)> triptonide 4 (7.5) [82]. In the same study, the anti-inflammatory activity order was: triptriolide 10 (>19) > 1(17) > 5(9.6) > 7 (9.0) > 2 (7.3) > 6 (6.6) > 4 (5.9).
793
Triptolide 1 (PG490) inhibits early cytokine gene expression in Jurkat T cells, effectively suppresses T lymphocyte activation, and is a potent inhibitor of interleukin (IL-2) transcriptional activation [83,84]. Compound 1 blocks IL-2 transcriptional activators Nuclear Factor of Activated T-cells (NF-AT) and Nuclear Factor-KB (NF-xB) in Jurkat cells. However, 1 inhibits the stimulation-dependent enhancement of DNA-binding activities at the NF-AT, but not at the NF-/tB, target DNA sequence and thus inhibits transactivation but not DNA binding of NF-*B. 1 inhibits NF-xB transcriptional activation without inhibiting nuclear NF-*B DNA-binding activity in a variety of cell types [82-86]. Inhibition of Activator Protein 1 (AP-1) transcriptional activation by 1 [85] involves inhibition of AP-1-specific DNA binding activity, similar to that observed for binding to the NF-AT target DNA sequences [82]. T-cell activation involving CD28 costimulation is sensitive to inhibition by 1, but resistant to CsA and FK506. The capacity of 1 to suppress costimulation-triggered T cell activation is consistent with the use of triptolide-based treatments in ailments that are resistant to CsA treatment and are known to involve T lymphocyte costimulation. Among these states are GVHD, systemic rheumatic and other autoimmune diseases, and allotransplant rejection [8792]. In 1981, Zhang et al. [93] first examined the antitumor activity of 1. Compound 1 injected i.p. at 0.25 mg/kg markedly prolonged the survival time of L6i5-bearing mice (by more than 140%) and inhibited the growth of S37, HCS and W256 by 38%, 47% and 51%, respectively, at 2 mg/kg. Recently it was reported that 1 inhibited the proliferation and colony formation of tumor cells at extremely low concentrations (2-10 ng/ml) and was more potent than taxol [94]. In vivo, treatment of mice with 1 for 2-3 weeks inhibited the growth of xenografts formed by four different tumor cell lines (B16 melanoma, MDA-435 breast cancer, TSU bladder cancer and MGC80-3 gastric carcinoma), indicating 1 has a broad spectrum of activity against tumors that contain both wild-type and mutant forms of p53. In addition, 1 inhibited experimental metastasis of B16F10 cells to the lung and spleen of mice. Importantly, tumor cells that were resistant to taxol attributable to the over expression of multidrug resistant gene 1 were still sensitive to the effects of triptolide [94]. PG490-88, the ester prodrug of 1,
794
retarded growth and caused regression of nude mouse xenograft tumors from a range of human tumor cell lines (H23 non-small cell lung cancer, COLO205 and HT-29 colon carcinoma, and PC-3 and DU145 prostate cancer) with both wild-type and mutant forms of p53 [95 and unpublished observations, Fidler J.M.; Li K.]. Compound 1 blocked NF-KB activation and sensitized tumor necrosis factor (TNF-oc)-resistant tumor cell lines to TNF-ccinduced apoptosis [85]. Compound 1 acted in synergy with CPT-11 (irinotecan), a topoisomerase I inhibitor, by blocking the S/G2/M arrest induced by CPT-11. As with doxorubicin, inhibition of p21 induction by CPT-11 drove cells into apoptosis rather than growth arrest [94]. PG490-88 enhanced the efficacy of the chemotherapeutic agents taxol, vinorelbine, 5-fluorouracil and CPT-11 and demonstrated enhanced anti-tumor activity with nonsmall cell lung cancer, colon carcinoma, prostate cancer and fibrosarcoma in nude mouse tumor xenografts [94 and unpublished observations, Fidler J.M.; Li K.]. Moreover, PG490-88 acted in synergy with CPT-11 to cause tumor regression [95]. PG490-88 is being evaluated in phase I clinical trials [96]. In cells treated with 1, the expression of p53 increased but the transcriptional function of p53 was inhibited. Additionally, 1 induced accumulation of cells in S phase, blocked accumulation of cells in G2/M induced by the topoisomerase II inhibitor doxorubicin, and prevented doxorubicinmediated induction of p2i wafl/cipl ; a p53-responsive gene. These data suggested that 1 enhanced apoptosis in tumor cells by blocking p21-mediated growth arrest [97]. 1 had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Whereas low concentrations of 1 inhibited cell proliferation and induced a senescence-like phenotype, higher concentrations induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p 21 wafl/cipl and hdm-2, were reduced, as was bcl-2. It was suggested that 1 might be an effective preventive as well as therapeutic agent against prostate cancer and might activate a functional p53 pathway in prostate cells [98]. In a different therapeutic area, compound 13 showed anti-HIV replication activity in H9 lymphocyte cells with an EC50 of 1 (ig/ml [31]. Many of the diterpene quinoid compounds showed significant inhibitory activities for IL-loc and IL-1(3 release from
795
LPS-stimulated peripheral blood mononuclear cells compared with the reference compound prednisolone. The inhibition by compounds 37-43 was 30-70% at a concentration of 1 x 10"5 g/ml, and for prednisolone at 3 x 10"7 g/ml was 87% for IL-la and 76% for IL-lp. At the same concentration triptophenolide compounds did not show any inhibition [42]. When several diterpenoids and triterpenoids, isolated from the herb, were examined for antifertility activity, 1 showed the most potent in vitro spermicidal activity [99]. In contrast, 4 had the most potent male in vivo antifertility effect in the modified MB-50 assay, and it was therefore suggested that 4 might be a good candidate to be developed into a male antifertility agent. The triterpenoid tripterine (celastrol) is reported to have anti-inflammatory and immunosuppressive activities. Celastrol inhibited the response of mouse splenocytes to SRBC both in vitro (at 0.1 - 1.0 |ig/ml there was a 27.1-75.3% inhibition) and in vivo (at 1-2 mg/kg qd x 6, there was a 30.9-44.3% inhibition) with either primary or secondary stimulation. This compound was also found to significantly inhibit cotton pellet-induced granuloma growth in rats and to depress the delayed-type hypersensitivity (DTH) reaction of mouse skin to dinitrochlorobenzene [100]. Celastrol showed potent inhibition of LPS-induced NF-KB activation in murine macrophage RAW264.7 cells transfected with an NF-KB reporter gene construct and on nitric oxide (NO) production in LPSstimulated RAW 264.7 cells. The IC50 value for NF-KB activation was 0.27 uM and for NO production was 0.23 uM [101]. In low nanomolar concentrations, celastrol was found to suppress the production by human monocytes and macrophages of the proinflammatory cytokines TNF-a and IL-p\ Celastrol also decreased the induced expression of class II MHC molecules by microglia. In macrophage lineage cells and endothelial cells, celastrol decreased induction but not constitutive NO production. Low doses of celastrol administered to rats significantly improved their performance in memory, learning and psychomotor tests. The potent antioxidant and anti-inflammatory activities of celastrol and its effects on cognitive functions suggest that the drug may be useful to treat neurodegenerative diseases accompanied by inflammation, such as Alzheimer's disease [102].
796
Most of the sesqueterpene alkaloids showed depressant effects on humoral immunity using the hemolysin reaction as an index. Wilfortrine 55 (160 mg/kg mg/kg/d x 9d, i.p.) suppressed the graft-vs.-host reaction (GVHR). 53 (80 mg/kg/d x lOd, i.p.) showed marked inhibitory effects on the DNCB-induced DTH reaction on mouse skin. Both alkaloids (80 mg/kg/d x 4d, i.p.) significantly decreased the clearance rate of charcoal particles and the weights of spleen and thymus [103]. However, the potency of the suppressive activities was not comparable with that of triptolide diterpenoids TISSUE CULTURE AS A SOURCE OF TRIPTOLIDE To avoid the limitation of plant sources, several groups initiated studies of tissue culture from the TW plant as a source of triptolide 1. Kutney's group first studied TW tissue culture. The shake-flask and stirred fermenter batch cultures of TW cell tissue line TRP 4a grown on modified PRL-4 medium were harvested after 6 weeks. Cell cultures were extracted and separated by column chromatography. Compounds 1 and 2 were separated in yields that were 3 and 16 times greater, respectively than those observed in the plant itself. Additionally, dehydroabietic acid, celastrol, oleanoic acid, polpunonic acid, p-sitosterol and other compounds were separated from the culture [104]. Zhu and his group cultivated tissue induced from TW leaves and stems, on the 67-v medium containing 0.1 mg/L, 2,4-D and 0.1 mg/L KT for 7 weeks. After separation of the cultivated material, the yield of 1, 2 and 4 was 12 times more than that in the plant (9.184 mg/L) [105]. CONCLUSION The chemistry of components from the TW plant has been well characterized. The biological activity of these components is under study for the development of clinically useful therapeutic agents. Among the components, triptolide type compounds are being developed for treatment of autoimmune diseases as well as cancer, and other components such as celastrol-type compounds and alkaloids also have prospects for further development. All of these are examples derived from the investigation of Traditional Chinese
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Medicines, where there is a wealth of promising compounds. Further exploration with modern scientific technology will lead to the discovery and development of even more useful medicines to treat cancer and other diseases in the future.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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BIO ACTIVE NATURAL COMPOUNDS FROM MEDICO-MAGIC PLANTS OF BANTU AREA BLANDINE AKENDENGUE1*, GUY JOSEPH LEMAMY1, HENRI BOUROBOU BOUROBOU2, ALAIN LAURENS3 1
Departement de Pharmacologie, Faculte de Medecine, Universite des Sciences de la Sante, B.P. 7464 Libreville, Gabon. 2 Centre National de Recherche Scientifique et Technologique, Herbier National du Gabon, B.P. 13354, Libreville, Gabon. Laboratoire de Pharmacognosie, UMR 8076 CNRS, Faculte de Pharmacie, Universite Paris XI, rue Jean-Baptiste Clement, 92296 Chatenay-Malabry, France. ABSTRACT: Ethnobotanical studies conducted in three Bantu countries, namely Central Africa, Gabon and Equatorial Guinea, led us to collect, among other medicinal plants, eighteen medico-magic plants belonging to thirteen families. Seven are trees (Distemonanlhus benthamianus, Erythrophloeum guineense, Guibourtia tessmanii, Monodora myristica, Musanga cecropioides, Polyalthia suaveolens, and Tetrapleura tetraptera), four are shrubs (Lippia multiflora, Microdesmis zenkeri, Tabernanthe iboga, and Securinega microcarpd), six are herbs {Aframomum stipulation, Costus afer, Costus lucanusianus, Pennisetum purpureum, Piper umbellatum, and Scoparia d ulcls) and two are liana {Adenia gracilis, and Adenia lobata). These plants are used for various activities such as antalgic, febrifuge, aphrodisiac, antiparasitic, neurotonic, emetic or oxytocic. Active compounds obtained from some of these medico-magic plants are reviewed. Among these active molecules are indole alkaloids such as ibogai'ne, tabernanthine and voacangine from Tabernanthe iboga; isoquinoline alkaloids such as oliverine from Polyalthia suaveolens; diterpenoids such as scopadulcic acid B, scoparic acid A and scopadulin from Scoparia dulcis; triterpenoids such as aridanin from T. tetraptera. Other natural active compounds isolated are coumarins, flavones, saponins and benzoxazolinones. These products showed various biological activities such as antiviral, antimicrobial, antiparasitic, cytotoxic, stimulating of central nervous system, analgesic, anti-inflammatory, H+, K+-ATP-ase inhibitor, (3-glucuronidase inhibitor, hypotensive and bradycardisant. Keywords: bioactive, medico-magic, Bantu
INTRODUCTION The Bantu are the people in Africa who call a human being a Muntu (singular; plural: Bantu) in their language. They live in an area that • : correspondence to Blandine Akendengue ([email protected])
804 extends from the Equator all the way to the Cape of Good Hope in the south. The common root of Bantu languages has been demonstrated [1,2]. Medicinal plants used by Bantu people have been reported herein [3,4]. The Bantu regard bad luck in life, marriage problems, childlessness, bad crops, d eterioration o f 1 iving c onditions, p rofessional p roblems, s uch a s illness, just as well as diseases which require treatment, like fever or tuberculosis. Plants are used in all these contexts to resolve problems and to cure. The sign of God is looked for in the plant, whether it is called upon for help or is kept for strengthening by the spirits; it is personalised and regarded as a treasure. Man lives in harmony with his remedies. In accordance with his cultural conception, he creates reciprocal relationships between him and the plant. These relationships become stronger during illness and weaker in times of health but never disappear. Medico-magic plants include also the esoteric part. They are not just consumed and thrown away when they seem useless. Rather, their use is subject to rules and regulations marked by the typical characteristics of magic: manipulation of certain materials, which can be learned and is backed by the firm conviction to be able to achieve or change something for the benefit of the user. There are procedures in all Bantu societies where the same plant can be used both as remedy and magic. Bantu medicine displays some magical character. The administration of medicomagic plants follows a defined ritual and above all, for the healer and the patient, it is clear that the efficiency of these magic plants is conditioned by the respect of rituals. We registered various ethnic groups' concepts of the effects and uses of medicinal plants in a cultural and social context. These species, which have therapeutic properties, can be used in specific rituals (Aframomum stipulatum, Tabernanthe iboga), or to free someone from a magic spell (Guibourtia tessmanii, Polyalthia suaveolens) or bring luck (Scoparia dulcis). G. tessmanii, especially, is an endemic big tree of primary big forests of Cameroon and Gabon [5]. T. iboga, the most famous in Gabon, is prized by the natives for its hallucinogenic and aphrodisiac properties. Traditional healers use iboga for its stimulating properties to enhance their psychic powers, increase inspiration and stave off fatigue. The strength of this study is that it combines both fieldworks and results from scientific investigations of listed plants which include biological activities of extracts and active compounds. Traditional uses of plants and extracts activities are presented, and active products obtained from some of these plants are reviewed. Fieldworks have been conducted among traditional healers in Central Africa (Bambari and Bangui areas) and in Bata and Malabo areas of Equatorial Guinea [3] by one of us (B.A.). Identification of plants was made via vernacular names and with the help of botanists. In Gabon, ethnobotanical studies have been led in the Estuaire and Ngounie provinces by the same author and by H.B.B. who also identified the plants. Eighteen medico-magic plants have been collected. Nevertheless,
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all Bantu medico-magic species are not presented in this paper. Traditional uses are listed in Table 1. Some plants have been quoted for treating different diseases. Five species have been recognised to possess a cicatrizant activity (Costus afer, C. lucanusianus, Erythrophloeum guineense, Musanga cecropioides, and Piper umbellatum). Costus species seem to be known also for their pectoral properties. Four plants are known for their antalgic activity (A. stipulatum, Monodora myristica, Musanga cecropioides, P. umbellatum), whereas three are febrifuge (Lippia multiflora, P. umbellatum, Tetrapleura tetraptera). Two plants are aphrodisiac; among them T. iboga is widely used as hallucinogen during rites and as a tool by hunters to increase awareness and allow them to remain very still for prolonged periods. The traditional healers also showed antihelmintic, antimalarial, antirheumatic, antifungal, emetic and oxytocic plants. The other cited diseases are cold, dermatosis, otitis, epilepsy, and gonorrhoea. The majority of listed plants showed a biological activity; some activities being associated with the empirical use. Lyophilizated preparations of C. lucanusianus showed a uterine relaxant activity producing a complete inhibition of oxytocin-induced contractions. This uterine relaxant activity was reversible. Barium chloride-induced contractions were not modified [6,7]. Chewing sticks of Distemonanthus benthamianus induced improvement of children's gingival health and oral hygiene during a clinical study [8]. The extracts and essential oil of L. multiflora showed in vitro antimalarial activities against FcBl chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum. The essential oil inhibited the growth of parasite by 50%, 24 and 72 hours after administration at dilutions of 1/12,000 and 1/21,000, respectively. Tested on a highly synchronised culture, the essential oil inhibited growth mostly at the trophozoite-schizont step, indicating a potential effect on the first nuclear division of the parasite [9,10]. The essential oil of L. multiflora also showed pediculocidal and scabicidal activities against body lice, head lice and scabies' mites [11] as well as antibacterial activity. The Gram-negative bacteria were the most sensitive [12].
806 Table 1. Bantu medico-magic plants use Family and specie
Use (route)
Annonaceae Monodora myristica Dun.
Constipation, headaches (Ea)
Polyalthia suaveolens Engl. et Diels
Antiparasitic, anti-inflammatory (E)
Caesalpiniaceae Distemonanthus benthamianus H. Baill.
Dermatosis (E)
Erythrophloeum guineense Don.
Cicatrizant (E)
Guibourtia tessmanii (Harms) J. Leonard
Antiseptic (E)
Apocynaceae Tabernanthe iboga Baill.
Neurotonic, hallucinogen, aphrodisiac (Ib)
Crassulaceae Kalanchoe crenala Haww.
Otitis, cold, antifungal (E), epilepsy (1)
Euphorbiaceae Microdesmis zenkeri Pax.
Oxytocic (E)
Securinega microcarpa (Blume) Pax et H.
Aphrodisiac (1)
Graminae Pennisetum purpureum Schum.
Dermatosis (E)
Mimosaceae Tetrapleura tetraptera Taub.
Febrifuge, antirheumatic (E), emetic (I)
Moraceae Musanga cecropioides R. Br. Tedlie
Antalgic, oxytocic, cicatrizant (E)
Passifloraceae Adenia gracilis Harms
Medico-magic (E)
807
Adenia lobata Engler
Cold (E)
Piperaceae Piper umbellatum L.
Antalgic, febrifuge, cicatrizant (E)
Scrophulariaceae Scoparia dulcis L.
Emetic, antihelmintic, oxytocic, gonorrhoea (I)
stomach
disorders,
Verbenaceae Lippia multiflora Mold.
Febrifuge, antimalarial (E)
Zingiberaceae Aframomum stipulatum K . Schum
Antalgic , tonic (E)
Costus afer Ker-Gawl.
Antirheumatic, cicatrizant (E), pectoral (1)
Costus lucanusianus J. Braun et K. Schum
Antihelmintic, pectoral (I), cicatrizant (E)
" E: external I : internal b
The essential oils from the leaves of A. stipulatum and that of the seeds of M. myristica also exhibited in vitro antibacterial activities. Tested by the diffusion method, the both essential oils (5 ul per disc) inhibited the growth of bacteria with a 10-18 mm zone diameter of inhibition area [13]. Alcoholic extract of T. tetraptera also showed antibacterial activities with an MIC of 4mg/mL against Pseudomonas aeruginosa and Staphylococcus aureus, whereas aqueous extract only inhibited the growth of S. aureus [14]. T. tetraptera extract also exhibited molluscicidal activities against Bulinus globosus, Schistosoma mansoni, S. bovis, and Lymnaea natalensis [15-18]. An anticonvulsant activity was observed with the fruit volatile oil of T. tetraptera. Intraperitoneally (i.p.) administered in mice, T. tetraptera oil (0.4 mL per mouse) protected 78 % of them against leptazol-induced convulsions [19]. Analgesic effects were observed with aqueous extracts of P. umbellatum and M. cecropiodes. Administered in rats (i.p.), P. umbellatum aqueous extract brought about an ataxia with a decrease in the animal's watchfulness, which lasts about 48 hours. A fall of rectal temperature and a decrease of spontaneous motor activity as well as an increase of analgesic activity were also observed [20]. The aqueous extract of the leaves of M, cecropiodes did not affect ache-induced
808
contractile response, but produced significant inhibition of the twitch and pendular movement of the rat and rabbit smooth muscles and reduced writhing induced by acetic acid in mice [21]. Aqueous and ethanolic extracts of S. dulcis and essential oil of L. multiflora also showed analgesic activities as well as anti-inflammatory effects. Aqueous and ethanolic extracts of S. dulcis prolonged the sleeping time induced by pentobarbital in mice; ethanolic extract but not water extract reduced writhings induced by acetic acid in mice and paw oedema induced by carrageenin in rats [22]. The essential oil of L. multiflora (2, 4 and 8 mL/kg) showed a dose-dependent analgesic effect on acetic acid-induced writhing in mice. At 8mL/kg, the essential oil also antagonised hyperexia induced by Brewer's yeast [23, 24]. Crude alkaloid extracts of M. myristica and Polyalthia suaveolens trunk barks collected in Gabon exhibited in vitro antiplasmodial activities against Kl chloroquineresistant strains of P. falciparum with IC50 values of 7.65 and 4.08 ug/mL respectively [25]. The active compounds listed in this paper have been isolated from the following species: L. multiflora, M. myristica, P. suaveolens, S. dulcis, T. iboga, and T. tetraptera. These products showed various biological activities such as antiviral, antimicrobial, antiparasitic, cytotoxic, stimulating of central nervous system (CNS), analgesic, antiinflammatory, H+, K+-ATP-ase inhibitors, P-glucuronidase inhibitors, hypotensive and bradycardisant. These compounds belong to the following groups: alkaloids, terpenes, coumarins, flavones, saponins and benzoxazolinones. ALKALOIDS Ibogai'ne (1), ibogaline (2), iboxygaine (3), noribogaine (4), and tabernanthine (5), five T. iboga indole alkaloids, showed a tremorproducing potency to mice when given subcutaneously (s.c.) and intravenously (i.v.). Pharmacokinetics parameters obtained in mouse brain after i.v. injections showed that the tremor-producing activity depends more on chemical structure than on lipid solubility of tested alkaloids. A methoxy group in the position of R2 (Figure 1) enhances tremorigenic potency, tabernanthine and ibogaline (s.c.) being the most active with ED50 values of 5 umol/kg and 8 umol/kg respectively. ED50 values of 35 umol/kg and 80 umol/kg were observed with ibogaine and iboxygaine respectively, noribogaine being less active [26]. Tabernanthine antagonised the impairment of brain catecholamines turnover observed at simulated high altitude but did not change the endogenous levels of brain dopamine and noradrenaline in rats at normal atmospheric pressure or in hypoxia [27]. Ibogaine (1), tabernanthine (5), voacangine (6) and conopharyngine (7) (two others iboga alkaloids), when given i.p. to mice, showed a CNS stimulating activity in the course of a habituation test using psychogalvanic reaction (PGR). Structural parameters were also
809 Alkaloids Indole alkaloids:
IBOGAINE(l) IBOGALINE (2) 1BOXYGAINE (3) NORIBOGAINE (4) TABERNANTHINE (5)
Rl
R2
R3
CH3O CH3O CHjO OH
H CH3O H H CH3O
H H OH H
Zetler, Singbartl, and Schlosser (1972)
OMe
COOCH,
VOACANGINE (6) CONOPHARYNGINE (7)
R= H R = CH3O
Bert, Marcy, Quermonne, Cotelle, and Koch (1988)
810 Aporphine
OMe
OLIVERINE (8)
Cave, Guinaudeau, Leboeuf, Ramahatra, Razafindrazaka (1978) [56]
Terpenoids
Monoterpenoid
CARVACROL (9) Bruneton(1993)
811 Diterpenoids
SCOPADULCIC ACID B (13): R = COOH SCOPADULCIOL(16) : R = CH2OH Hayashi, Asano, Mizutani, Takeguchi, Kojima, Okamura, and Morita (1991)
HOOC
SCOPARICACIDA(17)
Hayashi, Kawasaki, Okamura, Tamada, and Morita (1992)
812
HOOC
SCOPADULIN (18)
Hayashi, Kawasaki, Miwa, Taga, and Morita (1990)
Triterpenoid
COOH
NHCOCH
ARIDANIN (20) Adesina, and Reisch (1985) [57]
813 Flavones
GIUM
OH
8-HYDROXYTRICETIN 7-GLUCURON1DE (21) Kawasaki, Hayashi, Arisawa, Morita, and Berganza (1988)
OMe OMe
OMe
HYMENOXIN (23)
Hayashi, Uchida, Hayashi, Niwayama, and Morita (1988)
Coumarin
SCOPOLETIN (24) Ojewole, and Adesina (1983)
814 Miscellaneous
MeO. -N — H
6-METHOXYBENZOXAZOLINONE (25)
Chen, and Chen (1976) Fig. (1). Chemical structures of bioactive compounds.
observed in this test. IbogaTne was more active than tabemanthine, suggesting that a methoxy group in the Rl position seems very favourable for a CNS stimulating activity. Voacangine (6) and conopharyngine (7) were less active, indicating that the presence of a methoxycarbonyl group (Figure 1) is unfavourable [28]. Neither a locomotor hyperactivity nor stereotypy was observed, suggesting a non-amphetaminic-like stimulating activity and a direct cortical effect. These results are in accordance with the traditional use of plants as neurotonic. On isolated rat duodenum, ibogame and tabemanthine increased the hypertonic effect following the addition of calcium ions to the organ previously decalcified [29,30]. Tabemanthine showed a bradycardisant activity [31]. The anti-addictive properties of ibogaine have been described. Animal studies and noncontrolled observations in humans indicated that ibogaine significantly affects drug dependence phenomena such as drug withdrawal and intake of addictive drugs, ibogame attenuated many symptoms of naloxoneprecipitated withdrawal in morphine-dependent rats [32]. An intraperitoneal injection of ibogaine (40 mg/kg, single dose) in rats that previously r eceived a c ocaine s elf-administration p roduced a s ignificant decrease o f cocaine i ntake, w hich remained unaltered for m ore t han 4 8 hours. However, a more prominent inhibitory effect was observed in animals treated repeatedly with ibogaine (40 mg/kg i.p.), once a week for 3 consecutive weeks. Since the half-life time of ibogaine is short, these results indicated that the compound or its metabolite is a long-lasting interrupter of cocaine dependence [33]. IbogaTne is not itself addictive but it is claimed to cure the addiction of several hard drugs such as cocaine and heroine but even alcohol and nicotine. Oliverine (8), an isoquinoline alkaloid isolated from P. suaveolens collected in Cameroon, exhibited after 24 hours of incubation a microfilaricidal activity at 10-100 (xg/mL, but the compound showed a minimal lethal dose of 8 mg/kg of body weight in mice [34].
815
TERPENOIDS Monoterpenoids Carvacrol (9), isolated from a hexane extract of L. multiflora leaf collected in Nigeria, exhibited tremendous antimicrobial activity [35]. The essential oil of L. multiflora showed pediculocidal and scabicidal activities when tested comparatively with benzyl benzoate. A 20% v/v preparation of Lippia oil applied to scabietic subjects for 5 consecutive days gave 100% cure compared with 87.5% cure obtained for benzyl benzoate at the same concentration. The scabicidal activity of L. multiflora essential oil seems probably due to the presence of terpineol (10), a- and P-pinene (11; 12) which are known to be lethal to body and head lice [11]. Terpineol and a-pinene were also isolated from the oil of M. myristica fruit collected in Cameroon, as well as carvacrol isolated from the oil of M. myristica seeds collected in Nigeria [36]. Carvacrol has been suggested to be responsible for the antibacterial activity of the oil of M. myristica seeds collected in Democratic Republic of Congo [13]. The antibacterial activity of phenols, especially carvacrol, has been reported for others medicinal plants too [37]. Diterpenoids Various active diterpenoids have been isolated from S. dulcis, a herb growing in tropical and subtropical areas, used to treat stomach disorders, hypertension and blennorhagia. Scopadulcic acid B (13), a tetracyclic terpenoid isolated from 70 % ethanolic extract of a Paraguayan specie of S. dulcis, and its derivates diacetyl scopadol (14) and scopadulcic B methyl ester (15), as well as scopadulciol (16) isolated from a Taiwanese specie, prevented gastric secretion by inhibiting the H+, K+-adenosine triphosphatase (ATP-ase), which is responsible for the H+ ions secretion into the stomach [38-40]. Scopadulcic acid B and its debenzoyl derivate, diacetyl scopadol, had no effect on the related enzyme Na+, K+-ATP-ase. The inhibition mechanism of scopadulcic acid B involves the failure of both H+, K+-ATP-ase and the K+-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step [39]. A labdanetype diterpenoid, scoparic acid A (17), isolated from an ethanolic extract of S. dulcis collected in Paraguay, inhibited p-glucuronidase by binding specifically with the enzyme [41]. Scopadulcic acid B and scopadulin (18), another tetracyclic diterpene also isolated from a chloroform-soluble part of the 70 % ethanolic extract of Paraguayan specie, exhibited in vitro and in vivo antiviral activities against Herpes Simplex Virus Type 1 [42, 43]. Scopadulcic acid B inhibited the viral replication with an in vitro therapeutic index of 16.7. Single-cycle replication experiments showed that the compound interfered with considerably early events of virus growth. Orally (p.o.) or i.p. administered immediately after virus inoculation, scopadulcic acid B effectively prolonged both the appearance
816 of herpetic lesions and the survival time at a dose of 100 and 200 mg/kg per day [42]. In vitro and in vivo antitumour and cytotoxic activities were also observed with scopadulcic acid B. The compound showed an in vitro cytotoxic activity against human tumour tissues with IC50 values of 0.068-0.076 ug/mL. The normal human tissues were relatively more resistant, an IC50 of 0.245 ug/mL being observed against Chang Liver cells. The 5-fluorouracil (5-Fu), used as reference, exhibited in the same conditions against tumour tissues and Chang Liver cells IC50 values of 6.0-6.8 ug/mL and 11.1 (xg/mL respectively. In mice, inoculated with Ehrlich cell suspension, at 25 mg/kg (i.p.) the cytotoxic activity of scopadulcic acid B was lower than that of 5-Fu with respectively, 12.5% and 50% survival attained at 45 days after tumour implantation without detectable tumour ascites [44]. Scopadulcic acid B was proved to be a potent antitumour promoter as well as various natural terpenoids, inhibiting the effects of tumour promoter 12-O-tetradecanoylphorbol-13acetate (TPA) in vitro and in vivo. The compound inhibited TPAenhanced phospholipid synthesis in cultured cells, and suppressed in mice the promoting effect of TPA on skin tumour formation initiated with 7,12dimethylbenz[a]anthracene [45]. Triterpenoids An ethanolic extract of S. dulcis collected in Brazil as well as glutinol (19), a triterpenoid from which it has been isolated, showed analgesic activity p.o in mice and rats, reducing writhings induced by acetic acid. They exert analgesic effects through a peripheral mechanism. The ethanolic extract and glutinol also showed anti-inflammatory effects, inhibiting the carrageenin-induced paw oedema and pleurisy. Glutinol mainly exerts his action during the early phase of acute inflammatory process [22]. Aridanin (20), a triterpenoid glycoside isolated from T. tetraptera fruits collected in Nigeria showed a molluscicidal activity against Biomphalaria glabrata with a LQo of 0.88 ug/mL, Bayluscide® (70 % niclosamide), the reference, exhibiting in the same conditions a LC50 of 0.35 ug/mL [46]. At low concentrations of 0.125 to 1.0 ppm, aridanin caused a significant reduction in the egg production and growth of B. glabrata and Lymnaea columella, indicating that the compound could control schistosomiasis if used in slow-release formulations [47]. Aridanin was also active against Schistosoma mansoni and S. bovis miracidia, 100% mortality or immobility being observed after 30 min (50 ug/mL) or after 5 min exposure time at a dose of 100 ug/mL. At a low concentration of 0.25 ug/mL, aridanin reduced the production of cercariae in snails, revealing that the compound acts against the transmission of schistosomiasis at different stages of the schistosome development [16].
817
FLAVONES Two flavonoids, 8-hydroxytricetin 7-glucuronide (21) and isovitexin (22), also isolated from ethanolic extract of S. dulcis collected in Paraguay, were found to be P-glucuronidase inhibitors. The two compounds showed mild inhibitory activity against P-glucuronidase from bovine liver. The inhibitory activity of 8-hydroxytricetin 7-glucuronide was one-tenth of that of the well-known P-glucuronidase inhibitor, glucosaccharo-l:4-lactone. Tested in the presence of a large amount of bovine serum albumin (BSA), 8-hydroxytricetin 7-glucuronide showed almost the same degree of inhibition in the presence of 400 times of BSA, suggesting that it inhibits the activity of P-glucuronidase even in the presence of other proteins [48]. Hymenoxin (23), another flavone isolated from a chloroformic soluble fraction of ethanolic extract of S. dulcis, showed an in vitro cytotoxic effects against human tumour tissues such as HeLa 229, HeLa S3 and against normal tissues such as Chang liver and intestine cells. Like with scopadulcic acid B, a difference of susceptibility between the human cancer tissues and those from normal tissues was observed with hymenoxin, the rD50 against HeLa 229 cell growth and Chang liver cell growth being 0.097 ug/mL and 0.510 ug/mL respectively [49]. COUMARIN Hypotensive and neuromuscular actions were observed with scopoletin (24), a coumarin isolated from the fruit of T. tetraptera collected in Nigeria. Administered at 10-100 mg/kg i.v, the compound reduced the arterial blood pressure of anaesthetised rats. At a dose of 10"6-10"3 M, scopoletin produced negative chronotropic and inotropic responses in guinea-pig isolated atria, inhibited acetylcholine-induced contractures of the toad rectus abdominis muscle, and depressed electrically-evoked twitches of the chick isolated biventer-cervicis muscle and rat isolated phrenic-nerve hemidiaphragm muscle preparations. The pharmacological effects o f s copoletin w ere not a ltered b y a tropinisation [51]. S copoletin relaxes the smooth muscles and reduces electrical stimulation-evoked and exogenous, noradrenaline-evoked contractions of muscle preparations. Ojewole suggested that scopoletin probably produces hypotensive action in laboratory animals through its smooth muscle relaxant activity, dilating blood vessels, and by acting as a non-specific spasmolytic agent [50]. SAPONINS Saponins and ethanolic extracts of T. tetraptera stem bark, collected in Ivory Coast, inhibited in vitro the luteinising hormone-releasing, hormone (LHRH)-induced LH release. The decrease in the amount of immunoassayable hormone was both time and dose dependent [52,53].
818
MISCELLANEOUS The 6-methoxybenzoxazolinone (25) isolated from S. dulcis showed a hypotensive activity [54]. Hypotensive effects of methanolic extract of L. multiflora leaves collected in Ivory Coast seem to be due to a caffeic ester isolated from a phenolic fraction. The compound induced a marked and long-lasting tensional fall: at 2mg/kg i.v, the decrease in systolic arterial blood pressure was high as- 40% and lasted for more than 15 minutes [55]. ABREVIATIONS i.p. i.v. p.o s.c. E I ATP-ase BSA CNS 5-Fu IC ID LC TPA
= = = = = = = = = = = = = =
intraperitoneal intravenous per os subcutaneous external internal adenosine triphosphatase bovine serum albumin central nervous system 5-fluorouracil inhibitory concentration inhibitory dose lethal concentration 12-O-tetradecanoylphorbol-13 -acetate
ACKNOWLEGMENTS The authors gratefully acknowledge the International Centre for Bantu Civilizations (CICIBA) and European Union Bantu Cultural Program for financial support of fieldwork. They also thank the Ministry of Culture of Central Africa and the Ministry of Information, Tourism, Culture and Art of Equatorial Guinea for arranging meetings with the traditional healers. They are grateful to Dr. Kosh Komba, Mr. Boris Belle, Mr. Etienne Mokili and Mr. Bernard Ndonazi for having provided information in Central Africa. They also owe thanks to Mrs. Anasthasie Adomba and Mr. Odembet of Gabon for having shared their knowledge with them. Finally they would like to thank Mr. Cornelio Essono and all the traditional healers of Equatorial Guinea for information. REFERENCES [1] [2]
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Hayashi, T.; Okamura, K.; Kakemi, M.; Asano, S.; Mizutani, M.; Takeguchi, N.; Kawasaki, M.; Tezuka, Y.; Kikuchi, T.; Morita, N.; Chem Pharm Bull, 1990, 35(10), 2740-2745. Asano, S.; Mizutani, M.; Hayashi, T.; Morita, N.; Takeguchi, N.; The Journal of Biological Chemistry, 1990, 265(36), 22167-22173. Hayashi, T.; Asano, S.; Mizutani, M.; Takeguchi, N.; Kojima, T.; Okamura, K..; Morita, N.; JNat Prod, 1991, 54(3), 802-809. Hayashi, T.; Kawasaki, M.; Okamura, K.; Tamada, Y.; Morita, N.; Tezuka, Y.; Kikuchi, T.; Miwa, Y.; Taga, l.;JNat Prod, 1992, 55(12), 1748-1755. Hayashi, K.; Niwayama, S.; Hayashi, T.; Nago, R.; Ochiai, H.; Morita, N.; Antiviral Research, 1988, 9, 345-354. Hayashi, T.; Kawasaki, M.; Miwa, Y.; Taga, T.; Morita, N.; Chem Pharm Bull, 1990, 35(4), 945-947. Hayashi, K.; Hayashi, T.; Morita, N.; Phytotherapy Research, 1992, 6, 6-9. Nishino, H.; Hayashi, T.; Arisawa, M.; Satomi, Y.; Iwasima, A. Oncology, 1993, 50, 100103. Adewunmi, C O ; Awe, S.O.; Adesina, S.K.; Planta Med, 1988, 54(6), 550-551. Adewunmi, C O ; Furu, P.; Madsen, H.; Phytotherapy Research, 1989, 3 (3), 81 -84. Kawasaki, M.; Hayashi, T.; Arisawa M.; Morita, N.; Berganza L.H.; Phytochemistry, 1988, 27(11), 3709-3711. Hayashi, T.; Uchida, K.; Hayashi, K.; Niwayama, S.; Morita, N.; Chem Pharm Bull, 1988, 36(12), 4849-4851. Ojewole, J.A.O.; Adesina, S.K.; Planta Med, 1983, 49,46-50. Ojewole, J.A.O.; Adesina, S.K.; Planta Med, 1983, 49, 99-102. Benie, T.; El Izzi, A.; Tahiri, C ; Duval, J.; Thieulant, M.L.; C R Soc Biol, 1987, 181, 163167. El Izzi, A.; Benie, T.; Thieulant, M.L.; Duval, J.; Planta Med., 1990, 56, 357-359. Chen, CM.; Chen M.T.; Phytochemistry, 1976, 15, 1997-1999. Pam Huu Chanh; Koffi, Y.; Pam Huu Chanh, A.; Planta Med, 1988, 54(4), 294-296. Cave, A.; Guinaudeau, H.; Leboeuf, M.; Ramahatra, A.; Razafindrazaka, J.; Planta Med, 1978, 33, 243-250. Adesina, S.K.; Reisch, J.; Phytochemistry, 1985, 24(12), 3003-3006.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
821
BIO ACTIVE NON-ALKALOID AL CONSTITUENTS FROM THE GENUS ERYTHRINA RUNNER R.T. MAJINDA1*, CORNELIUS C. W. WANJALA2, BENARD F. JUMA1 Department of Chemistry, University of Botswana, P/Bag UB 00704, Gaborone, Botswana. 2Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya ABSTRACT: The genus Erythrina is very rich in secondary metabolites particularly of the flavonoids class. A literature survey of non-alkaloidal secondary metabolites from Erythrina showed the presence of flavanones, flavonols, chalcones, cinnamoylphenols, stilbenoids, isoflavones, isoflavans, isoflavanones, pterocarpans, isoflav-3-enes, 3-phenoxychromones, coumastans, 3-phenyl-coumarins, lignans, cinnamate esters, simple phenolics, triterpenes, sesquiterpenes, long-chain carboxylic acids, and long-chain alcohols. The documented bioactivities of some of the isolated metabolites range from antimicrobial, anti-inflammatory, inhibition of platelet aggregation, tyrosinase inhibition, phospholipase A2 (PLA2) inhibitors, cyclooxygenase inhibitors, antioxidant, inhibitors of NA/H + exchange system, phospholipase C inhibitors, behavioural depression, muscle relaxation, (3-adrenergic inhibition, diuretic, anticancer, cytotoxic, DNA-repair properties, oestrogenic or proestrogenic activities, antitrypanosomal, antiplasmodial, and anti-HIV activities. The bioactivity profile represented the various classes fairly reasonably but it became apparent that a number of these isolates have not yet been tested for biological activity. A very close agreement between ethnomedical use of the various Erythrina extract preparations (Table 1) and the results of biological activity (Table 2) was found, with the reported activities of pure isolates strongly supporting the documented ethno-medical uses and reported pharmacological activity. The most common activity exhibited by extracts and pure isolates was mostly microbial related. It was also interesting to note that some compounds showed high efficacy against resistant organisms, a very important aspect, since most used drugs especially antibiotics tend to produce resistance to certain strains of organisms. The survey showed furthermore that certain structural features were essential for certain activities and also that compounds that were found active were usually effective against not just one but several disease functions. One cannot help but surmise that more activities are yet to be reported for these same isolates. The challenge remains for researchers in the various pertinent disciplines to carry out more work on these to fill in the knowledge gaps that still exist. I. INTRODUCTION The genus Erythrina comprises around one hundred and ten species of orange or red flowered trees, shrubs and herbaceous plants. It is divided into five sub-genera and twenty-six sections, largely on the basis of morphology, especially the colour and shapes of the flowers and fruits and of inflorescence characteristics. They are found throughout the tropical and semi-tropical
822 regions of the world. In the southern Africa, it occurs in scrub forest, dry woodland and coastal bush throughout the four northern provinces of South Africa, and also in Mozambique, Botswana, Zimbabwe, Angola, Zambia and Malawi. The genus Erythrina is part of the Papillionaceae sub-family of the Leguminoseae (Fabaceae). There are twenty-eight species and three sub-species in Mexico, twenty-six species in Central America, twenty-two species in South America, five species and two varieties in West Indies, thirty species and sub-species in tropical Africa, six species in South Africa seven species in continental Asia and seven species in Malaysia and the Pacific [1-2]. II. SOME ETHNOBOTANICAL AND PHARMACOLOGICAL INFORMATION ON ERYTHRINA Traditionally, Erythrina species have found use amongst different communities for various ailments. The bark of E. fusca and E. indica, for example, has been used for the treatment of fever, malaria, rheumatism, toothache, boils and fractures. Boiled roots of the same were taken internally as a remedy for beriberi. In Kenya, a concoction of dried bark of E. abyssinica has been used for the treatment of trachoma (oral) and elephantiasis (external) and its roots against malaria and syphilis, while the pounded green stem bark is taken as an antihelminthic [3]. The water extract of flowers of E. americana have been used in Mexico for the treatment of insomnia. The rest of other documented traditional medicinal uses are listed in Table 1. The seeds of Erythrina species are known to contain toxic alkaloids, as well as blood clotting substances, which may be of value in the treatment of thrombosis. The seeds, stem and root bark of some Erythrina species have been used also to stupefy fish, as narcotic, purgative, and diuretic [1,4], while crushed seeds of some have been used as rat poison. All the Erythrina species examined have yielded alkaloids, mostly of the erythrinaline type, having the curare like poisoning action [1,4]. Several of the more common species are used for decorative purposes in gardens and city streets. A number of biological activity tests have been carried out on some species of this genus, which corroborate the reported ethnomedicinal values. Table 2 [5] gives a summary of some of the results obtained. Table 2 also includes a very recent report of antitrypanosomal and antiplasmodial activities of a 90% ethanolic extract of E. senegalensis from Cote d'lvoire. A noteworthy observation from this report is that antitrypanosomal activity is reported for the first time from the genus [5b]. III. PHYTOCHEMICAL WORK ON NON-ALKALOIDAL CONSTITUENTS OF ERYTHRINA Extracts of the leaves, bark and roots of the genus Erythrina have a significant history of medicinal use for the treatment of diseases such as female infertility, stomach pain and gonorrhoea [6] and others shown in Table 1, and biological tests on Erythrina plant extracts [5] showed that these have various pharmacological activities (Table 2). In spite of this, earlier work on Erythrina tended to concentrate on the alkaloidal content of the plants. It was perceived, then, that alkaloids could be the only active constituents in these plants. It was for this reason perhaps that there was initially very little interest in the neutral non-alkaloidal constituents of the genus. It was not until the late seventies and early eighties that serious work started on the non-alkaloidal constituents of some Erythrina species [7-9]. Subsequently more reports started to appear on non-alkaloidal Erythrina content [10-12]. The research on these neutral constituents has since intensified and a number of studies have been conducted on the biological activity of these constituents. Nakanishi and co-workers [2] did some work
823 which could be the first to mark the beginning to serious biological activities of the neutral constituents of Erythrina. The following sections give an account of the biologically active neutral metabolites of the genus Erythrina. The major class of neutral metabolites that the genus Erythrina elaborates is the flavonoids in a very broad sense and these are divided into flavonoids sensu strictu [i.e. in a narrow sense], Fig. (1) and isoflavonoids, Fig. (2) depending on their biogenetic origin. Table 1: Ethnomedicinal Application of Some Erythrina Species (NAPRALERT, 2004) Plant
Part
Remedy
E. flabelliformis
seed
Toothache contraceptive
E. folkersii
bark
inflammation of womb (oral) diuretic
seed E. fusca
bark seeds
infested sores (washed externally) fungal dermatosis anti-inflammation agent skin infections
E. glauca
not specified
malaria (oral decoction) narcotic and purgative
E. humeana
bark
tuberculosis (oral decoction)
E. indica
bark leaf leaf
antihelminthic (oral) expectorant(oral) aphrodisiac (oral) menstrual troubles
E. mulungu
bark
fevers (oral)
E. sacleuxii
leaf bark
malaria (oral) malaria (oral) serious wounds
E. senegalenses
bark bark + leaf entire plant
oracular infections (oral) bronchial affections (oral) jaundice (oral) female infertility (oral) dysentery (oral) venereal disease (oral)
E. strictu
bark
epilepsy (oral)
E. subumbrans
leaf
menorrhagia (oral)
E. variegata
bark + coconut oil flowers leaf
swellings and inflammations (external application) skin ulcers antiasthmatic (oral) augmenting milk flow
E. zeyheri
bark
tuberculosis
824 Table 2: Reported Bioactivities for Some Erythrina Species (NAPRALERT, 2004) Plant species E. abyssinicci E. berteronna
Part bark root root bark root back
E. caffra
Bark
E. crisla-galli E. flnbelliformis
Leaf Seed
E. fusco E. latissima E. lysistemon
Seed Bark root
E. sacleuxii E. senegalensis E. suberosn
leaf flowers stem bark leaf
E. vnriegaln
bark
Activity antibacterial antifungal smooth muscle relaxation antifungal anticoagulant antibacterial cyclooxygenase inhibition antibacterial larvicidal diuretic CNS depressant antibacterial antiviral cytotoxicity antimalarial Molluscicidal Antiplasmodial, antitrypanosomal antitumour hypotensive antiulcer plant germination inhibition Smooth muscle relaxation Antifeedant
p-Hydroxycinnamoyl-CoAl 3x malonyl-CoAli
Figure 1: Biosynthetic relationships amoung different classes of tlavonoids
Extract CHC13 Ethanol 60% Methanol CH2C12 Acetone EtOAc
H2O EtOAc MeOH:CH2Cl2 1:1 EtOAc. H2O Ethanol 90% [5b] EtOAc/H2O 1:1 EtOAc. MeOH Acetone
825 •COOH
\
H
f T L* ^J T V°!l ^ 0^ 5 ^) Pterocarpans
6 a-hydroxypterocarpans
1 1 Pterocarpenes
Figure 2: Major isoflavonoid classes and their biosynthetic relationship [ adapted fromTahara and Ibrahim (13)]
IV. BIOACTIVE COMPOUNDS FROM ERYTHRINA A survey of non-alkaloidal secondary metabolites from Erythrina revealed the presence of flavanones, flavonols, chalcones, cinnamoyl phenols, stilbenoids, isoflavones, isoflavans, isoflavanones, pterocarpans, isoflav-3-enes, 3-phenoxychromones, coumastans, 3phenylcoumarins, lignans, cinnamate esters, simple phenolics, triterpenes, sesquiterpenes, squalene, C-18 unsaturated hydroxycarboxylic acid, and «-octacosanol. The documented bioactivity of some of the isolated metabolites includes antimicrobial activity [9,14-32], antiinflammatory [33-35], inhibition of platelet aggregation [9,15,32], tyrosinase inhibition [32], phospholipase A2 (PLA2) inhibitors [34,36], cyclooxygenase inhibitors [23], antioxidant [13,33,35,37-40], inhibitors of NA+/H+ exchange system [41], phospholipase C inhibitors [42], behavioural depression [15], muscle relaxation [15], P-adrenergic inhibition [15], diuretic [15], anticancer [13,43], cytotoxic [44-45], DNA-repair properties [44], oestrogenic [13] or proestrogenic activities [13], antiplasmodial [5b,46], and anti-HIV [47, 47b] activities. V. BIOACTIVE FLAVONOIDS FROM ERYTHRINA SPECIES In this section, flavonoids will be referred loosely to as compounds whose biogenetic origin is depicted in Fig. (1). Thus, in this section, chalcones, 1,3-diphenylpropanes (cinnamoylphenols) and stilbenoids (not generally considered as flavonoids) will be included as well as other 'conventional' flavonoid classes. Bioactive compounds will therefore be discussed according to class rather than common activity, and as such compounds that exhibit the same activity but belong to different classes will be discussed together with those from the same class albeit displaying different bioactivities. The same reasoning will be used in
826 discussing isoflavonoids. Bioactive flavonoids have been reported in this genus and their breakdown, first according to class (or subclass) and then type of activity, is given below. 1. Bioactive Flavanones: Liquiritigenin Derivatives Liquiritigenin [40] (5-deoxynaringinin) 1 and its derivatives seem to be restricted to very few Erythrina species and are found predominantly in E. abyssinica [9,46,48] and E. latissima [24,38-39] with reports of one or two metabolites of the class each in E. lysistemon [40], E. sigmoidea [49-50], and very recently E. fusca [32b]. In a screen for phytoestrogens, based on the binding affinity for the bovine uterine receptor, 1 was found to have affinity similar to that of the common dietary phytoestrogens genistein and daidzein [13b]. Nakanishi and coworkers [9] did some work that led the isolation and biological activity assessment of the novel neutral metabolites from E. abyssinica [9], viz abyssinones I 2, II 3, III 4, IV 5. The driving force for this work came probably from an ethno-medical observation that the roots of the plant were used to treat malaria and syphilis [3] and from an earlier finding by the same group that 60% aqueous methanolic extract of these roots was found to possess strong activity against fungi and Gram-positive bacteria [51]. Abyssinone II 3 was found to be active against Gram-positive bacteria Staphylococcus aureus & Bacillus subtilis (MIC 50 ug/ml each), Micrococcus lysodeikticus (25 ug/ml); against yeasts Saccharomyces cerevesiae & Candida utilis (100 ug/ml each) and against fungi Sclerotinia libertiana (12.5 ug/ml), Mucor mucedo (50 ug/ml), and Rhizophus chinensis (100 ug/ml). Abyssinone II also inhibited rabbit platelet aggregation induced by 38 ug/ml of collagen (IC50 3.5 ug/ml), and 0.57 uM sodium arachidonate (IC50 74 ug/ml, 40 % inhibition), but no inhibition against that induced by 10 uM ADP. Abyssinone I 2 was active against S. aureus & B. subtilis (MIC 50 ug/ml each); S. cerevesiae & C. albicans (100 ug/ml each), S. libertiana (12.5 ug/ml) & M. mucedo (50 ug/ml) but inactive against R. chinensis (>100 ug/ml). Abyssinone III 4 was not active against any bacteria, yeasts or fungi up to 100 ug/ml and was not tested for inhibition of platelet aggregation, while abyssinone IV 5 showed activity against both S. aureus (25 ug/ml) and B. subtilis (12.5 ug/ml) but was inactive (>100 ug/ml) against yeasts and fungi. Abyssinone IV further showed some antiplasmodial activity against the chloroquine-resistant W2 (IC50 7.7 fig/ml; chloroquine 0.093 ug/ml, quinine 0.21 ug/ml) and chloroquine-sensitive D6 (9.0 ug/ml) strains of Plasmodium falciparum [46]. Abyssinone VII 6 and erylatissin C 7 are new metabolites from E. abyssinica [49] and E. latissima [38,39] respectively, which have displayed free radical scavenging properties (IC50 27.7 and 710 ug/ml respectively) against 2,2-diphenyl-l-picrylhdrazyl (DPPH) radical. These compounds showed some weak preliminary antibacterial (0.1-0.5 ug) and strong antifungal (0.01-0.lug) activity using TLC bioautography technique. Table 3: Liquiritigenin Derivatives from Erythrina Species Compound Liquiritigenin 1 Abyssinone I 2 Abyssinone II 3 Abyssinone III 4 Abyssinone IV 5
Abyssinone VII 6 Erylatissin C 7
Erythrina source E. lysistemon [40] E. abyssinica [9] E. abyssinica [9] E. latissima [38,39] E. abyssinica [9] E. abyssinica [9,48,46] E. latissima [24] E. sigmoidea [27,30] E. abyssinica [48] E. latissima [38,39]
Activity Phytoestrogenic activity Antibacterial, antifungal Antibacterial, antifungal, antiyeast, inhibits platelet aggregation Antibacterial; antiplasmodial W2 (chloroquine resistant strain-7.7|j.g/ml), D6 (chloroquine sensitive strain -9.0 ug/ml) DPPH radical scavenging properties Weak antioxidant(DPPH), antibacterial (Gram-positive), antifungal.
827
No
R1
R2
R3
Name
1
H
OH
H
Liquiritigenin
2
3 ,4 (2,2-dimethylchromeno)
H
Abyssinone I
3
3,3-DMA
H
Abyssinone II
4
3,3-DMA
5
3,3-DMA
6 7
OH
4 ,5 (2,2-dimethylchromeno)
Abyssinone III
OH
3,3-DMA
Abyssinone IV
OH
OH
3,3-DMA
Abyssinone VII
OH
OMe
3,3-DMA
Erylatissin C
2. Bioactive Flavanones: - Naringenin Derivatives Naringenin [38-39] 8 and its derivatives seem to be more widely distributed among Erythrina species than liquiritigenin derivatives. Abyssinone V 9, first reported by Nakanishi and coworkers from E. abyssinica [9], showed antimicrobial activity against Gram-positive bacteria, S. aureus (50 ug/ml), B. subtilis (25 ug/mi) & M. lysodeikticus (12.5 ug/ml) and was non active (>100 ug/ml) against yeasts and fungi. It also showed a weak to moderate phospholipase A2 (PLA2) inhibition activity with IC50 value of 6 uM (trifluoropirazine at 20 uM showed >80% inhibition under assay conditions). The report of a PLA2 inhibitor, 6prenylabyssinone 10, first isolated from the methanolic extract of the stem bark of the Samoan medicinal plant E. variegata [36] and which has demonstrated strong PLA2 activity, is perhaps a good example of how ethno-medical data can provide clues or leads to certain putative pharmacological activities. The Samoan healers prepare extracts of the bark in coconut oil and apply the extract externally for swellings and inflammations. The extract is also used to treat skin ulcers that appear as boils or burns which burst and suppurate. It thus appeared, from this data, that E. variegata extractives in Samoa could be effective against inflammation or inflammation-like processes. Compound 10 was isolated from this bark extract and found to inhibit PLA2 with IC50 of 10 uM. Burttinone 11, first isolated from the stem bark of E. burttii [53] and later from E. lysistemon [43], was evaluated by the National Cancer Institute (NCI) as an anticancer agent through the 60 cell panel screen. The IC50 values for 11 were less than 50 uM against 43 cell lines. Burttinone further showed maximum toxicity against colon cancer cell line, HCC 2998 (IC50 = 20 uM), while the IC50 were higher than 50 uM in all the five tested leukaemia cell lines. It was, however, not selective in its action against any tested panel, indicating that it is a general cytotoxic agent which may probably have no clinical value.
828
Sigmoidins A 12 and B 13, first isolated from the stem bark of E. sigmoidea [11-12,32], exhibit weak but significant in vitro activity against both Gram-positive and Gram-negative bacteria. These two compounds were very recently [33] analysed for their ability to inhibit the stable l,l-diphenyl-2-picrylhydrazyl (DPPH) free radical and arachidonic acid metabolism. These were further studied in two experimental models of inflammation induced in mouse ears by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phospholipase A2-induced rat paw oedema. Both compounds proved to be potent scavengers of DPPH, while arachidonic acid metabolism inhibition studies showed them to be selective inhibitors of 5-lipoxygenase with no effect on cyclooxygenase-1 activity. Sigmoidin A, 12 gave IC50 of 31 uM. In the phospholipase A2-induced mouse paw oedema assay, only sigmoidin B 13 inhibited oedema formation at 60 minutes, showing a percentage inhibition below that obtained with cyproheptadine (59% vs 74%). In the TPA test, Sigmoidins A and B decreased the induced oedema by 89% and 83% respectively. The data for the two compounds seem to suggest that they have different mechanisms of action and this depending on whether the compound has one or two prenyl (or y,y-dimethylallyl or 3,3-dimethylallyl) units in the B-ring. Sigmoidins A and B also display oestrogen-like activity and thus displayed low but significant binding affinities on bovine uterine oestrogen receptor [60], albeit at much lower activity level than for 17P-ostradiol, a putative oestrogen. Compounds 14-17, recently isolated from the stem bark of E. fusca, displayed weak to moderate antibacterial activity (Table 5) against S. aureus, B. subtilis and E.faecalis [32b]. Table 4: Bioactive Naringenin Derivatives from Erythrina Species Compound Naringenin 8 Abyssinone V 9
6-pvenylabyssinoneV 10
Erythrina source E. latissima [38,39] E. abyssinica [9,48,52] E. burttii [53] E. eriotriocha [54] E. sigmoidea [55] E. variegata [36] E. variegata [36]
Activity Antibacterial; Phospholipase A2 inhibitor IC50 6 u-M
Phospholipase A2 (PLA2) inhibitor 1C5O 10 U.M
Burttinone 11
E. burttii [53] E. lysistemon [43]
Sigmoidin A 12
Sigmoidin B 13
Erysenegalone
14
Citflavanone Lonchocarpol A Lupinifolin
15 16 17
Anticancer-against 60 cell panel lines but not selective therefore not useful therapeutically. Antibacterial, anti-inflammatory.
E. abyssinica [52] E. eriotriocha [21 ] E. sigmoidea [11,12] E. latissima [24,56] E. abyssinica [48,52] E. berteroana [ 16] E. latissima [24,56] E. sigmoidea [11,12,57,58] E. suberosa [59] E, senegalensis [15] E. fusca [32b] E. fusca [32b] E. fusca [32b] E. fusca [32b]
Antibacterial, anti-inflammatory
Antibacterial Antibacterial Antibacterial Antibacterial
829
OH
O
No
R1
R2
R3
R4
R5
R6
Name
8
H
OH
H
H
OH
H
Naringenin
9
H
OH
H
3,3-DMA
OH
3,3-DMA
Abyssinone V
10
3,3-DMA
OH
H
3,3-DMA
OH
3,3-DMA
6-prenyl-
11
H
OH
H
a
OMe
3,3-DMA
Burttinone
12
H
OH
3,3-DMA
OH
OH
3,3-DMA
Sigmoidin A
13
H
OH
H
OH
OH
3,3-DMA
Sigmoidin B
abyssinone V
OH
O No
R1
R3
R2
R4
14
OH
3,3-DMA
7,8(2,2dimethylchromeno)
15
OH
H
7,8(2,2dimethylchromeno)
16
OH
3,3-DMA
17
OH
6,7(2,2dimethylchromeno)
OH
Name Erysenegalone Citflavanone
3,3-DMA
Lonchocarpol A
3,3-DMA
Lupinifolin
830 Table 5: In vitro Antibacterial Activity of Compounds from E.fusca [32b] Compound
Staphylococcus aureus
Bacillus subtilis
Enterococcus faecalis
MIC"
MBC"
MIC
MBC
MIC
>100
>100
50
50
100
100
12.5
>100
12.5
12.5
>100
>100
Lonchocarpol A
6.25
>100
3.13
3.13
6.25
6.25
Lupinifolin
12.5
>100
6.25
6.25
50
50
Tetracycline
<0.25
<0.25
16
16
0.13
0.13
Erysenegalone Citflavanone
MBC
"Minimum inhibitory concentration in 'Minimum bactericidal concentration in
3. Chalcones from Erythrina Species There have been so far only four chalcones reported for the genus Erythrina (Table 6) and all of them are reported active. Isobavachalcone 18 first isolated from Morus cathayana root showed binding affinity to bovine uterine oestrogenic receptor [60]. Isobavachalcone also showed broad antioxidative activity in rat liver microsomes and mitochondria by inhibiting NADPH, ascorbate, t-BuOOH and CC14 lipid peroxidation in microsomes [37]. It also prevented NADH-dependent and ascorbate-induced mitochondrial lipid peroxidation [37]. Abyssinone VI19, first reported from E. abyssinica [9] by Nakanishi and co-workers, showed very strong inhibition to platelet aggregation induced by 10 uM ADP (IC50 8.6 ug/ml), 38 ug/ml collagen (IC50 5.5 ug/ml), and 0.57 uM sodium arachidonate (IC50 37 ug/ml). Licoagrochalcone A 20, first isolated from Glycyrrhiza glabra, showed weak oestrogenic activity as manifested by its binding affinity on bovine uterine oestrogenic receptor [60]. It also showed antiplasmodial activity against the chloroquine resistant W2 (IC50 12.8 ug/ml, chloroquine 0.093 ug/ml, quinine 0.21 ug/ml) and the chloroquine sensitive D6 (IC50 19.5 ug/ml, chloroquine 0.0008 ug/ml, quinine 0.042 ug/ml) strains of Plasmodium falciparum [46]. The compound 3-methylbutein 21 showed strong 2,2-diphenyl-l-picrylhydrazyl radical (DPPH) scavenging properties with MIC value of 52.0 ug/ml within 30 minutes [48]. Table 6: Bioactive Chalcones from Erythrina Species Compound
Erythrina source
Activity
Isobavachalcone 18
E. burttii [61]
Abyssinone VI 19
E . abyssinica [9,48] inhibits platelet aggregation E. sigmoidea [50]
oestrogenic activity, broad antioxidative activity
Licoagrochalcone A 20
E. abyssinica [46,48] Antiplasmodial, oestrogenic E. latissima [39,39]
3-methylbutein 21
E. abyssinica [48]
radical scavenging DPPH
831
R1 OH
O
No
R1
R2
R3
Name
18
3,3-dimethylallyl
H
H
Isobavachalcone
19
H
H
H
Abyssinone VI
20
H
H
3,3-dimethylally]
Licoagrochalcone A
21
H
H
OCH3
3-methylbutein
4. Bioactive Stilbenoids from Erythrina Seven stilbenoids have, so far, been reported from the genus Erythrina, namely oxyresveratrol [62-63], dihydrooxyresveratrol [62], burttinol D [64], latissimbenzofuran [24,65], bidwillol B [11], eryepogin F [29] and eryvarin L [30].
22: Dihyrooxyresveratrol: 23: Oxyresveratrol:
7,8-dihydro A 7,8
Stilbenes are a class of biologically active components found in plants and have been shown to possess various medicinal properties [63]. These have been shown to have, among others, antioxidant properties, especially against human low-density lipoprotein (LDL). The most potent of these compounds, (Z)-piceatanol(3,5,3,4 -tetrahydroxystilbene)-3-O-(3-Dglucopyranoside, also called (Z)-astringin, was found in red wines [66]. Oxyresveratrol 23 showed some significant radical scavenging properties, as well as strong tyrosinase inhibition. Tyrosinase is responsible for the moulting process in insects, undesirable browning in fruits and vegetables and colouring of skin, hair & eyes in mammals [67]. The inhibitory effects of oxyresveratrol on tyrosinase from mushroom and murine melanoma B-16 showed it to have potent inhibitory effect (IC50 1.2 uM) on mushroom tyrosinase, a 32-fold stronger inhibition than kojic acid, which is a depigmenting agent used as the cosmetic
832 material with skin-whitening effect and a medical agent for hyperpigmentation disorders [67]. At 100 uM oxyresveratrol gave an IC50 value of 52.7 [iM on L-tyrosine oxidation by tyrosinase activity. The kinetics and mechanism for inhibition of the mushroom tyrosinase exhibited the reversibility of oxyresveratrol as a non-competitive inhibitor with L-tyrosine as substrate [67]. The interaction between oxyresveratrol and tyrosinase exhibited a high affinity reflected in a K, value of 3.2-4.2 xlO"7 M. Oxyresveratrol does not seem to have any effect on the promoter activity of the tyrosinase gene in murine melanoma at 10 and 100 uM. The depigmenting effect of oxyresveratrol appears to work through reversible inhibition of tyrosinase activity rather than suppression of the expression and synthesis of the enzyme [67].
R1
No
R1
R!
R3
R4
R5
R6
R7
Name
24
OCH 3
H
H
OH
CH3
H
H
Latissimbenzofuran
25
H
OCH 3
3,3-DMA
OH
H
H
CHO
Eryepogin F
26
H
H
H
OCH3
H
OCH3
H
Eryvarin L
Three of the five reported benzofurans from Erythrina genus have been shown to exhibit biological activity. Latissimbenzofuran 24, isolated from E. latissima [24,65], showed very strong preliminary antibacterial activity against Gram-positive, Gram-negative bacteria and fungi [65], It has turned out to be, to date, the most active compound we have ever isolated, with activities lower but comparable to antibacterial (chlorampenicol) and antifungal (miconazole) standards in the TLC bioautography technique. Eryepogin F 25, first reported from£. poeppigiana [29] (first report of 3-formyl-2-arylbenzofuran in the genus Erythrina), is a very rare 2-arylbenzofuran in the plant kingdom possessing a formyl group, making it so far the third example of 2-aryl-3-fromylbenzofuran isolated as a natural product [68]. The biosynthesis of these interestingly involves the C-ring opening in a coumastan as the possibility of D-ring formation from a 3-arylcoumarin has been ruled out by previous studies [69].
OCH3
Fig. 3: Proposed biogenetic hypothesis for formation of 25
Eryepogin F showed very potent anti-methicillin resistant Staphylococcus aureus (MRSA) against 13 resistant strains. According to the criteria of resistance of MRSA to methicillin and
833
oxacillin compounds that do not inhibit MRSA strains at or above 12.5 ug/ml are considered inactive. Eryepogin F inhibited all 13 strains of MRSA with activity based on bactericidal action [29]. Eryvarin L 26, recently isolated from the roots of E. variegata [30], showed antiMRSA, albeit weaker, with IC50 value of 25 ug/ml. Eryvarin L also inhibited the growth of 5 strains of vancomycin-resistant enterococci at 50 ug/ml. 5. Bioactive Cinnamoylphenols from Erythrina Cinnamoylphenols or 1,3-diphenylpropanoids are relatively rare in the genus with only seven compounds, viz erypostyrene [27], eryvariestyrene [18,70], eryvarinols A & B [71], and erycristanols A, B and C [70], so far reported. Erypostyrene 27 exhibited strong anti-MRSA against 13 MRSA strains [27] with an IC50 value of 6.25 ug/ml. It also inhibited 6 strains of Candida albicans at 50 ug/ml. This observed antimicrobial pattern could be advantageous in treating mixed infections by C. albicans and MRSA. Furthermore, the antimicrobial activity of 27 against both species is based on fungicidal and bactericidal actions. Thus 27 could be a leading candidate in the development of new antimicrobial agents, possessing both anticandidal and anti-MRSA activities [27].
R'O
27: R ] =CH 3 , R 2 =OH 2 8 : R 1 = H , R2 = H
Erypostyrene Eryvariestyrene
Eryvariestyrene 28, first reported from E. variegata [18] and later on in E. crysta-galli [70], showed weak to moderate antibacterial activity against a variety of organisms [18] (S. aureus 50 ug/ml, E. coli 50 ug/ml, Klebsiella pneumoniae 100 ug/ml and Mycobacterium smegmatis 50 ug/ml) but was inactive (>100-1000 ug/ml) against Salmonella gallinarium, Candida albicans and Pseudomonas aeruginosa [18]. Erypostyrene 27 and eryvariestyrene 28 inhibited HIV-1-induced cytopathogeneicity in MT-4 cells in a dose-dependent fashion (Table 7). Eryvariestyrene achieved a 52 to 70% inhibition of the HIV-1 replication at a concentration of 20 uM. Its 50% effective concentration was 11 to 18 uM. On the other hand, erypostyrene displayed a 28 to 55% inhibition of the HIV-1 replication at a concentration of 4 uM. Both compounds were cytotoxic to the host cells at concentrations 20 and 100 uM, respectively. The 50% cytotoxic concentrations were 7.4 to 8.9 uM for 27 and 38 to 45 uM for 28. Thus, both compounds were found to be selective inhibitors of HIV-1 replication, though modestly so [47b].
834 Table 7: Inhibitory Effects of Erypostyrene (27) and Eryvariestyrene (28) on HIV-1 Replication in MT-4 Cells Experiment 1
Experiment 2
Compound
EC50 (nlVI)
CC50 (nM)
ECso (HM)
CC50(nM)
Erypostyrene
>7.4*
7.4
3.1
8.9
38
11
45
Eryvariestyrene
18
EC50: 50% effective concentration CC50: 50% cytotoxic concentration * Twenty-eight percent inhibition was achieved at concentration of 4 \M. HO-
OCH,
29 Angolesin (a-methyldeoxybenzion) Angolesin 29, a 1,2-diphenyl propane and not strictly speaking a cinnamoylphenol, will, for convenience sake, be discussed with the cinnamoylphenols. Angolesin 29, reported from the roots of E. poeppigiana [27,72] , showed anti-MRSA activity against 13 strains of MRS A with an IC50 value of 50 (J.g/ml. It however failed to inhibit the growth of C. albicans (> 100 Hg/ml) [27]. VI. ISOFLAVONOIDS FROM ERYTHRINA SPECIES Most of the reported metabolites from Erythrina belong to the general class of isoflavonoids, whose biogenetic relationship is shown in Fig. (2). Under this general class are compounds reported to fall into the following classes: isoflavones (biggest), pterocarpans, isoflavanones, isoflavans, isoflav-3-enes, coumastans and 3-phenylcoumarins, and phenoxychromones. 1. Bioactive Isoflavones from Erythrina Species Isoflavones form the largest class of metabolites ever reported from the genus Erythrina with pterocarpans trailing close at second. They however form the second largest class of bioactive compounds from the genus after the pterocarpans. This class shows the widest diversity of activity among all reported bioactive compounds. b. Genistein (5-hydroxyisoflavones) Derivatives
OH
835
No 30 31 32 33 34 35
R1 H
36
3,3-dimethylallyl
3,3-dimethylallyl 3,3-dimethylallyl 6,7(2,2-dimethylchromeno) 3,3-dimethylallyl
R2 H OH OH
R3 H a H H a
OH
3,3-dimethylallyl
c
OH
R4 H b H H H H
H
d
Name Genistein Indicanine D Wighteone (Erythrinin B) Alpinumisoflavone Erysenegalensein E 8-prenylerythrinin C (Isosenegalensin) (Euchrenone bio) (revised structure) Senegalensin (revised structure)
CH 2 OH v
JL
*c
=
HO
b =
These are 5-hydroxyisoflavones, having genistein 30 as the simplest member, and are reported to have several reported bioactivities. Genistein, a precursor of 5-hydroxyisoflavonoids, has been implicated in a variety of biological activities including allelopathic, oestrogenic or proestrogenic, antihaemolytic, antioxidant and anticancer activities [13]. It is also a potent inhibitor of enzymes such as catechol-O-methyltransferase, DOPA decarboxylase, dopamine P-hydrolase, histadine decarboxylase and lipase [13]. It is also a common dietary phytoestrogen [13b, 13c], that is, any plant substance or metabolite that induces biological responses in vertebrates and can mimic or modulate the actions of endogenous oestorgens usually by binding to oestrogen receptors. As plant oestrogen whose richest source has so far been Soya bean, genistein has been shown to block the action of a transcription factor, known as CCAAT binding factor, neutralising it before the switch is tripped, so that the cancer cell starves, withers and dies [13c]. Thus genistein, commonly consumed as a component of Soya bean, is a flavonoid capable of stopping cancer growth and angiogenesis. Crucially, it has been reported to have no harmful effects on normal healthy cells [13d]. The dichloromethane-methanol (1:1) extract and the methanol extracts of the stem bark of E. indica were tested for in vitro cytotoxicity against human KB cells and proved active (Table 8) with IC5o values of 10 and 36 ug/ml respectively. Bioassay-guided fractionation of the CH2Cl2-MeOH (1:1) extract led to, among others, active compounds 31-36 (Table 6). Wighteone 32, also called erythrinin B, was the most potent followed by alpinumisoflavone 33 and erysenegalensein E 34, whereas 8-prenylerythrinin C (or isosenegalensin or euchrenone bio) 35 displayed weak cytotoxic activity. It is important to note that the structure of senegalensin 36 has been written as 35 until 2001 when Tanaka and coworkers [82] revised it to 36 based on extensive 2D NMR and X-ray structure of its p-bromobenzoyl derivative. The structure of euchrenone b ]0 (or isosenegalensin or 8-prenylerythrinin C) was consequently revised from 36 to 35. Isosenegalensin was also evaluated as an anticancer agent through the 60 cell panel [43] and gave IC50 less than 50 uM only against four cell lines: ovarian cancer IGROV1 (45 uM), non-small cell lung carcinoma NCI-H322M (46 uM), colon cancer HCC2998 (48 uM), and renal cancer UO-31 (49 uM). The IC50 values were higher than 100 uM against nine cell lines. Isosenegalensin 35 was found non selective in its action against any tested panel indicating that it was of general toxicity and could have no clinical value [43].
836
Compounds 32 and 35, isolated by bioassay-guided separation of the methanolic extract of the Indonesian E. variegata, were also found to be moderate inhibitors of the Na"7H+ exchange system of the arterial smooth muscle cells with MIC's of 1.25 |ig/ml each [41]. Table 8: Cytotoxicity Against Human KB Cells off. indica Extracts and Isoflavone Isolates compounds
Erythrina source
EDso (Hg/ml)
CH2Cl2-Me0H extract (1:1)
10.00
E. indica [45]
MeOH extract
36.00
E. indica [45]
Indicanine D 27
12.50
E. indica [45]
Wighteone(erythrinin B) 28
0.78
E. indica [45], E. lysistemon [23,40,43], E. orientalis [73], E. variegata [74], E. suberosa var glabrescenses [75]
Alpinumisofiavone 29
4.13
E. arborescens [76,77], E indica [45,62], E. lysistemon [40,43], E. senegalensis [78], E. suberosa var glabrescenses [75], E. variegata [74]
Erysenegalensein E 30
6.25
E. indica [45], E. lysistemon [40], E. senegalensis [79],
8-prenylerythrinin C 31
13.00?
E. indica [45], E. lysistemon [40,43], E. senegalensis [80], E. eriotriocha [81]
13.00
E. indica [45], E. senegalensis [80,83], E. suberosa var glabrescenses [82]
(Euchrenone bio> (Isosenegalensin) Senegalensin
32
Table 9: Bioactive Isoflavone Derivatives from Erythrina Species
Compound
Erythrina source
Auriculatin 37
E. eriotriocha [84]
Activity Antibacterial, diuretic, PLA2 inhibitor,
E. senegalensis [15,78,83,85,86] cytotoxic E. variegata [87] Warangalone 39
E. addisonae [34]
(Scandenone)
E. arborescens [77]
Anti-inflammatory
E.eriotriocha [88] E. sigmoidea [21,50] E. variegata [18] 6,8-diprenylgenistein 40
E. senegalensis [78,79]
Antibacterial, diuretic, p-adrenergic
E. sigmoidea [21,50]
stimulation, platelet aggregation inhibitor
E. variegata [18,90,91] E. vogelii [92] 8-prenylluteone 38
E. eriotriocha [88]
PLA2 inhibitor, cytotoxic
E. lysistemon [40] E. senegalensis [42,78,79]
837
OH
No
R1
37 38 39 40
R2
O
R3
R4
Name
6,7(2,2-dimethylchromeno)
3,3-dimethylallyl
OH
Auriculatin
3,3-dimethylallyl
3,3-dimethylallyl
OH
8-prenylluteone
6,7(2,2-dimethylchromeno)
3,3-dimethylallyl
H
Warangalone (scandenone)
3,3-dimethylallyl
3,3-dimethylallyl
H
6,8-diprenylgenistein
OH
OH
Auriculatin 37, first isolated from Milletttia auriculata [93], was subsequently reported in several Erythrina species (Table 9). A detailed pharmacological screen on some isolates from E. senegalensis revealed the compounds auriculatin 37 and 6,8-diprenylgenistein 40, to be non-toxic, exhibiting antimicrobial activity against Gram-positive bacteria and having diuretic properties [15]. In addition, 40 also exhibited some P-adrenergic stimulation and platelet aggregation properties [15]. Auriculatin 37, along with 8-prenylluteone 38, inhibited phospholipase C (PLC) in vitro and the formation of total inositol phosphates (IPj) in plateletderived growth factor (PDGF)-stimulated NIH3T3yl cells. These compounds exhibited potent activity on phosphoinositides (PI)- turnover in response to PDGF stimulation [42]. These two compounds, both with at least one prenyl group, were shown to have an inhibitory effect on the Pi-turnover in the NIH3T3yl cells and the enzymatic activity of PLC with an IC50 of 20 (J.M. When the anti-PLC of 37 and 38 was compared with that of common flavonoids (luteolin, quercetin, hesperetin and genistein), the common flavonoids were found to exhibit very weak inhibitory activity with IC50 values of around 250 uM against PLC [42]. It appears that the presence of a prenyl group at position 8 was related to the inhibitory action against PLC-yl. These compounds further showed cytotoxicity against several human tumour cell lines viz PC-3 (prostate), NCI-H226 (lung), CRL1579 (melanoma) in vitro (Table 10), with moderate cytotoxicity (IC50 range 9.0-20 uM), with 37 showing more potent inhibition than 38 on PC-3 and CRL1579 cell lines [42] (Table 10). Since PLC plays an important role in intracellular signal transduction and cell proliferation, 37 and 38, as PLC inhibitors, maybe cell growth inhibitors and could find application in cancer treatment or as tools for exploring the mechanism of PLC-mediated signal transduction. Table 10: Cytotoxicity of 37 and 38 Against Some Human Tumour Cell Lines
Compound
Human Cell Line (ICJO values in \M) PC-3 (prostate)
NC1-H226 (lung)
Auriculatin 37
9.4
12.1
CRL1579 (melanoma) 9.0
8-prenylluteone 38
20.0
15.1
16.4
838 Warangalone or scandenone 39, first described by Pelter and Stainton [94], has been reported in a number of Erythrina species [18,21,34,50,77,88]. Its anti-inflammatory activity was discovered through a bioassay-guided fractionation of an EtOAc extract of the bark of E. addisonae, which showed activity on a model of ovalbumin hind paw oedema [34]. The antiinflammatory activity of the extract and the warangalone (the main active constituent of the extract) was assayed on phospholipase A2 (PLA2) and carrageenin-induced mouse paw oedema, 12-O-tetradecanoylphorbol 13 acetate (TPA)-induced mouse ear oedema, and a model of chronic dermatitis caused by repeated administration of TPA. Warangalone showed marked effectiveness as an anti-inflammatory agent on the PLA2-induced paw oedema (Table 11) and on the TPA-induced ear oedema (Table 12) in mice after systemic and local administration respectively [34]. Table 11: Effect of Test Drugs on PLA2-Induced Mouse Paw Oedema" Test drug
Time 30min
%I
60 min
%I
Control
6.50
E. addisonae EtOAc extract
3.50
46
3.80
39
Warangalone 39
5.16
21
2.00
69
Cyproheptadine
2.16
66
2.32
63
6.30
"E. addisonae EtOAc extract (50 mg/kg), warangalone (5 mg/kg), and cyproheptadine (5 mg/kg) were intraperitoneally administered 30 min before subcutaneous plantar injection of PLA2 and oedema was measured 30 and 60 min later. Table 12: Effect of Test Drugs on TPA-induced Ear Oedema"
Test drug
Control E. addisonae EtOAc extract Warangalone 39 Indomethacin
Ear swelling
%1
MPO activity
14.25 1.80 3.07 2.57
87 78 82
155 n.d 91 99
41 36
"E. addisonae EtOAc extract, warangalone, and indomethacin were administered at 1, 0.25, and 0.5 mg/ear respectively, on the ear surface together with TPA. b
MPO = myeloperoxidase activity.
(b). Bioactive Daidzein Derivatives from Erythrina Genus It is interesting to note that most common activity that daidzein and its derivatives exhibit seems to be predominantly antimicrobial (Table 13). Daidzein 41, a possible precursor for 5deoxyisoflavones, has been reported in several Erythrina species [38-40,76,94-96] and is a well-known dietary phytoestrogen [13b]. It has been reported also to exhibit weak to moderate antibacterial activity and weak radical scavenging properties on the DPPH radical [38-39]. 8prenyldaidzein 42, so far reported only in two Erythrina species [21,95], was found to be active against S. aureus (MIC 10.5 ug/ml, tetracycline 0.1 ug/ml, erythromycin 0.2 ug/ml), B. subtilis (10.5 ug/ml, tetracycline 0.2 ug/ml) and Mycobacterium smegmatis (12.5 ug/ml,
839
erythromycin 0.78 ng/ml). Neobavaisoflavone 43, first isolated from the seeds of Psoralea corylifolia [100], and later in a few Erythrina species (Table 13), exhibits weak radical scavenging properties on the DPH radical [38-39]. It also showed antifungal properties against Cryptococcus neoformans (MIC 50 ug/ml) and Aspergillus fumigatus (50 ug/ml), was found inactive against Candida albicans (> 100 ug/ml) and showed antibacterial activity against Gram-positive S. aureus (2.5 ug/ml) [50,97]. It may be worth noting that a comparison between 41, 42 43 and 44 seems to point to the fact that the presence of a 3,3-dimethylallyl (y,Y-dimethylallyl or loosely referred to as prenyl) enhances activity towards bacteria and fungi and that the presence of a prenyl unit at 3 - position (as opposed to 8-position) enhances activity against bacteria and also broadens the spectrum wide enough to make the compound fungitoxic. When a prenyl unit is cyclised as in corylin 44, the activity against fungi (C. neoformans, A. fumigatus and C. albicans) is lost completely [97]. Erylatissin A 45 was only weakly antibacterial and radical scavenging [38-39], while erysubin F 47 was only very weakly anti-MRSA with MIC range of 100 ug/ml and with all 13 MRSA resistant strains [75]. Bidwillon C 46 exhibited activity against Streptococcus mutants (IC50 25 ug/ml), Prevotella intermedia (50 ug/ml) and Porphyromonas gingivalis (50 ug/ml) [19]. Ulexone A 48, first reported from gorse (Ulex europeaeus [101]), later in Ulex jussiaei [102] and so far reported only in E. vogelii [99], showed insect feeding deterrent activity against Costelytra zealandica (Coleopteran) larvae [101] with an FD50 (concentration which reduces feeding by 50%) of 17 ppm. Table 13: Bioactive Isoflavones (Daidzein Derivatives) from Erythrina Species Compound
Erythrina source
Daidzein 41
E. arborescens [76 ]
Estrogenic,antibacterial, weakly DPPH
E. indica [95]
radical scavenging
Activity
E. latissima [38,39] E. lysistemon [40] E. orientalis [94,96] 8-prenyldaidzein 42
E. eriotriocha [21 ]
Antibacterial
E. indica [95]
Neobavaisoflavone 43
E. abyssinica [48] E. latissima [24,56,65] E. sigmoidea [50,97]
Antibacterial, antifungal, DPPH radical scavenging
Corylin
E. sigmoidea [97]
Antifungal
44
Erylatissin A 45
E. latissima [38,39]
Bidwillon C 46
E. x bidwillii [19]
Antibacterial, antifungal
E. variegata [30] E. poeppigiana [29] Erysubin F 47
Antibacterial (anti-MRSA)
E. suberosa var glabrescenses [75] E. variegata [26,98]
E. vogelii [99] Ulexone A* 48 *genistein derivative but included here for convenience
Insect feeding deterrent
840
R1
No
R1
R2
R3
R4
R5
41
H
OH
H
H
OH
42
H
OH
3,3-DMA
H
OH
H
8-prenyldaidzein
43
H
OH
H
3,3-DMA
OH
H
Neobavaisoflavone
44
H
OH
H
3 ,4 (2,2-dimethylchromeno)
H
Corylin
45
H
OH
H
OH
OCH3
3,3-DMA
Erylatissin A
46
H
7,8(2,2dimethyl
H
OH
H
Bidwillon C
OH
H
Erysubin F
H
Ulexone A
R6 H
Name Daidzein
chromeno) 47
H
OH
3,3-DMA
3,3-DMA
48
OH
OH
H
3 ,4 (2,2-dimethylchromeno)
Bioactive Pterocarpans from Erythrina Species Pterocarpans form the second largest class of metabolites reported from Erythrina species after isoflavones but represent the largest number of bioactive compounds in any given subclass (Tables 14-19). A large number of pterocarpans are phytoalexins, that is, antibiotic chemicals that are produced in response to microbial challenge [103]. The activity profile spans "normal" pterocarpans, through 6a-hydroxypterocarpans to 6a, 11 a-dehydropterocarpenes, Fig. (2). The major activity exhibited is antimicrobial (Tables 14-16), but other activities such as inhibition to rabbit platelet aggregation [9] (Table 17), cytotoxicity and potential antitumour [44] (Table 18), antiplasmodial [46] (Table 19), phospholipase A2 inhibitors [36], and antiHIV [47]are shown by this class. The antimicrobial profile (Tables 14-16) can be divided into general antibacterial and antifungal properties (Table 14), antibacterial activity against resistant strains thus giving anti-MRSA properties (Table 15) and activity against cariogenic oral bacterial strains (Table 16). What seems to emerge (though more structure activity work needs to be done) is that for general activity against bacteria, the presence of a 3 or 9- hydroxy (preferably both) and of both 2- and 10-prenyl in a pterocarpans nucleus is essential for the activity (erycristagallin 53, erycristin 54, and erythrabyssin II 56), while for general fungitoxicity, the non-availability of a 9-hydroxy either through a cyclisation process with 10prenyl (phaseollidin 58 vs phaseollin 59) or through methyl ether formation (erythrabyssin I or cristacarpin 55 vs erythrabyssin II 56). It appears that for anti-MRSA, (Tablel5) a general pterocarpans nucleus (erycristagallin 53, orientanol B 65, orientanol C 66 and 2-prenyl-6ahydroxyphaseollidin/demethylerystagallin A 67, sandwicensin [27] 69), with either 3/9-
841 dihydroxy, or 2/10-diprenyl units (53 and 67) or presence of at least one of 3/9 hydroxy and at least one of 2/10 prenyl units (65, 66 and 69 [27]) is essential. The activity against various mutants streptococci strains of cariogenic bacteria [31] (Table 16) reveals the essence of a pterocarpans nucleus. Erycristagallin 53 and sigmoidin K 68 are structurally very similar except that the presence of a 6-oxo functionality makes the latter a coumastan as opposed to a pterocarpan. Sigmoidin K was found to be totally inactive (>100 ug/ml) against nine strains of mutants streptococci and one strain of Lactobacillus casei and showed a very wide variability in activity (6.25->100 ug/ml) against the seven strains of other streptococci but gave good activity range (1.56-6.25 ug/ml) against Actinomyces. Erycristagallin conversely was very active against all organisms with very narrow activity range (mutant streptococci 6.25 ug/ml, other streptococci 3.13-6.25 ug/ml, Actinomyces 1.56 ug/ml, Lactobacillus casei 6.25 ug/ml). The other more active compounds are erystagallin A 60, demethylerystagallin A 67 and orientanol B 65 in that order. The presence of either one or both 3/9 hydroxyls and one or both of 2/10 prenyl units seems important for activity. It appears that activity is greatly enhanced by the presence groups (2,10-diprenyl, 3,9-dihydroxy) (53, 67), attenuated by the presence of both a single 2-prenyl and a 9-hydroxy (65, 60), while the absence of both of these groups reduces or removes the activity completely (55, 61). The antibacterial effect of erycristagallin to mutant streptococci was based on bactericidal action. Erycristagallin (6.25 ug/ml: MIC) also completely inhibited radio-labelled incorporation of thymidine into Streptococcus mutants cells, as well as the incorporation of radio-labelled glucose into bacterial cells. This points to the potential that erycristagallin has as a phytochemical agent for prevention of dental caries by inhibiting the growth of cariogenic bacteria and by interfering with the incorporation of glucose responsible for the production of organic acids. It is important to note that according to criteria for resistance of MRSA to methicillin and oxacillin strains are defined as resistant if they are not inhibited at a concentration of above 12.5 ug/ml, and as sensitive if they are inhibited at/or below this concentration [26]. Cristacarpin 55, erythrabyssin II 56, phaseollidin 58 and phaseollin 59, were tested for their inhibitory effect on rabbit platelet aggregation [9] (Table 17) and only erythrabyssin II showed significant activity. An important feature of many tumour cells is that they have defects in their ability to repair damage to DNA as compared to normal cells. Therefore, agents with selective toxicity towards repair-deficient cells might be potential antitumour agents. In this respect, DNA repair-deficient and repair-proficient yeast mutants can be used to screen for potential antitumour agents. The chloroform extract of the bark of E. burana showed selective activity against the repair-deficient rad 52 yeast strain. Activity-guided fractionation of the chloroform extract [44] yielded compounds cristacarpin 55 and phaseollidin 58. Both compounds showed selective activities in rad 52 and rad 321 assays compared to with the wild-type RAD+ strain. Cytotoxicity activities of cristacarpin and phaseollidin were determined in three cell lines (Table 16). Phaseollidin was found to be moderately active in the wild-type CHOC cytotoxicity assay, while cristacarpin, interestingly, was inactive against wild-type CHOC but showed activity against a P-glycoprotein overproducing cell line [44], showing unusual reversal of activity. It is also interesting to note that the roots of £. abyssinica have long been used in ethno-medicine to treat various ailments such as malaria and syphilis [3], Recent work by Yenesew and co-workers [46] on the acetone extract of the roots of E. abyssinica showed potent activity against the chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum with IC50 values of 0.49 and 0.64 ug/ml respectively, explaining its wide traditional use as an antimalarial [3]. This extract yielded, among others, pterocarpans isolates [46], erythrabyssin II 56, erycristagallin 53 and eryvarin D (we found it to be identical to 3-hydroxy-9-methoxyl0-prenylpterocarpene) 62, with antiplasmodial activity against W2 and D6 strains of Plasmodium falciparum (Table 19). Erythrabyssin II was found to be the
842 most active of the pterocarpans tested [46]. Erycristagallin was also reported as a pterocarpans isolate from a Samoan E. variegata whose bark extract exhibited strong PLA2 inhibition activity [36]. Three isolates were obtained from this plant and erycristagallin gave the strongest inhibition of the three with an IC50 value of 3 uM (trifluoropirazine at 20 \xM showed >80% inhibition under these assay conditions) [36]. The ethyl acetate extract from E. mildbraedii, in the carrageenin induced mouse paw oedema, showed anti-inflammatory activity [35], and erycristagallin was isolated as the active ingredient. The erycristagallin from this extract, in vivo, significantly inhibited the PLA2-induced mouse paw oedema, as well as the mouse ear oedema induced by TPA (ID50 < 10 ug/ear). It further reduced significantly the chronic inflammation and leukocyte infiltration induced by repeated application of TPA. In vitro, erycristagallin inhibited the arachidonic acid metabolism via the 5-lipoxygenase pathway in rat polymorphonuclear leukocytes (IC50 23.4 (xM), but had no effect on cyclooxygenase-1 metabolism in human platelets, while showing antioxidant activity in the DPPH test. The anti-inflammatory activity of erycristagallin, as with other phenolics, may be based on its capacity to inhibit arachidonic acid metabolism via the 5-lipoxygenase pathway [35]. HIV inhibitory pterocarpans 3-0- methylcalopocarpin (we found to be identical to orientanol B) 65 and sandwicensin 69 were isolated from an organic bark extract of a Guatemalan E. glauca that showed preliminary anti-HIV activity [47]. The two compounds inhibited the cytopathic effects of in vitro HIV-1 infection in human T-lymphoblastoid cell line (CEM-SS). Orientanol B (3-O-methylcalopocarpin) was cytoprotective over a modest concentration range (EC50 0.2 ng/ml; IC50 3.0 ug/ml) with a maximum of 80-95% protection, while sandwicensin was less effective (EC50 2 ug/ml; IC50 7 ug/ml), with a maximum protection of only 50-60%. Compound 69 was also very recently found to be weakly anitbacterial against S. aureus, B. subtilis and E. faecalis with MIC values of 50, 100 and 50 ug/ml respectively [32b], Table 14: Erythrina Pterocarpans with Antibiotic Activity Bacteria (MIC in ug/ml)
Yeast
Compound
Fungus
(Ug/ml) S.a
Erybraedin A 49
B.s
Ms
12.5
6.25
Erybraedin C 50
12.8
12.5
Erybraedin D 51
78.3
Erybraedin E 52
22.1
Erycristagallin 53
1.56
Erycristin
54
6.25
6.25
1.56
Eryabyssin I 55
12.5
6.25
3.13
3.13
S.m
Pg
A.a
6.3
6.3
6.3
M.I
(H-g/ml)
S.c
C.u
S.I
M.m
R.c
50
50
6.25
25
*
*
*
*
(cristacarpin) Erythrabyssin 56
II
0.78
3.13
*
Isoneorautenol 57
25.0
Phaseollidin 58
50
25
25.0 *
*
#
*
*
Phaseollin 59
12.5
6.25
25
50
12.5
12.5
12.5
S.a = Staphylococcus aureus, B.s = Bacillus subtilis, M.s ~Mycobacterium smegmatis, S.m = Streptococcus mutant, P.g.= Porphyromonasgingivalis,
A.a = Actinobacillus actinomycetencomitans, M.I = Micrococcus lysodeikticus, S.I =
Saccharomyces cerevesiae, C.u = Candida utilis, S.I = Sclerolinia libertiana, M.I = Mucor mucedo, R.c = Rhizopus chinensis. * = > 100, # = >50
843 Table 15: Anti-MRSA Activity of Pterocarpans from the Genus Erythrina
Antibiotic parameter (in ng/ml)
Proportion
Compound
Anti-MRSA range
MIC50
MIC,o
of resistant strains
Cristacarpin SS (Erythrabyssin 1)
100
100
100
13/13
Erycristagallin
53
3.13-6.25
6.25
6.25
0/13
Erystagallin A
60
12.5-25
25
25
9/13
Eryvarin A
61
>100
>100
>100
13/13
Eryvarin D
62
12.2-25
25
25
9/13
Eryvarin E
63
25-M00
>100
>100
13/13
Folitenol
64
6.25->100
12.5
50
6/13
Orientanol B 65 (3-O-methylcalocarpin)
3.13-6.25
6.25
6.25
0/13
Orientanol C
66
6.25-25
12.5
12.5
2/13
Phaseollidin
58
25-50
50
50
13/13
Phaseollin
59
25
25
25
13/13
Demethylerystagallin A 67 (2-prenyl-6a-hydroxyphaseollidin)
6.25-12.5
12.5
12.5
0/13
Methicillin
12.5->100
>100
>100
13/13
Oxacillin
12.5->100
>100
>100
13/13
IC50 and IC90 cone, needed to inhibit 50% (seven strains) and 90% (12 strains) of strains tested Table 16: Activity of Erythrina Pterocarpans Against Cariogenic Oral Bacteria [31].
MIC range in ng/ml
Compound Mutants streptococci (9 strains)
Other streptoc
Actinomyces
Lactobncillus
occi (7 strains)
(3 strains)
casei (1 strain)
Erycristagallin 53
6.25
3.13-6.25
1.56
6.25
Cristacarpin
100
50-100
25
100
Demethylerystagallin A 67
12.5-25
6.25-12.5
3.13
12.5
Erystagallin A
60
6.25-12.5
6.25-12.5
3.13
12.5
Eryvarin A
61
>100
>100
100->100
>100
Orientanol B
65
6.25-12.5
6.25-12.5
6.25
6.25
>100
6.25->100
1.56-6.25
>100
55
(3-O-methylcalocarpin) Sigmoidin K. 68
844 Table 17: Inhibition of Rabbit Platelet Aggregation (IC50 in (xg/ml) by Pterocarpans from E. abyssinica [9|.
Compound
Inhibition induced by ADP (10 uM
Collagen (38 ng/ml)
"
74 (slight inhibition)
74
Erythrabyssin II 56
23
9
34
Phaseollidin
58
30
23
34
Phaseollin
59
30
36
80
Arachidonic acid (0.57 u,M)
Erythrabyssin I 55 (cristacarpin)
Table 18: Bioactivity Data for Pterocarpans 55 and 58 Isolated from E. burana |44|
Compound
Organism (IC]2 in )xg/ml RS322YK
Cristacarpin 55
RS167N
RS321
Cell line (IC50 in jalVI) RS188N
P-388
CHOC
CHOC-
+
rad52
rad 6
rad 321
RAD
80
540
108
330
>10.0
>20.0
4.0
500
>1000
400
5500
>10.0
4.0
7.6
PGO
erythrabyssin 1 Phaseollidin 58
P-388= wild type P-388 murine leukaemia cells; CHOC = wild type Chinese hamster ovary cells; CHOC-PGO = Pglycoprotein overproducing Chinese hamster ovary cells. Table 19: In vitro Antiplasmodial Activity of Pterocarpans from Root Bark of E. abyssinica [46] Against Strains W2 and D6of Plasmodium falciparum.
IC50 in uM
Compound W2 (chloroquine-resistant) Erycristagallin
D6 (chloroquine-sensitive) 19.0
53
20.1
Erythrabyssin II 56
6.5
8.1
Eryvarin D
20.6
21.9
Chloroquine
0.093
0.008
Quinine
0.21
0.042
62
845
No
R1
R2
R3
R4
R5
49
H
OH
3,3-DMA
H
OH
50
H
OH
3,3-DMA
3,3-DMA
OH
H
Erybraedin C
51
H
OH
3,3-DMA
8,9(2,2 -dimethyl-
H
Erybraedin D
R' 3,3-DMA
Name Erybraedin A
chromeno) 52
2,3-furano
H
H
OH
3,3-DMA
Erybraedin E
54
3,3-DMA
OH
H
H
OCH3
3,3-DMA
Erycristin
56
3,3-DMA
OH
H
H
OH
3,3-DMA
Erythrabyssin II
57
H
OH
H
8,9(2,2-dimethyl
H
Isoneorautenol
3,3-DMA
Phaseollidin
chromeno) 58
H
OH
H
H
OH
59
H
OH
H
H
9,10(2,2dimethyl-
Phaseollin
chromeno) 64
3,3-DMA
OH
H
H
9,10(2,2dimethyl-
Folitenol
chromeno) 65
3,3-DMA
66
2,3(2,2-dimethylchromeno)
69
H
OCH3
H
H
OH
H
Orientanol B (3-O-
H
H
OH
3,3-DMA
Orientanol C
H
H
OCH3
3,3-DMA
Sandwicensin
methylcalopocarpin)
OH
846
No
R1
R!
R3
R4
R5
55
H
OH
H
H
OCH3
60
3,3-DMA
OH
H
H
OCH3
61
H
OCH3
H
H
67
3,3-DMA
OH
H
H
R« 3,3-DMA
Name Cristacarpin (Erythrabyssin I)
3,3-DMA a
OH
Erystagallin A Eryvarin A
3,3-DMA
Demethylerystagallin A 2-prenyl-6a-hydroxyphaseollidin
a =
o
R1
No
R1
R2
R3
R4
R5
53
3,3-DMA
OH
H
H
OH
62
H
OH
H
H
OCH3
R6 3,3-DMA 3,3-DMA
Name Erycristagallin Eryvarin D (3-hydroxy-9-methoxy-l 0pren ylpterocarpan)
63
3,3-DMA
OH
H
H
OCH3
3,3-DMA
Eryvarin E
68
3,3-DMA
OH
H
H
OH
3,3-DMA
Sigmoidin K
3. Bioactive Isoflavanones from Erythrina Species Eriotrichin B 70 (also called Bidwillon A) was found to be antibacterial against S. aureus with MIC50 of 8.3 ug/ml [20], while 2,3-dihydropratensein (5,7,3 -trihydroxy-4 methoxyisoflavanone) 71 showed antifungal activity against Cladosporium cucumerinum (2 jj.g) in a TLC bioautography preliminary assay [12]. Orientanol F 72 showed Anti-MRSA
847 against 13 strains with a range of 3.13-12.5 ug/ml, MIC50 of 6.25 ug/ml and MIC90 of 12.5 Hg/ml [26], while eryzerin A 76 showed sensitivity to 3 out of 13 strains (MIC50 = MIC90 =25 ug/ml) and eryzerin B 77 was inactive with all 13 tested strains resistant (MIC50 = MIC90 =>50 ug/ml) [28]. The organic extract from the roots of a Tanzanian strain E. lysistemon was found to be active in the NCI's primary anti-HIV screen [47], and the activity-guided isolation gave modestly active 5-deoxyglasperin F 73 (EC50 11.5 ug/ml) and 2,3- -dihydro-2hydroxyneobavaisoflavone 75 (EC50 7.6 ug/ml) and barely active 5-deoxylicoisoflavanone 74 together with three other inactive isoflavonoids. Table 20: Bioactive Isoflavanones from Erythrina Species
Compound
Erythrina source
Eviotrichin B 70
E. eriotriocha [20]
(Bidwillon A)
E. orientalis [105]
2,3-dihydropratensein 71
E. berteroana [12]
Orientanol F 72
Activity Antibacterial (S. aureus 8.3 |ag/ml)
Antifungal (Clndosporium cucumerinum 2
E. latissima [24]
Hg)
E. orientalis [105]
Anti-MRSA (MICso 6-25 fig/ml)
E. variegata [26] E. lysistemon [47 ]
Anti-HIV (IC5o 11.5 ug/ml)
flavanones 74
E. lysistemon [47]
Anti-HIV (weak)
2,3-dihydro-2 -hydroxy
E. lysistemon [47]
Anti-HIV (lC5o7.6|ig/ml)
5-deoxyglasperin F 73 5-deoxylicoiso-
-neobavaisoflavone 75 Eryzerin A 76
E. zeyheri [28]
Anti-MRSA (M1C5O 25 ug/ml)
Eryzerin B
E. zeyheri [28]
Anti-MRSA (M1C50>50 ug/ml)
77
848
No
R1
R2
R3
R4
70
H
3,3DMA
OH
3,3DMA
OH
71
OH
H
OH
H
H
72
H
3,3DMA
7,8(2,2-dimethylchromeno)
H
73
H
H
OH
H
74
H
H
OH
H
R5
R' H
R7
R8
Name
OH
H
Eriotrichin B
OH
OCH3
3,3DMA
2,3-dihydropratensein
OH
OH
H
Orientanol F
2,3 (2,2-dime
OH
H
5-deoxyglaperin F
3 ,4 (2,2-dime-
H
5-deoxylicoisoflavanone 2,3-dihydro-2-hydroxy
thylchromeno) OH
thylchromeno) 75
H
H
OH
H
OH
76
H
3,3DMA
OH
H
77
H
3,3DMA
OH
3,3DMA
3,3DMA
OH
H
OH
3,3DMA
OH
H
Eryzerin A
OCH3
H
OH
H
Eryzerin B
neobavaisoflavone
4. Bioactive Isoflavans from Erythrina Eight isoflavans have (i.e erythribidin A, phaseollinisoflavan, methoxyphaseollinisoflavan, 2 O-methylphaseollidinisoflavan, eryzerins D and E, eryvarin C, and 4 -O-methylglabridin), to date, been reported in Erythrina genus and only three viz eryzerin C 78, eryzerin D 79 (both isolated from E. zeyheri [28] ) and eryvarin C 80 (from E. variegata [26,98]) have been reported to have some anti-MRSA activity. Eryzerin C gave MIC50 and MIC 90 values of 6.26 ug/ml each, inhibiting all 13 strains tested, while eryzerin D was slightly less active with MIC50 and MIC90 values of 12.5 ng/ml each, again inhibiting all 13 strains tested.
OH R1
No
R1
R2
R3
Name
78
3,3-dimethylallyl
OH
3,3-dimethylallyl
Eryzerin C
79
6,7(2,2-dimethylchromeno)
3,3-dimethylallyl
Eryzerin D
80
6,7(2,2-dimethylchromeno)
H
Eryvarin C
849 5. Bioactive isoflav-3-enes from Erythrina Seven isoflav-3-enes viz eryepogin A [27,72] (burttinol B [64]), eryepogin B [72] (burttinol C [64]), burttinol A [64], 7,4 -dihydroxy-2,5 -dimethoxyisoflav-3-ene [46], bidwillol A [19,106], eryvarins H and I [30], have to date been reported in the genus, and of these only two have been reported to be biologically active. 7,4 -dihydroxy-2 ,5 -dimethoxyisoflav-3-ene 81 isolated from E. abyssinica [46] was reported to have antiplasmodial activity against chloroquine-resistant (W2: IC50 27.2 uM) and chloroquine-sensitive (D6: IC50 18.2 uM) strains of Plasmodium falciparum. Bidwillol A 82, reported from E. x bidwillii [19] and E. orientalis [106], was found to be weakly active against Lactobacillus fermentum (IC50 25 ug/ml) and Actinomyces naeslundii (IC50 25 ug/ml [19]. OMe
81: R1 = H, R2 = OCH3
7,4 -dihydroxy-2 ,5 -dimethoxyisoflav-3-ene
82: R' = 3,3-dimethylallyl, R2 = H
Bidwillol A
6. Bioactive Coumastans from Erythrina Five coumastans (4-hydroxycoumasterol [50], Indicamines A [95] and B [22], robustic acid [95] and sigmoidin K [26,104]) have been reported to date in the genus, and only two of these have been reported to have biological activity. Indicamine B 83 was isolated from the root bark of E. indica [22] and was found moderately active against S. aureus (MIC50 9.7 (ig/ml, streptomycin 5.5 ug/ml) and M. smegmatis (18.5 ug/ml, streptomycin 1.7 ug/ml) but inactive against E. coli (>1000 ug/ml, streptomycin 5.0 p.g/ml). Sigmoidin K 68 has been discussed under bioactive pterocarpans. ^0. 6
1^ OMe
l
OH
J3 \ ^
L "OH
83: Indicamine B
VII.
BIOACTIVE CINNAMATE ESTERS FROM ERYTHRINA
Cinnamate esters are the only non-flavonoid (word used in a very general sense) and nonalkaloidal, bioactive, identifiable secondary metabolites reported in the genus. Cinnamate esters have been reported in several Erythrina species [24,48,56,90,107-108]. Several cinnamate esters have been reported in the genus and these include erythrinasinate (octacosanyl E isoferulate), erythrinasinate B (octacosanyl E ferulate), erythrinasinate C (tetradecanyl E ferulate), erythrinasinate D (hexacosanyl E isoferulate), hexacosanyl E
850 ferulate, and triacontanyl 4-hydroxycinnamate. The three esters 84-86, isolated from E. senegalensis and E. excelsa, were thoroughly screened for pharmacological activity [107], involving over fifty tests. The two esters, erythrinasinate B 84 and erythrinasinate 86 were isolated from E. senegalensis [21,108] while 85 hexacosanyl E ferulate was reported from E. excelsa [107]. The three esters exhibited activity that could be broadly described as having CNS, cardiovascular and metabolic. Compound 84 was completely non toxic when administered orally or peritoneally. Its CNS activity was manifested by reflex depression, behavioural depression, muscle relaxant and antielectric shock properties. It showed antiarrhythmic effects (cardiovascular agent) as well as aquaretic properties (metabolic agent). Compound 85 was non toxic when administered orally or peritoneally. It exhibited reflex depression, behavioural depression, muscle relaxation, cholinergic activation, antiarrhythmic and aquaretic properties. Compound 86 was completely non toxic regardless of whether it was administered orally or peritoneally and it showed reflex depression, behavioural depression, muscle relaxant, cholinergic activation, anti-electric shock, antiarrhythmic and aquaretic properties.
O
No
R1
R2
R3
Name
84
H
OCHj
-(CH 2 ) 27 CH 3
Erythrinasinate B
85
H
OCH 3
-(CH 2 ) 25 CH 3
Hexacosanyl E-ferulate
86
CH3
OH
-(CH 2 ) 27 CH 3
Erythrinasinate
VIII.
CONCLUSION
The survey revealed that the genus Erythrina was very rich in secondary metabolites particularly of the flavonoids class. The bioactivity profile represented the various classes fairly reasonably but it became apparent that a number of these isolates have not yet been tested for biological activity. The challenge remains for researchers to carry more work on these to fill in the knowledge gaps that still exist. The survey revealed very close agreement between ethno-medical use of the various Erythrina extract preparations (Table 1) and the results of biological activity (Table 2). The reported activities of pure isolates strongly support the documented ethno-medical uses and reported pharmacological activity on the various extracts which are, by and large, mostly microbial related. It was also interesting to note that some compounds showed high efficacy against resistant organisms - a very important aspect - since most used drugs especially antibiotics tend to produce resistance to certain strains of organisms. It also emerged from the survey that certain structural features were essential for certain activities. Compounds that were found active were usually effective against not just one but several disease functions. One cannot help but surmise that more activities are yet to be reported for these isolates.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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CHEMICAL CONSTITUENTS AND PHARMACOLOGY OF ARISTOLOCHIA SPECIES TIAN-SHUNG WU*, AMOORU G. DAMU, CHUNG-REN SU, AND PING-CHUNG KUO Department of Chemistry, National Cheng Kung University, Tainan, Taiwan; Tel: 886-6-2747538. Fax: 886-6-2740552 E-mail: [email protected] ABSTRACT: Aristolochia species have long been known for their wide use in traditional medicine and have attracted intense research interest because of their numerous biological activity reports and unique constituents, aristolochic acids. Aristolochia species are sources of a number of physiologically active compounds of different classes. Aristolochic acid derivatives with various carbon skeletons, aporphines, benzyliso- quinolines, isoquinolines, protoberberines, protopines, amides, chlorophylls, mono-, sesqui-, and diterpenoids, lignans, biphenyl ethers, flavonoids, tetralones, benzenoids, and steroids have been identified from different Aristolochia species. The major focus of recent research is on the negative aspects of aristolochic acids because of the nephro- toxicity of some aristolochic acids. This report addresses the complex array of the biologically active and chemically diverse metabolites identified from Aristolochia species during the past seventy years, in addition to biosynthetic studies, ecological adaptation and chemotaxonomy to show the rapid development in the phytochemistry and pharmacology of the Aristolochia species.
INTRODUCTION The family Aristolochiaceae with seven genera: Apama, Aristolochia, Asarum, Euglypha, Holostylis, Saruma and Thottea is nested with in the magnoliid (or eumagnoliid) clade of primitive angiosperms, which include Piparales, Magnolidales, Laurales, Wintarales, Chloranthaceae and monocotyledons.There are about 600 to 700 species, most of them in the genus Aristolochia (ca. 500 species) and most of the rest in Asarum (ca. 100 species). The majority of the Aristolochiales are tropical, though a number of them range as far north as Canada, Scandinavia, and Northern Japan. They may grow as climbing vines, as short creeping herbs and a few are shrub-like [1-3]. Aristolochia species are herbaceous perennials, under shrubs or shrubs, often scandent, scrambling, twining, sometimes lianas, usually with prostrate or tuberous rhizomes or rootstocks, and alternate, pinnate, polymorphic or lobed leaves bearing essential oils. The
856 flowers are axillary, bilaterally symmetric, tubular and calyx usually mixture of purple, brown, green or red with fetid odors. Fruits are dry capsules with flattened or rounded seeds. Species of Aristolochia were widely distributed in tropical, subtropical and temperate regions of the world. They are known to occur in Asia, Africa, North and South America and Australia but there is a wide distribution across tropical Asia. Members of Aristolochia have been cultivated in gardens as ornamentals due to their attractive leaves and flowers, often with bizarre colors or patterns [4-7]. Some oddly shaped flowers have been given names like "bird's head" and "Dutchman's pipe". Many species of Aristolochia have been used in the folk and traditional medicines as medicaments and tonics (Table 1) [8-14]. Especially, the use of Aristolochia species in Chinese popular medicine has a long tradition [15-19]. Some species have been used in the form of crude drugs as anodynes, antiphlogistics and detoxicants in Mainland China. The mature fruits of A. debilis, known in China by the name, Madouling are still used for the treatment of snakebite, tuberculosis and as antihypertension [20]. The dry roots of A. indica are reputedly used in Indian folk medicine as an emmenagogue or abortifacient [21-24]. A. albida, native of West Africa has been used in traditional medicine for various ailments including skin diseases, dysentery, gastrointestmalcolics and snakebites [25-27]. The roots of A. argentina, popularly known as Charrua or Charruga are used in the Argentinean folk medicine [28,29] as an emmenagogue and for treating arthritis, poisoning and pruritus. A. bracteata, an Indian medicinal plant [30] is reputed for its efficacy as anthelmintic, purgative, emmenagogue, and for expelling round worms. The rhizomes of A. brevipes, commonly known as guaco are used by the local Tarasc people [31] to treat arthritis and diarrhea and also applied to cure wounds from snakebites. A. chilensis is known by the vernacular names ' Oreja de zorro' (fox ear) and hierba de lavirgen maria (virgin mary's herb) and a decoction of its roots [32,33] was drunk at least well in to the second half of last century to reduce abundant lochia (puerperal secretions). The roots of A. cinnabarina are used as painkiller in Chinese folk medicine [34]. The aerial parts of A. constricta, a medicinal plant forward in Equador and South America are empirically used in folk medicine [35-37] as antispasmodic, anthelmintic, emmenagogue and against snakebites. The aerial parts of A. grandiflora are said to have antimicrobial, uterotonic, and cytotoxic properties and also used for treating snakebites [38,39]. The fruits and roots of A. zollingeriana, an endemic species [15] of Taiwan have been used as an alternative for the famous Chinese medicine 'Madouling', as an analgesic,
857
Table 1. Traditional/folklore medicinal uses of Aristolochia species Botanical name A. acutifdia
Trivial name Jiquiro, Jarrinha
A. albida
A. argentina
Charrua, Charruga
A. baetica A. birostris
A. bracleala
A. brevipes
A. chamissonis A. chilensis
Part used whole plant
Papular use treatment of erysipelas
References 47
rizhomes
ailment for skin diseases; treatment of dysentery, gastrointestinal colics and snakebites; as an adjuvant as emmenagogue, antiseptic, diaphoretic and diuretic; treatment of arthritis, poisoning, and pruritus as emmenagogue; in cancer in cancer
25,26,27
as emmenagogue, anthelmintic, purgative, mosquitoes repellent, antidote, anodyne and insecticide treatment of arthritis, wounds, snakebites and toothache in debility treatment of abundant lochia (Puerperal secretions); as emmenagogue as analgesic and pain killer treatment of cancer, menstrual troubles, legulcer, wound and tumor; as depurative and insect repellent in cancer
30
aerial parts
roots Capivara, Angilico, Jarrinha, Ukulwe, Bracteated birthwort
Guaco
Oreja de zorro, Hierba de la virgin maria
roots
whole plant
rhizomes
whole plant roots
Sichuan Zhusalian Upright birthwort
roots
roots
A. constricia
Pipevine, Ma dou ling, Tian xian teng Saragez
A. cucurbitifolia
Qing muxiang
A. cinnabarina A. clematits
A. contorta
roots
aerial parts
fruits, roots
as antispasmodic, analgesic, anticancer, antimalarial and antiinflammatory; as emmenagogue; treatment of snakebites as anodynes, antiphlogistics, expectorant, antitussive
28,29
48,49 50,51
31,52 53
32,33
34 54
55
35,36,37
56
858
A. debilis
A. gibertii
Ma dou ling, Qing muxiang, Seimokkou, Birthwort long, Chinese fairly vine, Birthwort Mil hombres hembra, Contrayerba, Patito
roots
and antiasthmatic; treatment of snakebites and lung inflammation as bronchiectatic; to decrease high blood pressure
20
whole plant
treatment of swelling; in stomachpain; as ailments
57
A. gigantea
whole plant
58,59
A. grandiflora
aerial parts
as emmenagogue, abortive and antiseptic; treatment of wounds and skin diseases as uterotonic, cytotoxic and antimicrobial; treatment of snakebites; as flies and maggots repellent as expectorant, antitussive, antiasthmatic and analgesic; treatment of snakebites as emmenagogue, cardiotonic, diuretic, antiinflammatory, abotifacient and mild sedative; against intestinal worms poisonous bites and stings treatment of asthma, cough and piles; as antivenomous, antibacterial, antipruritic, hypotensive, expectorant, and emetic as expectorant and antitussive, analgesic and antiasthmatic as antiphlogistic, detoxicant and anodyne treatment of cancer, sclerosis, uterus tumor and nose cancer treatment of snakebites and rheumatism; as circulatory stimulant, antiinflammatory, antiseptic and abortive as bronchiectatic;
A. helerophylla
Yellowmouth, Dutchman's pipe
fruits, roots
A. indica
Indian birthwort
roots
A. kaempferi
Yellowmouth, Dutchman's pipe
whole plant
A. kankauensis
fruits, roots
A. liukiuensis
roots
A. longa
Tian xian teng
whole plant
A. macroura
Mil hombers, Patito Coludo, Jarrinha, Isipomilhombres
whole plant
A. manshuriensis
Manchurian
whole plant
15
15
21,22,23,24
60
15
61 62,63
64
65,66,67,68,
859
A. maurorum
birthwort, Manchurian Duchman's pipe, Guan mu tong, K.an-mokutsu, Mokuboi, Kwangbanggi Zarand
A. mollissima
Xun gu feng
fruits, roots
A. paucinervis
Barraztam
whole plant
roots
whole plant
A. rotunda A. rodriguesii
Sangue-de-Cristo
roots, aerial parts
A. triangularis
Mil hombres
bark
roots
A. tuberosa
A. yunnanensis
Yunnan ma douling, Nan mu xiang
roots
A. zollingerinnn
Ma douling
fruits, roots
to decrease high blood pressure
69,70
as antiseptic; in wound healing; for scab of sheep as analgesic, anticancer, antimalarial, antiinfiammatory, antirheumatic and bronchiectatic; to decrease high blood pressure; treatment of stomach ache and abdominal pain treatment of skin infections, gas gangrene, abdominal pain and infections of the upper respiratory tract in cancer; as depurative as abortifacient and antiinfiammatory; treatment of snakebites as antirheumatic, antifertility, diaphoretic, diuretic, antiseptic, emmenagogue, antidote, and abortive; treatment of wounds and skin diseases treatment of sore throat, venomous snakebites, and tuberculosis treatment of gastrointestinal diseases, tricomoniasis, and various pains as expectorant, painkiller, antitussive, antiasthmatic, and analgesic; Treatment of snakebites
71,72
40
73, 74, 75
76 77
41,42,43,44
78
45,46
79
expectorant, antitussive, antiasthmatic and also for the treatment of snakebite and lung inflammation. The roots and fruits of A. mollissima [40] ("Xun Gu Feng" in Chinese) are employed as analgesic, anticancer, antimalarial and anti-inflammatory agents and also for the treatment of
860 stomach ache, abdominal pain and rheumatism. A. triangularis, a medicinal plant [41-44] found in South America was used in the treatment of wounds and skin diseases, as emmenagogue, antidote, abortive, antirheumatic, antiseptic and also as tonic agent by local people. A. yunnanensis [45,46] (Yunnan Ma Do Ling) is a Chinese crude drug, Nan Mu Xiang recommended for gastrointestinal diseases, trichomoniasis, and various pain conditions in traditional medicine. ETHNOPHARMACOLOGY Several bioactivity studies have been reported to assess the traditional uses of Aristolochia species. Table 2 summarizes the biological activities of the members of Aristolochia. Lee et al. reported that the extract of A. debilis [80] showed potent inhibition of COX-2 activity and iNOS activity in lipopolysaccharide (LPs)- induced mouse macrophages RAW 264.7 cells. Extracts of the whole plant of A. grandiflora [81] showed moderate neutralization ability against the heamorrhagic effect of Bothrops atrox venom. Some of Aristolochia species have been reported to possess insecticidal and repellent activity. For example, A. clematitis used as insect repellent, A. grandiflora used against flies and maggots [82] and A. bracteata extracts showed clear activity against mosquitoes [83]. The essential oil of A. indica [84] found to show antibacterial activity. An extract of A. indica showed reproducible tumor inhibitory activity [85] against the adenocarcinoma 755 test system. The crude petroleum ether, chloroform, and alcoholic extracts of the roots of A. indica [89-92] were found to exert 100 % abortifacient activity in mature female mice. Methanolic extract of A. macroura [93] showed cytotoxicity against a human hepatocelluar carcinoma cell line, HepG2. The deffated chloroform fraction obtained from the rhizomes of A. paucinervis [94] has a high bacteriostatic activity against bacterial strains like Clostridium perfringens ATCC13124 and Enterococcus faecalis ATCC 29212. Another study indicated that the defatted chloroformic rhizome fraction of A. paucinervis [95] was most active against Clostridium perfringens, Clostridium difficile, Enterococcus faecalis, Micrococcus lutens and Bacillus subtilis. The defatted chloroform fraction of leaves of this plant also possessed a high bacteriostatic activity against both anaerobic and aerobic strains. Results of this study support the use of A. paucinervis in Moroccan traditional medicine to treat skin and soft-tissue infections, especially gas gangreen and intestinal diseases. Bio-screening study reported by Coussio et al. [96] indicated that A. triangularis contained
861
Table 2. Ethnopharmacological reports on Aristolochia species Botanical name A. albida
Plant material rhizomes
A. argentina
aerial parts
A. bracteata
seeds
A. bracteolate A. constricta A. debilis
whole plant whole plant whole plant
A. elegans A.fructus A. grandiflora A. indica
A. macroura A. manshuriensis
A. mollissima A. (iff. orbicularis A. papillaris A. paucinervis
A. taliscana A. triangularis
A. trilobata
whole plant
Bioactivity insecticidal antifeedant an ti spasmodic insecticidal, antibacterial, antifungal antiinflammatory, antibacterial, analgesic; toxicity to goat antiplasmodial anti spasmodic inhibition of testosterone-5ccreductase; inhibition of COX-2 activity, inhibition of iNOs activity; inhibition of lipid peroxide formation; inhibition of melanin formation
antimitotic; antiviral whole plant leukotriene B4 receptor antagonist activity whole plant neutralizing ability against the haemorrhagic effect aerial parts antibacterial; roots tumor inhibitory; abortifacient activity; interceptive activity; antispermatogenic effect cytotoxicity leaves cell suspension culture cardiotonic, whole plant antihypoxic activity; synergistic effect for pesticides; inhibition mutagenicity of Trp-P-1 whole plant antirheumatic arthritis; conctraceptive roots repellent effect against the corn borer Sitophilus zeamais smooth muscle relaxant activity whole plant bacteriostatic, bactericidal; rhizomes, leaves antibacterial; smti-Helicobacter pyroli acitivity; antidermatophytic, antifungal trypanocidal roots cytotoxicity, whole plant inhibition of growth of crown gall tumors; typical c-mitotic action antibacterial; leaves, bark antiphlogistic, antiinflammatory
Ref. 100 99 101 102
104
105 106 107 108 80
109, 110 111 112 113 81 84 85 86,92 89,91 90 93 114 115 116 117 118 119 120 94 95 95 121 122 96
123 97 98
862 cytotoxicity against KB cells and inhibited the growth of crown gall tumors. The hexane extracts of A. trilobata leaves [97] and bark were most active against Staphylococcus aureus and validated its traditional use to heal the deep and surface wounds. The chloroform extract of A. trilobata leaves [98] showed anti-inflammatory effect with an ID50 value 108 )ug/cm2 comparable to that of indomethacin (93 ug /cm2). The phytochemistry and pharmacology of Aristolochia has evoked a great deal of interest because of their numerous traditional uses and number of enthopharmacological reports on their crude extracts. Consequently, Aristolochia has become one of the most intensely investigated genera and a large number of papers have been published on the production of physiologically important metabolites by Aristolochia. Thus the primary aim of this chapter is to bring up-to-date, in graphical form, those data with the metabolites of Aristolochia which have appeared in the literature up to June 2004, concerning the isolation, structural elucidation, biological activity and literature references. CHEMICAL CONSTITUENTS Over the last seventy years over sixty species of Aristolochia have been exploited for chemical examination by research groups throughout the world and a variety of compounds have been isolated. The spectrum of physiologically-active metabolites from Aristolochia species covers 14 major groups based on structure: aristolochic acid derivatives, aporphines, amides, benzylisoquinolines, isoquinolones, chlorophylls, terpenoids, lignans, biphenyl ethers, flavonoids, tetralones, benzenoids, steroids, and miscellaneous. The aristolochic acid derivatives, host of phenanthrene derived metabolites were further classified into aristolochic acids, sodium salts of aristolochic acids, aristolochic acid alkyl esters, sesqui- and diterpenoid esters of aristolochic acids, aristolactams, denitroaristolochic acids, and aristolactones. The terpenoids can further be subdivided into 4 groups: mono-, sesqui-, di- and tetraterpenoids. Aristolochic acids
The constituents of the Aristolochia have became the active subject of a phytochemical and pharmaceutical work since the discovery of compounds belonging to the aristolochic acids group. The naturally occurring aristolochic acids, with 3,4-methylenedioxy-10-nitrophenanthrenic-1-acid skeleton are typical constituents of the Aristolochia, as well
863 as the butterflies that feed on such plants and are claimed to be responsible for the biological activity of Aristolochia species. The first paper on the aristolochic acids from the genus Aristolochia which appeared in 1943 by Rosenmund and Reichstein concerned A. clematitis, a plant used in China and Taiwan for the treatment of cancer, menstrual troubles, leg ulcer and tumors [124]. It was named aristolochic acid A by Tomita and Sasagawa and its structure was elucidated by Pailer et al. in 1956 by means of chemical reactions [125,126]. Table 3 lists the sixteen aristolochic acids that have been isolated so far from Aristolochia. Aristolochic acid I (5) is the abundant aristolochic acid found in almost all species of Aristolochia studied with few exceptions and has been reported to be the major constituent responsible for the high antitumor activity of Aristolochia samples [99,127-131]. Since its first isolation in 1943, the aristolochic acid I (5) is without a doubt the most extensively studied of the hundreds of known secondary Aristolochia metabolites due to its potent antitumor activity and have recently attracted intense research interest because of their nephrotoxicity. The major focus of much recent research has been on the negative aspects of these compounds because of the Chinese Herb Nephropathy [224]. Recently health food supplements containing aristolochic acid have been prohibited for use in weight reduction, with full scientific support [225, 226].The aristolochic acid II (1) is a simple and second abundant aristolochic acid derivative devoid of oxygenation in ring-C [227]. A general structural feature of these aristolochic acids which exhibited simple substitutions by hydroxyl, methoxyl, ethoxyl and/or methylenedioxy groups is that oxygenated substituents are usually present at the C-6 or C-8 position of the lower aromatic ring. When two substituents were present, both C-6 and C-8 were substituted. However, aristolochic acids V (13) [130,230] and Va (9), and Vila (11), E (12) and 7-methoxy aristolochic acid I (15) have been reported to be oxygenated at C-6 & 7 and C-7 & 8, respectively. Aristoloside (16) [65] and aristolochic acid IIIa-6-0glucoside (7) are the only two glucosylated aristolochic acids in nature. Aristoloside (16) was first isolated from A. manshuriensis and was then also found in A argentina [130], A cinnabarina [142,143] and A elegans [168]. Peter and Muzaffer reported 9-methoxy aristolochic acid II (6), as only aristolochic acid with oxygenation at C-9 from A. ponticum [207]. Aristolochic acid Via (8) found in A. argentina is the only example to be oxygenated at C-2 position [130]. Tseng and Ku isolated an unusual aristolochic acid namely debilic acid (17) with a nitrophenanthrenyl acetic
864 acid skeleton from A. debilis [242,243]. Later it was also found in the A. mashuriensis [193] and A. tubflora [220]. Table 3. Aristolochic Acids Isolated from Aristolochia Species Compound
Name
Source
Ref.
A. argentine! A. auricularia A. baetica A. cinnabarina A. clematitis
127,130 132 48,49 142, 143 127, 144, 146, 147, 149, 153, 227 157 158 127, 163, 166 127 228 229 62, 127 127, 193, 195,241 144 203 206 207 127,211 127 220 223
0
c -V
OH NO2
R5 R
R4
2 R,
Ri
H
R2 H
H
R. H
R5 H
Aristolochic acid 11(1)
A. contorta A. cucurbitifolia A. debilis A. esperanzae A. heterophylla A. kaempferi A. longa A. manshuriensis A. mollissima A. moupinensis A. pallida A. ponticum A. rotunda A. sipho A. tubflora A. zollingeriana
H
OH
H
H
H
Aristolochic acid Ilia (Aristolochic acid C) (2)
A. argentina A. cinnabarina A. clematitis A. contorta A. cucurbitifolia A. debilis
130 142, 143 146 230 159 127, 162,
865 A. fangchi A. heterophylla A. indica A. kaempferi A. longa A. manshuriensis A. rotunda A. tagala A. tuberosa A. tubflora A. watsonii A. zollingeriana
H
H
H
OH
H
Aristolochic acid la (3)
H
OCH3
H
H
H
Aristolochic acid III (4)
H
H
H
OCH3
H
Aristolochic acid I (Aristolochic acid A; Aristolochic acid) (5)
166 127, 172, 230 228, 232 180 223,233 62 195 127,211 216 218,219 220 127 223 129,130 A. argentina 127, 140 A. chilensis 158 A. cucurbitifolia 231 A. fangchi 127, 130 A. argentina 132 A. auricularia 52 A. brevipes 127,146 A. clemalitis 163, 166, A. debilis 234 A. esperanzae 127 A. longa 62 A. versicolar 222 A. zollingeriana 223 A. acuminata 127, 128 A. albida 99 A. argentina 127, 129, 130, 131 A. auricularia 132 A. austrozechuanica 133 A. badamae 127 A. baecita 48, 49, A. bracteata 127, 134 A. brevipes 127,135A. championii 137 A. chilensis 52 A. cinnabarina 138, 139 A. clematitis 127, 140 A. contorta 141-143 A. cucurbitifolia 127,144A. debilis 153 A. elegans 154-157 A. esperanzae 56, 158, A. fangchi 159 A.fimbriata 127,160A. foveolata 166 A. griffithii 127, 167A. heterophylla 171 A. indica 127 127, 172, 173 A. kaempferi
866
A. ponticum
127 174 175 176,177 92, 127, 178-185 61, 127, 156,158, 164, 186 187 127, 188190 62, 127, 183,191, 192 65, 127, 193-196, 241 127, 197 127 127, 198204 176,205 127,128 127 206 127 207 208 127,209 210 127,211, 212 127, 183 127,213215 216 217 218,219 220 221,222 127 223 207
A. baetica A. cinnabarina
49 142,235
A. argentina
130
A. argentina A. debilis A. fangchi
130 127, 165 127, 172
A. kunmingensis A. kwnngsiensis A. longa A. manshuriensis A. maurorum A. maxima A. mollissima A. moupinensis A. multiflora A. ornithocephala A. pallida A. pandurata A. ponticum A. pubescens A. reticulata A. rodriguesii A. rotunda A. serpentaria A. sipho A. tagala A. triangularis A. luberosa A. tubflora A. versicolar A. westlandii A. zollingeriana
H
H
H
H
OCH3
H
Ogle
H
H
H
OH
H
H
OCH3
H
H
OH
OCH3
H
H
9Methoxyaristol ochic acid-II (6) Aristolochic acid-IIIa-6-OP-glucoside (7) Aristolochic acid-Vla (8) Aristolochi acid-Va (Aristolochic
867
H
OH
H
OCH3
H
acid-B) (9) Aristolochic acid IVa (Aristolochic acid D) (10)
A. acuminata A. albida A. argenlina A. clematitis A. cucurbitifolia A. debilis A. elegans A. esperanzae A. helerophylla A. indica A. kaempferi A. longa A. manshuriensis A. mollissima A. moupinensis A. multiflora A. rigida A. triangularis A. zollingeriana A. argenlina A. contorta A. cucurbitifolia A. debilis A. foveolata', A. heterophylla A. longa A. pubescens A. tagala A. tubflora
H
H
OH
OCH3
H
Aristolochic acid Vila (11)
H
H
OCH,
OH
H
H
OCH3
OCH3
H
H
H
OCH3
H
OCH,
H
Aristolochic acid-E(12) Aristolochic acid-V (13) Aristolochic acid IV (14)
A. contorta A. debilis A. argentina A. pallida A. argentina A. auricularia A. clematitis A. cucurbitifolia; A. debilis A. esperanzae A. kwangsiensis A. longa A. manshuriensis A. moupinensis A. pallida A. rigida A. versicolar A. zollingeriana
H
H
OCH3
OCH3
H
7-
A. contorta
127,128 99 127 127, 146 56, 158 127,166 168, 169 127 92, 177, 178,179 92, 127, 178, 179 229,233 62 65, 127, 194-195 199,202204,237 205 127, 128 238 239 223 130 230 56 127, 162, 163 174 232 62 208 216 220 230 166 130 206 127,130 132 127,146 56, 158 163 127 190,240 62 127, 194, 196 205 206 238 222 223 230
868
H
Ogle
H
c
OCH3
H
Methoxyaristol ochic acid-1 (15) Aristoloside (16)
Debilic acid (17) N0 2
A. debilis A. longa A. argentine! A. cinnabarina A. elegans A. manshuriensis A. debilis A. manshuriensis A. tubflora
127, 162, 163 62 130 142,143 168 65, 127, 194,241 127,242, 243 127,193 220
cr
[
"OCH,
Sodium Salts of Aristolochic Acids It is interesting to note that Formosan Aristolochia species including A. cucurbitifolia, A. foveolata, A. heterophylla, A. kaempferi and A. zollingeriana are the only species [159,228,244-246] from which sodium salts of aristolochic acids were isolated. However, very recently Lopes and co workers have also isolated sodium aristolochate I (21), II (18), Ilia (19) and IVa (22) from Brazilian species, A. pubescens [208]. Isolates of this class from various Aristolochia species were listed in Table 4. Aristolochic Acid Alkyl Esters Table 5 summarizes the occurrence of fourteen alkyl esters of aristolochic acids in Aristolochia species. It is noted that all the reported aristolochic acid alkyl esters are methyl esters except 32 which is an ethyl ester of aristolochic acid la with an ethoxy group at C-8 [129]. Compounds 25-31 are methyl esters of reported aristolochic acids whereas compounds 33-38 are methyl esters of non reported aristolochic acids. Among alkyl esters of aristolochic acids reported so far aristolochic acid I methylester (28) and aristolochic acid IV methylester (30) are frequently encountered in Aristolochia species. Formosan Aristolochia species are known to be rich source for alkyl esters of aristolochic acids, particularly A. zollingeriana [171,223] and A cucurbitifolia [56,159]. Sesqui- and Diterpenoid Esters of Aristolochic Acids Table 6 shows the eleven sesquiterpene esters of aristolochic acids isolated from Aristolochia species. Isolation of the aristoloterpenate I (40) from the radix of A. mollissima is the first report of such compounds in
869 Table 4. Sodium Salts of Aristolochic Acids Isolated from Aristolochia Species Compound
Name
Source
Ref.
A. cucurbitifolia A.foveolata A. heterophylla A. kaempferi A. pubescens A. zollingeriana A. foveolata A. heterophylla A. kaempferi A. pubescens A. zollingeriana A. cucurbitifolia A. cucurbitifolia A. foveolata A. heterophylla A. kaempferi A. manshuriensis A. pubescens A. zollingeriana
159 174 228 233, 244 208 245 174 228,232 244 208 245 159 56, 159 174,246 228,232 229,233, 244 241 208 245 159 174 228 244 208 245 56 174 228
0
cXT
•^^I^^O-Na*
i
R. R?
R, H
R, H
R3 H
Sodium aristolochate II (18)
OH
H
H
Sodium aristolochate Ilia (19)
OCH3 H
H H
H OCH3
Sodium aristolochate III (20) Sodium aristolochate I (21)
OH
H
OCH3
Sodium aristolochate IVa (22)
H
OH
OCH3
H
OCH3
OCH3
Sodium aristolochate Vila (23) Sodium aristolochate VII (24)
A. cucurbitifolia A. foveolata A. heterophylla A. kaempferi A. pubescens A. zollingeriana A. cucurbitifolia A. foveolata A. heterophylla
which the carboxylic acid group of aristolochic acid forms ester linkage with a sesqui- or diterpene [204,250]. It is interesting to note that, among eleven compounds of this type from Aristolochia species, we discovered eight sesquiterpene esters of aristolochic acids from the roots and stems of
870
Table 5. Aristolochic Acid Alkyl Esters Isolated from Aristolochia Species Compound
Name
c Ri
Source
Ref.
0 II OR NO2
A R?
R CH3
R, H
R2 H
R3 H
CH3
H
H
OH
CH3
OCHj
H
H
CH3
H
H
OCH3
CH3
OH
H
OCH3
Aristolochic acid IVa methyl ester (29)
A. elegans A. indica A. kwangsiensis A. manshuriensis A. moupinensis A. versicolar
CH3
OCH3
H
OCH3
Aristolochic acid IV methyl ester (30)
A. brevipes A. championii A. cucurbitifolia A. elegans
Aristolochic acid 11 methyl ester (25)
Aristolochic acid la methyl ester (26) Aristolochic acid III methyl ester (27) Aristolochic acid I methyl ester (28)
A. argentina A. cucurbitifolia A. heterophylla A. longa A. manshuriensis A. zollingeriana A. kaempferi
129 159 228 62, 192 241 223 244
A. cucurbitifolia A. kaempferi A. argentina A. cucurbitifolia A. debilis A. elegans A. heterophylla A. indica A. kaempferi A. longa A. manshuriensis A. mollissima A. sipho A. versicolar
159 244 129 159 163, 165 168 228 127, 179 244 62, 192 65,241 203 209, 215 221 168 127, 178 247 241 205 222 52 138, 248 56, 159
871 A. kaempferi A. kwangsiensis A. longa A. manshuriensis A. moupinensis A. versicolar A. zollingeriana
CH3 CH2CH3
H
OCH3
OCH3
H
H
OCH2CH3
Aristolochic acid VII methyl ester (31) Aristolochic acid-la ethyl ester ethyl ether (32) O
A. cucurbitifolia A. argentina
168 244 127, 188, 190, 240 62, 192 65 205 221, 222 223 56 129
OCH3 R2O^~
.NO 2
5c
)
R4
CH3
R3 H
R4 H
CH3
CH3
H
H
H CH3 H CH3
CH3 CH3 CH3 CH3
OH OH H H
H H OCH3 OCH3
Ri
R2
H
Aristolochic acid All methyl ester (33)
A. auricularia A. kaempferi A. zollingeriana
Ariskanin-A (Aristolochic acid BII methyl ester) (34) Ariskanin-B (35) Anskanin-C (36) Ariskanin-D (37) Ariskanin-E (38)
A. manshuriensis A. zollingeriana A. A. A. A.
zollingeriana zollingeriana zollingeriana zollingeriana
132 61, 158, 244 171, 223 195 223 223 223 223 223
A. heterophylla [249,251] and a diterpene ester of aristolochic acid from the roots and stems of A. elegans [252] and thus, A. heterophylla is considered to be a rich source of sesquiterpene esters of aristolochic acids. Aristoloterpenate I (40) and III (42) are esters of aristolochic acid I (5) with the sesquiterpenes, madolin M (305) and L (306), respectively, whereas aristoloterpenate II (39) and IV (41) are esters of aristolochic acid II (1) [249]. In these four esters C-ll of the aristolochic acid and C-4' of sesquiterpene were involved in the ester linkage. The stereochemistry at
872 Table 6. Sesqui- and Diterpenoid Esters of Aristolochic Acids Isolated from Aristolochia Species Compound Aristoloterpenate II (39) Aristoloterpenate I (40)
Aristoloterpenate IV (41) Aristoloterpenate III (42)
Aristophyllide C (43) Aristophyllide A (44) Aristophyllide D (45) Aristophyllide B (46) Aristolin (47) Aristoloin I (48) Aristoloin II (49)
Occurrence A. heterophylla A. heterophylla A. kaempferi A. mollissima A. heterophylla A. cucurbitifolia, A. heterophylla; A. kaempferi A. mollissima A. heterophylla A. heterophylla A. mollissima A. heterophylla A. heterophylla A. elegans A. pubescens A. pubescens A. pubescens
Ref. 228,249 228, 249 244 204,250 228,249 159 228, 249 244 204 228,251 228,251 204 228,251 228,251 252 208 208 208
C-4' of aristoloterpenate I-IV (39-42) was determined as R by circular dichroic studies. Aristophyllide A (44) and B (46) are aristolochic acid I (5) esters of the rearranged e«/-elemane type sesquiterpenes, aristophyllene and its stereoisomer, respectively [251]. Aristophyllide C (43) and D (45) are aristolochic acid II (1) esters of aristophyllene and its stereoisomer, respectively [251]. In these esters aristolochic acid and sesquiterpene were bound by an ester linkage between C-ll and C-12'. The absolute configuration of C-5' and C-12' of these four esters were determined by application of the circular dichroic exciton chirality method. Aristophyllide A (44) and C (43) have R and S configurations at C-5' and 12' whereas aristophyllide B (46) and D (45) possessed opposite stereochemistry S and R at C-5' and 12' centers. Aristolin (47) is the first example of an ester composed of aristolochic acid and a diterpenoid, in which C-16 hydroxy group of e«f-kauran-16(3, 17-diol involves in the ester linkage with C-ll carboxylic acid group of aristolochic acid [252]. Very recently, Isabele et al have reported the isolation of two more diterpene esters of aristolochic acids, aristoloin I (48) and II (49) together with aristolin (47) from the tubercula of A. pubescens [208]. In aristoloin I (48) and II (49), aristolochic acid I (5) and II (1) were bound to a kaurane diterpene, 16a,17-en?-kauranediol by an ester linkage through C-ll and C-17' centers. Thus, from these reports it appears that a general theme running through the chemistry of Aristolochia is the ability to form esters
873 .CHj
R 43 H 44OCH,
R 45 H 46 OCH3
Fig. (1). The Structures of Sesqui- and Diterpenoid Esters of Aristolochic Acids
of aristolochic acids with a number of different types of secondary metabolites. Denitroaristolochic Acids The twelve denitroaristolochic acids isolated from Aristolochia species are presented in Table 7 and endemic Formosan Aristolochia species, A. cucurbitifolia was noted for profuse production of denitroaristolochic acids [56,159,254]. Research work by our group revealed that majority of
874 Table 7. Denitroaristolochic Acids Isolated from Aristolochia Species Compound
Name
Source
Ref.
0 OR
] T
R3
R, R H
R> H
R2
H
R3 H
H
H
H
CH3
H
H
A. manshuriensis
241,253
OCH3
Demethylaristofolin E (Aristolic acid II) (50) Aristolic acid I (51)
A. albida A. cucurbilifolia A. helerophylla A. indica A. kaempferi A. reticulata A. versicolar
H
H
Aristofolin E (52)
H
OH
OCH3
Aristofolin B (53)
H CH3
OH H
H H
OCH3 OCH3
Aristofolin D (54) Aristolic acid methyl ester (55)
H
OCH3
H
OCH3
H
Ogle
H
OCH3
6-Methoxy aristolic acid (56) Aristofolin A (57)
CH3
OCH3
H
OCH3
6-Methoxy aristolic acid methyl ester (58)
Na
Ogle
H
OCH3
Sodium aristofolin A (59)
CH3 Na
H H
OCH3 OH
OCH3 OCH3
Aristofolin C (60) Sodium aristofolin B (61)
A. kaempferi A. manshuriensis A. cucurbilifolia A. helerophylla A. kaempferi A. cucurbitifolia A. cucurbitifolia A. heterophylla A. indica A. kaempferi A. kwangsiensis A. versicolar A. cucurbitifolia A. kaempferi A. kwangsiensis A. versicolar A. championii A. cucurbitifolia A. heterophylla A. cucurbitifolia A. cucurbitifolia
99 56, 159, 254 228,232 179,182 229, 244 209 222 244 241 56, 254 228,232 229 56, 254 56, 254 232 179, 182 244 247 222 56, 254 229,233 188, 190 222 138,248 56, 254 232 56, 254 159
denitroaristolochic acids were the constituents of Formosan Aristolochia species [254]. Aristolic acid I (51) is the abundantly reported denitroaristolochic acid in Aristolochia species and has been shown to be
875 extensively studied compound after aristolochic acid I (5) and II (1). The effect of aristolic acid I (51) on a diverse array of physiological process has been examined, as it is a denitro derivative of an active ingredient aristolochic acid I (5) to understand the role of nitro group in the bioactivity. Aristofolin A (57), B (53), C (60), D (54), and E (52) [244,254] reported as metabolites of Aristolochia species of Taiwan origin were denitro derivatives of aristoloside (16), aristolochic acid Vila (11), 7-methoxy aristolochic acid I (15), aristolochic acid FVa (10) and aristolochic acid II methyl lester (25), respectively. Compounds 52, 55, 58 and 60 were isolated as methyl esters of denitroaristolochic acids. Aristofolin A (57) and sodium aristofolin A (59) are the only examples of denitroaristolochic acid containing glucosyl units [233,254]. Compounds 59 and 61 were isolated as sodium salts of denitroaristolochic acids from A. cucurbitifolia [254] and A. heterophylla [232], and A. cucurbitifolia [159], respectively. Aristolactams Aristolactams are reduction products of aristolochic acids and regarded as biogenetic intermediates in the biosynthetic pathway of aristolochic acids. They are supposed to originate in the plants by oxidation of aporphines. From Tables 8 and 9, it is evident that thirty six aristolactams have been reported so far from Aristolochia species. Aristolactams are by no means confined to Aristolochiaceae; at least species of Annonaceae, Menispermaceae and Monimiaceae have been reported to contain one or more of the over sixty known aristolactams [255]. Occurrence of aristolactams in these four families is of taxonomic significance and supports the view that the Aristolochiales is related to Magnoniales and Ranunculales [256]. The majority of aristolactams also possess oxygenated substituents at C-3 and C-4, C-6 and/or C-8, as in aristolochic acids. Aristolactams 62-82 showed similar substitution pattern to that of the accompanying aristolochic acids. However, aristolactams 83-97 differ in that they lack methylenedioxy substituents, instead they possess methoxyl or hydroxyl groups at C-3 and C-4. The seven aristolactam glycosides that have been described to date in Aristolochia include compounds 68-73, and 85. Compound 73 was found to possess a bioside on C-5, whereas the remaining all are iV-glycosides of aristolactams [239]. Compound 72 is the only representative of acylated glycoside of aristolactam, with ?ra«s-/?-coumaroyl moiety as acyl group on C-6 of glucose unit [260]. A. argentina was found to be rich in aristolactams
876
Table 8. Aristolactams Isolated from Aristolochia Species Compound
R H
Ri
c
Name O
Source
Ref.
257 132 56, 158, 159 158 174 158 158,229, 244 158,241 210 158 239 220 158 223 257 48,49 158, 159 158 158,228, 232 158,229, 244 158 158 218 158 257 191 210 239 257 132 49 207 223 99 127,257
\ NR
it
R3
H
R2 H
H
Aristolactam II (Cepharanone A) (62)
A. argentina A. auricularia A. cucurbitifolia A. debilis A. foveolata A. heterophylla A. kaempferi A. manshuriensis A. rodriguesii A. tagala A. triangularis A. tubftora A. westlandii A. zollingeriana
H
OH
H
H
Aristolactam Ilia (Aristolactam C) (63)
A. argentina A. baetica A. cucurbitifolia A. debilis A. heterophylla A. kaempferi A. manshuriensis A. tagala A. tuberosa A. westlandii
H
H
OH
H
Aristolactam-Ia (64)
H
OCH3
H
H
Aristolactam III (65)
H
H
OCH3
H
Aristolactam I (Aristolactam A)
A. argentina A. longa A. rodriguesii A. triangularis A. argentina A. auricularia A. baetica A. ponlicum A. zollingeriana A. albida A. argentina
877 (66)
A. auricularia A. baecita A. bractenta A. brevipes A. chilensis A. contorta A. cucurbitifolia A. debilis A. elegans A.fangchi A.foveolata A. heterophylla A. indica A. kaempferi A. manshuriensis A. mollissima A. ponticum A. reliculata A. rotunda A. tagala A. tubflora A. westlandii A. zollingeriana
H
H
H
OCH3
glc
H
H
H
glc
OH
H
H
Aristolactam C W-p-D-gluco- side (69)
glc
H
OH
H
glc
H
OCH3
H
Aristolactam la NP-D-gluco- side (70) Aristolactam I A^P-D-gluco- side (71)
9-Methoxyaristolactam-II (67) Cepharanone A N(5-D-gluco- side (68)
A. auricularia
A. cinnabarina A. contorta A. foveolata A. heterophylla A.mollissima A. cinnabarina A. contorta A. cucurbitifolia A. elegans A.foveolata A. heterophylla A. indica A. kaempferi A. mollissima A. pubescens A. rotunda A. tuberosa A. zollingeriana
132 48 258 52 259 260, 262 59, 158, 159 127, 158, 234 168, 174 127,172 246 158,228 127, 178, 181,182 158 241 202 207 209 127 158 220 158 223,245 132
A. foveolata A. pubescens
142,261 262 246 228,232 203 142, 143, 261 262 56 168, 169 246 228 127, 180 244 204 208 263 218 223 174 208
A. cinnabarina A. clematitis A. contorta A. cucurbitifolia
142, 143 153 230, 260 158
878 A. debilis A. foveolata A. helerophylla A. indica A. knempferi A. manshuriensis A. mollissima A. pubescens A. rotunda A. tagala A. tuberosa A. wesllnndii A. zollingeriana 6'-pcoumarylglc
H
OCH3
H
c H
H
glc-glc
H
OH
H
OCH3
H
H
OCH3
OH
H
H
H
OCH3
OH
Aristolactam-/V(6'-trans-pcoumaroyl)-|3-Dglucoside (72)
158, 163 174 158,228, 232 92, 127, 178, 180 158,244 158 203, 204 208 263 158 218 158 223,245
A. conlorta
260
A. triangularis
239
A. cinnabarina
142,261
A. cucurbitifolia
159
A. brevipes A. foveolata
52 174
0 NH
(
c
R4 R3
R2 Triangularine-B (73) 2-Hydroxy-8methoxycepharanone-A (74) Cepharanone B (75) 9Hydroxyaristolact am-I (76) 0 NH
R4 Ri
R3 R2
879 OCH3
OCH3
H
H
OCH3
H
0CH3
H
5,6-Dimethoxyaristolactam (77) Aristolactam-IV (78)
A. mollissimn
202
A. argentina
127, 130 178 202
A. indica A. mollissima H OCH3 9OCH3 A. auricularia Methoxyaristolactam A. cucurbitifolia 1(79) A. elegans A. heterophylla A. kaempferi A. manshuriensis A. mollissima A. ponticum A. zollingeriana H OCH3 OCH 2 CH 3 9A. mollissima Ethoxyaristolactam-I (80) H OCH3 9OCH3 A. auricularia Methoxyaristolactam A. cucurbitifolia IV (81) OCH3 Aristored (82) A. bracteata O A. reticulata
H
H
OCH3
y
CT
H3CO
NH
132 56, 159 169 228 244 241 204 207 223 202
132 159 127 127, 183, 209 183
A. serpentaria
1
^N)CH,
followed by A. heterophylla and A. cucurbitifolia. Aristolactam I (66) followed by aristolactam All (83), aristolactaml-A^- P-glucoside (71) and aristolactam II (62) were the frequently encountered aristolactams in the Aristolochia species. Aristolactam II (62) found in several species of Aristolochia is a simple aristolactam without substitution on ring B and C. Unusual aristolactams which have C-2 substitution, namely 2-hydroxy-8methoxycepharanone A (74), aristored (82), aristolactam DII (92), Dili (96), CII (93), CIII (97), CIV (95) and aristoliukine A (94) were described from Aristolochia species. Among these, compounds 92 and 96 reported from A argentina [130,266] contain -COOH group on C-2, where as 93, 95, 97 from A. argentina [228,266] and 94 from A. kaempferi [229,244] possess hydroxymethyl group at C-2. The presence of a carboxyl group in 92 and 96 suggest that they might be biogenetically derived from 3carboxyphenylalanine or 3-carboxy-4-hydroxyphenylalanine [266]. On the other hand, aristolactams 93 and 97 might either arise from
880 Table 9. Aristolactams Isolated from Aristolochia Species Compound
Name
Source
Ref.
A. aculifolia A. argentina A. baetica A. contorta A. cucurbitifolia A. debilis A. elegans A. foveolata A. heterophylla A. indica A. kaempferi A. manshuriensis A. mollissima A. rodriguesii A. tagala A. triangularis A. westlandii A. zollingeriana A. cucurbitifolia A. elegans A. heterophylla A. kaempferi A. cucurbitifolia
210 127,257,264 49 262 56, 158, 159 158 168, 169, 171 246 158,228,232 92, 127, 181 61, 158,229,244 158,241 204 210 158 239 158 223 159 168, 169 228,232 244 159
A. argentina A. baetica A. cucurbitifolia A. debilis A. heterophylla A. kaempferi A. manshuriensis A. rodriguesii A. tagala A. triangularis A. westlandii A. argentina A. elegans A. foveolata
127,257,264 49 56, 158, 159 158, 163 158,228 61,244 158,265 210 158 239 158 257 168, 169 174
0 RO
/ \ \ NH
X R, H
R3 H
R4
CH3
H
Aristolactam All (83)
CH3
H
H
H
Piperolactam A (84)
CH3
glc
H
H
CH3
CH3
H
H
Piperolactam AO-P-D- glucoside (85) Aristolactam BII (86)
H
CH3
OH
H
R2
Aristolactam AHIa (87)
881
H
CH3
H
OH
H
CH3
OCH3
H
CH3
CH3
OCH3
H
CH3
CH3
H
Aristolactam- Ala (88) Aristolactam AIII (89)
Aristolactam Bill (90)
OCH3 Aristolactam-Bl (taliscanine) (91)
A. heterophylla A. kaempferi A. mollissima A. triangularis A. argentina A. triangularis A. argentina A. baetica A. cucurbitifolia A. debilis A. heterophylla A. kaempferi A. manshuriensis A. tagala A. westlandii A. zollingeriana A. acutifolia A. argentina A. zollingeriana A. argentina A. taliscana
228, 232 158,229,244 204 239 257 239 127,257,264 49 158 158 158,232 61, 158,244 158 158 158 223 210 127,257,264 223 264 127
0 R2O——. NH
CO2H
OH
OCH3
H
CH2OH
OCH3
OCH3
H
CH2OH
OH
OCH3
OH
CH2OH
OH
OCH3
OCHj
CO2H
OH
OCH3
OCH,
CH2OH
OCH3
OCH3
OCH3
R4 Aristolactam DO (92) Aristolactam- CO (93) Aristoliukine A (94) Aristolactam CIV (95) Aristolactam DIN (96) Aristolactam- CIII (97)
A. argentina A. foveolata A. manshuriensis A. argentina A. triangularis A. kaempferi A. mollissima A. heterophylla
130,266 174 265 266 239 229, 244 204 228
A. argentina
130,266
A. argentina
266
aristolactams DII (92) and Dili (96) by reduction and methylation reactions or directly originate from the ammo acids 3hydroxymethylphenylalanine and 3-hydroxymethyl-4-hydroxyphenylalanine [266]. The 9-oxygenated aristolactams are rare in Aristolochia with compounds 67 [132], 76 [52], 79 [132], 80 [202] and 81 [132] being the examples with hydroxy or methoxy or ethoxy group at C-9.
882 Compound 80 is the only example of 9-ethoxy aristolactam found in A. mollissima [202]. Aporphines Twenty aporphine alkaloids have been isolated so far from Aristolochia species (Table 10). Glaziovine (98) was detected as the sole proaporphine component from Chilean species, A. chilensis [259]. Aporphines, magnoflorine (105) followed by cepharadione A (107) and 4,5-dioxo-dehydroasimilobine (110) are found in several species of Aristolochia. Aporphines with iV-formyl substitution, ./V-formylnornantenine (102), and its 6ot,7-dehydro derivative, 6a,7-dehydro-./Vformylnornantenine (103) were described by Touche et al. from A. brevipes [273]. The latter aporphine (103) was reported to exist in a 19:1 ratio of E/Z conformers due to stereoisomerism of the jV-formyl group. An 7V-acetyl aporphine derivative, ./V-acetylnornuciferine (99) besides magnoflorine (105) has been encountered in A. bracteata, an Indian medicinal plant by Pakrashi et al. [258]. Magnoflorine (105) and N,Ndimethylindcarpine (106) were polar quaternary aporphines with N,Ndimethyl group found in Aristolochia species. Aporphines 107-114 were representatives of 4,5-dioxoaporphines known to Aristolochia. The 4,5dioxoaporphines are a small group of aporphinoids found mostly among members of the botanical families Menispermaceae, Berberidaceae, Annonaceae, Fumariaceae and Aristolochiaceae [276]. Since aporphine alkaloids have been postulated as precursors of aristolactams and aristolochic acid in plants, 4,5-dioxoaporphines may be considered as possible intermediates. Tuberosinone iV-glucoside (109) is the only Nglucoside of aporphine found in Aristolochia species, A. tuberosa [218] and A. cinnabarina [141]. Lysicamine (115) from A. contorta [272] and isomoschatoline (116) and oxonuciforine (117) from A. elegans [169] were fully aromatic aporphines with a rare 9-oxo function. An unusual aporphine, dimethylsonodione (118) with 5,8-dioxo function was also identified from A. manshuriensis [241]. Most of the aporphines found in Aristolochia species possess 4,5-tetrahydro basic skeleton. Protoberberines Sixteen protoberberine type alkaloids reported from Aristolochia species were listed in the table 11. Occurrence of this type alkaloids were rare in Aristolochia, they were found only in four species, A. arcuata
883
Table 10. Aporphines Isolated from Aristolochia Species Compound
Name (-)-Glaziovine (98)
Source A. chilensis
Ref. 259
(-HV-Acetylnornuciferine (99)
A. bracteatn
258
Isoboldine (100)
A. papillaris
120
(-)-Isocorydine (101)
A. pubescens
208
H,CO
H,CO
884
I \
/V-Formylnomantenine (102)
A. brevipes
273
6a,7-Dehydro-JVformylnornantenin
A. brevipes
273
Asimilobine (104)
A. cucurbitifolia
159
Magloflorine(105)
A. argentina A. austrozechuanica A. baetica A. bracteata A. clemntitis A. conlorta A. cymbifera A. debilis A. elegans
131 133 49, 134 135 153 154, 155 267 268,269 167, 168, 169, 170 231 176, 177,236 92 164 270 194 176,205 239
\
r
0
\—o
e(103) |/
CHC
H3CO'"'
I
105 106
R, CH 3 H
TH
R2 H CH 3
R3 H H
CH3
R4 CH 3 CH 3
W.W-Dimethyllindcarpine (106)
A. fangchi A. helerophylla A. indica A. kaempferi A. macedonica A. manshuriensis A. moupinensis A. triangularis A. triangularis
239
885 0 o
JK.
\
/ ~~—
x
f/
.0
Cepharadione A (107)
J
1\
1
Tuberosinone (108) R CH3 H glc
107 108 109
Tuberosinone-A'-pD-glucoside (109)
R, H OH OH
4,5Dioxodehydroasim ilobine (110)
A. chilensis A. cucurbitifolia A. foveolala A. heterophylla A. indica A. kaempferi A. mollissima A. tagala A. Iriangularis A. zollingeriana A. cinnabarina A. kaempferi A. tuberosa A. cinnabarina A. tuberosa
A. chilensis A. contorta A. cucurbitifolia; A. elegans; A. heterophylla; A. indica A. kaempferi
0 /
^ N
s.
^ ^
1 T
R2O
Aristoliukine B (112)
r
/
J
1 3
R2 R. H CH3 CH 3 H CH3 H CH3 H H CH3
R
110 111 111 113 114
Aristolodione (111)
H H CH 3 H H
Triangularine-A (113) Aristoliukine C (114)
Lysicamine (115) Isomoschatoline (116)
I
H3CO"
r
J
1 R 115 H 116 OH
R2 H H
246
228, 232 92, 181 229,244 204 158 239
223,245 141,142,143 244
219,274,275 141,142,143 218,219,220
271 272
56, 159 169
228,232 181
61, 158,229,244 204 239 271 232 244 232 204
229, 244 239
A. kaempferi
244
A. contorta A. elegans
168, 169
R3 H OH H OH OCH
1H3CO^
A. mollissima A. iriangularis A. chilensis A. heterophylla; A. kaempferi A. heterophylla A. mollissima A. kaempferi A. triangularis
271
56, 159
R3 H H
272
886 Oxonuciforine (117) Demethylsonodion
A. elegans
168,169
A. manshuriensis
241
H3aX^>''x)
R 120 H 121 glc
H,CO.
Ogle
122
123
R,O.
H,CO' H3CO
R2 R
124 125 126 127 128 129
i
CH 3 H H H Ac Ac
R
2
H OH OH OH H OAc
R3 H OH Ogle Oxyl OAc OAc
R4 H H H H glc-Ac4 H
R2 Rs H H
130
H H Ac Ac
131 132 133 134
Fig. (2).The structures of Proto berberines
Ri H H H Ac Ac
R2 OH H H OAc H
R3 OH Ogle Oxyl Oglc-Ac4 OAc
R4 H OH
Rs CH 3
OH H
H Ac
OAc
glc-Ac
H
887 Table 11. Protoberberines Isolated from Aristolochia Species Compound Cyclanoline (119) 2,10-Dihydroxy-13-oxidodibenzo[a,g]quinoiizinium (120) 2-Hydroxy-10-O-[glucopyranosyl]-13oxidodibenzofa,gl-quinolizinium (121) 8-Benzylberbine-A (122) jV-Oxide 8-benzylberbine-B (123) (-)-8p-(4'-Hydroxybenzyl)-2,3-dimethoxyberbin10-ol(124) (-)-8p-[4'-Hydroxybenzyl]-2-methoxyberbin3,10,11-triol (125) (+)-10-O-[p-Glucopyranosyl]-8|3-[4'hydroxybenzyl]-2-methoxyberbin-3,ll-diol (126) (+)-8p-[4'-Hydroxybenzyl]-2-methoxy-10-O-[Pxylopyranosyllberbin-3,11 -diol (127) 3,10-Diacetoxy-8p-[4'-O-(a-glucopyranosyltetraacetate)-benzyll-2-methoxyberbine (128) 3,10,11 -Triacetoxy-8p-[4'-acetoxybenzyl]-2methoxyberbine (129) (-)-8a-[4'-Methoxybenzyll-2-methoxyberbin3,10,11-triol (130) (+)-10-O-[-p-Glucopyranosyl]-8a-[4'hydroxybenzyl]-2-methoxyberbin-3,9-diol (131) (+)-8a-[4'-Methoxybenzyl]-10-O-[Pxylopyranosyl]berbin-3,9-diol (132) (-)-3,ll-Diacetoxy-8a-[4'-acetoxybenzyl]-10-O-[pglucopyranosyltetraacetate]-2-methoxyberbine (133) 3,9,10-Triacetoxy-8a-[4'-O-(p-glucopyranosyltetraacetate)benzyll-2-methoxyberbine (134)
Source A. debilis A. nrcuala
Ref. 268 277
A. arcuata
277
A. gigantean A. gigantean A. constricta
278, 279 278,279 280
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
A. gigantea
279
[277], A. constricta [280], A. debilis [268] and A. gigantea [278,279]. Among them, A. gigantea is rich in this type alkaloids. Out of sixteen protoberberine type alkaloids found in Aristolochia, ten were from A. gigantea [278,279]. Seven of them were occurred as glycosides. Compounds 122-134 are 8-benzyltetrahydroprotoberberine type alkaloids [278,279]. Their unusual carbon skeleton had been obtained by introduction of a benzyl group at C-8 of berberine. On the basis of proposed biosynthetic pathway, where the berberine bridge carbon is considered to be derived from formate, Lopes suggested that the CHCH2Ar unit of 8-benzyltetrahydroprotoberberine alkaloids of Aristolochia comes from p-hydroxybenzaldehyde through a biosynthetic step involving two molecules of ^-hydroxybenzaldehyde condensing with one of L-tyrosine [279]. It is reported that rings B and C of the
888 tetrahydroprotoberberine alkaloids adopt a half-chair conformation and B/C cis configuration [278]. The 8-benzyl group was in P-orientation in alkaloids 124-129, whereas in alkaloids 130-134 it is in a-orientation. Compound 123 is the only N-oxide among these alkaloids. Compounds 119-121 were quaternary alkaloids. Compounds 120 and 121 reported from A. arcuata [277] are the 13-oxidodibenzo[a,y]-quinolizinium alkaloids. It is known that oxyberberinium and oxyprotoberberinium salts were generally obtained from natural products, which were deoxygenated in both A and D rings, having the L-tyrosine and p-hydroxybenzaldehyde as common biogenetic precursors. The largest difference between 13oxidodibenzo[a,y]-quinolizinium and oxyberberinium and oxyprotoberberinium alkaloids is the presence of only one oxygenated function at A and D rings. Since, 13-oxidodibenzo[a,y]-quinolizinium alkaloids from A. arcuata [277] are presumably biogenetically related to 8-benzylberberine and benzylisoquinoline alkaloids isolated from Aristolochia species, Ltyrosine and p-hydroxybenzaldehyde are considered to be their biogenetic precursors. Protopines Protopine alkaloids are apparently not very common in Aristolochia and however, constrictosine (135), 3-0-methylconstrictosine (136), 3,5di-O-methyl-constrictosine (137), 5,6-dihydroconstrictosine (138) and 5,6-dihydro-3,5-di-0-methylconstrictosine (139) are the only examples of the protopine alkaloids [280]. Their occurrence in Aristolochia was also strictly confined to A. constricta [280]. Protopine alkaloids are usually reported as C-2 substituted on the basis of biogenetic considerations. The unusual absence of C-2 substitution was noticed in the protopines of Aristolochia. R,O
R| 135 H 136 CH3 137 CH3
R,O.
R2 H H CH 3
Fig. (3). The Structures of Protopines
R. 138 H 139 CH,
R2 H CH,
889 Benzylisoquinolines Fourteen benzylisoquinoline type alkaloids have been identified in Aristolochia species (Table 12). Coclaurine (140), oblongine (141) and reticuline (142), respectively found in A. papillaris [120], A. triangularis [239] and A. reticulate [281], were simple benzylisoquinoline alkaloids [169]. Aristoquinoline A (143), B (145), C (144) isolated from A. elegans were novel benzoyl benzyltetrahydroisoquinoline ether iV-oxides. Compounds 143 and 145 were conformational isomers which differ in the conformation of C-ring and configuration of iV-oxide. Alkaloids 146 and 147 from A. elegans [167,282] and 148-150 from A gigantea [283] were bis-l-benzyltetrahydroisoquinoline alkaloids with one diphenyl ether link between rings C and C , usually through C-ll and C-12' centers. These alkaloids usually have \R,VR absolute configuration, however, 146 reported to possesses IS,\'R configuration. Compounds 151 and 152 from A. fangchi [231] and 153 and 154 from A. debilis [161,284] were bisbenzyltetrahydroisoquinoline alkaloids with two diphenyl ether links. These ether linkages were usually between rings C and C , and B and B', except in aristolochine which contained two ether links between rings B and C , and C and B'. Benzylisoquinolines and benzoyl benzyltetrahydroisoquinoline ether JV-oxides were assumed to be biogenetic intermediates in the catabolic process of bisbenzyltetrahydroisoquinoline alkaloids. Table 12. Benzylisoquinolines Isolated from Aristolochia Species Compound Coclaurine (140) Oblongine (141) Reticuline (142) (-)-(-ft)-Aristoquinoline A (143) (-Hfl)-Aristoquinoline C (144) (-)-(/?)-Aristoquinoline B (145) (-)-Temuconine (146) (-)-(«,«)-7'-O-Methylcuspidaline(147) (-)-Pedroamine (148) (-)-Pampulhamine (149) (-)-Geraldoamine (150) Fangchinoline (151) (-)-Obamegine(152) Tetrandrine(153) Aristolochine (154)
Source A. papillaris A. triangularis A. reticulata A. elegans A. elegans A. elegans A. elegans A. elegans A. gigantea A. gigantea A. gigantea A. fangchi A. fangchi A. debilis A. debilis
Ref. 120 239 281 169 169 169 282 167 283 283 283 231 231 284 161
890 H,CO.
R,0'
140 141
RT R3 OCH, H H H CH, OH
R4 H H
142
OCH, H
OH OCH, H
H
Rj OH H
H OH
~CH,
H,C
147 148 149 150
R H CH, CH, CH,
R, CH, H CH, CH,
R2 CH, H H CH,
^.OCH, H,CO. .OCH,
H,CO 151 152 153
R, H H CH,
R, H CH, CH 3
Fig. (4). The Structures of Benzylisoquinolines
Isoquinolines The occurrence of isoquinoline alkaloids in the genus Aristolochia is limited to three species, A. arcuata, A. elegans and A. gehrtii. Isolates of this calss were listed in the Table 13. Most of these alkaloids reported from Aristolochia were tetrahydroisoquinoline type, except 168 which is fully aromatic. It is interesting to note that all the alkaloids isolated from A. arcuata [285] were isoquinolines whereas all the alkaloids from A. elegans were isoquinolones with a carbonyl function at C-l. The
891 isoquinolines (156-160) isolated from A. arcuata [285] were reported to contain rare substituents, like TV-ethyl, TV-fructosyl and TV-glyceryl. Iwasa et al. [286] proposed that salsolinol (155), a simple isoquinoline alkaloid is formed by the condensation of dopamine with acetaldehyde, and it has been suggested that the O-methylating enzymes of salsolinol may be different from those of dopamine. On the basis of this proposal, Francisco et al. [285] suggested that the alkaloids 157-160 come from acetoacetate condensation with a dopamine derivative followed by decarboxylation. Isoquinoline alkaloids were also considered as biogenetic intermediates in the catabolic process of bisbenzyltetrahydroisoquinoline alkaloids. Amides The amides are another class of compounds isolated from several Aristolochia species (Table 14). Most of the amides from Aristolochia species, on structural investigation were found to contain a tyramine unit connected to phenolic acids like coumaric, ferulic, cinnamic acids, and rarely with acetic acid through an amide linkage. 7V-/?-/ra«.s-Coumaroyltyramine (178) and N-p-trans-iQuxXoyXtyrdsmne (179) are more frequently encountered amides in Aristolochia species. Their cis isomers, N-p-ciscoumaroyltyramine (174) and TV-cw-feruloyltyramine (175) were also isolated from Aristolochia species. Aurantiamide acetate (172), a rarely occurring modified phenylalanine dipeptide alkaloid was reported from only one species, A. tubflora [288]. A^raws-Cinnamyltyramine (177) was described only from A. cucurbitifolia [159] and A. elegans [169]. Aristomanoside (181) isolated from A. manshuriensis [241] is a 4,4'diglucoside of /V-feruloyl-3-methoxytyramine. Its aglycone, N-transferuloyl-3-methoxytyramine (180) was also found in A. manshuriensis. The last mentioned amide (180) and its cis isomer (176) were also reported from A. gehrtii [241]. The co-occurrence of biosynthetic derivatives, isoquinolones and alkamides in Aristolochia spcies, which could be formed by at least one unit C6-C2 is remarkable. Nacetyltyramide (171) from A. cucurbitifolia is the only example of an aliphatic acid amide of tyramine [56]. In addition, three simple amides, namely pyridine 3,5-dicarbozamide (169), 3,4-dimethoxy-Af./V-dimethylbenzamide (170) and cinnamamide (173) were also described from A kaempferi [61,244]. The occurrence of the tyramine amides and the modified dipeptides in these plants is of considerable biogenetic and chemotaxonomic significance.
892 Table 13. Isoquinolines Isolated from Aristolochia Species Compound
Name
Sourece
Ref.
Salsolinol (155) 6,7-Dihydroxy-l -methyl-A'-(6'fructopyranosyl)-l ,2,3,4tetrahydroisoquinoline (156)
A. arcuata A. arcuata
285 285
6,7-Dihydroxy-l, l-dimethyl-1,2,3,4tetrahydroisoquinoline (157) 6,7-Dihydroxy-l ,1 -dimethyl-/V-ethyll,2,3,4-tetrahydroisoquinoline(158) 6,7-Dihydroxy-l,l-dimethyl-A'-(2'glyceryl)-l,2,3,4-tetrahydroisoquinoline (159) 6,7-Dihydroxy-l ,1 -dimethyl-A'-(6'fructopyranosyl)-l ,2,3,4tetrahydroisoquinoline (160)
A. arcuata
285
A. arcuata
285
A. arcuata
285
A. arcuata
285
CH3 R 155 H 156 6'-Frc
H3C
CH3
R 157 H 158 CH2CH3 159 CH(CH2OH)2 160 6'-Frc
K
JL '
2
O
161 162 163 164 165 166 167
R F1 FI FI FI CH3 CH 3 C H3
K.]
IV2
OH OH OCH3 OCH3 OH OCH3 OCH3
OH OCH3 OH OCH3 OCH3 OH OCH3
Pericampylinone-B (161) Pericampylinone-A (162) Northalipholine (163) Corydaldine (164) 3,4-Dihydro-6-hydroxy-7-methoxy-2methylisoquinolin-1-one (165) Thalipholine (166) yV-Methyl corydaldine (167)
yV-Methyl-6,7-dimethoxyisoquinolone (168)
A. A. A. A. A.
elegans elegans elegans elegans elegans
168 169,206 169 169 168
A. gehrtii A. elegans A. elegans
287 169 169
A. elegans
168
0
Amino Acids Fifteen amino acids, alanine (182), glycine (183), leucine (184), proline (185), vallme (186), asparagine (187), glutamine (188), senne (189), threonine (190), aspartic acid (191), glutamic acid (192), tyrosine.
893 Table 14. Amides Isolated from Aristolochia Species Compound Pyridine-3,5-dicarbozamide (169) 3,4-Dimethoxy-A',A'-dimethylbenzamide(170) yV-Acetyltyramide (171) Aurantiamide acetate (172) Cinnamamide (173) yV-p-m-Coumaroyltyramine (174)
7V-/>-c/s-Feruloyltyramine (175)
c/s-TV-Feruloyl-3 -O-methyldopamine (176) yV-Jrans-Cinnamyltyramine (177) Af-/7-(r««i-Coumaroyltyramine (178)
,¥-/ra/j.s-Feruloyltyramine (179) (Moupinamide)
(ra/ts-Ar-Feruloyl-3-O-methyldopamine (N-transFeruloylmethoxytyramine) (180) Aristomanoside (181)
Source A. kaempferi A. kaempferi A. cucurbitifolia A. tubflora A. kaempferi A. gehrtii A. mollissima A. moupinensis A.Zollingeriana A. elegans A. gehrtii A. heterophylla A. zollingeriana A. gehrtii A. elegans A. cucurbitifolia A. elegans A. grandiflora A. heterophylla A. gehrtii A. kaempferi A. manshuriensis A. mollissima A. moupinensis A. zollingeriana A. acutifolia A. elegans A. gehrtii A. heterophylla A. manshuriensis A. moupinensis A. papillaris A. pubescens A. zollingeriana A. gehrtii A. manshuriensis A. manshuriensis
Ref. 244,245 244 56 288 61,229 287 204 205 223 168, 169 287 228 223 287 168, 169 159 168, 169 289 232 287 229 241 204 205 223 210 168, 169 287 228 241 205 120 208 223 287 241 241
(193), histidine (194), lysine (195), and y-aminobutyric acid(196) were found in A. clematitis which is known by its vernacular name Upright birthwort growing in Mainland China [297,298]. Asparagine (187) is also isolated from A kaempferi [233] and A. rotunda [212]. Chlorophylls The naturally occurring chlorophylls with a porphyrin skeleton are another group of constituents present in Aristolochia species. Aristophyll
894
OCH3
N 169
174 175 176
R, H OCH3 OCHj
H OCHj
R, 177 H 178 H 179 OCH, 180 OCH, 181 OCH,
R, R3 H H OH H OH H OH OCH, Ogle OCH3
R4 H H H H gle
Fig. (5). The Structures of Amides
A (197) and B (198) were reported as new chlorophyll derivatives from A. elegans [168,292] and aristophyll C (199) was also found as a new chlorophyll in A. heterophylla [232]. Methyl pheophorbide-a (200) and methyl 21-hydroxy-(21i?)-pheophorbide-a (201) were isolated from A. cucurbitifolia [56,159]. A known chlorophyll, pheophytin-a (202), was isolated from A. kaempferi [229]. Another new pheophytin-a derivative described from A. heterophylla is 132-hydroxy-(1325)-pheophytin-a (203) [232]. Incidentally, all these chlorophylls were isolated by our group from Aristolochia species collected in Taiwan.
Table 15. Phenanthrenes Isolated from Aristolochia Species Compound 2-(Phenanthro[3,4-d]-l,3-dioxole-6-nitro-5carboxamido)propanoicacid (204) 9-Ethoxyaristolactone (205) Aristolophenanlactone 1 (206) Aristolide-B (207)
Aristolide-A (208) Aristolide-C (209) Aristolamide (210) Argentinine (211) Aristololide (212)
Source A. longa
Ref. 62
A. tnollissimn A. tubflora A. cucurbitifolia A. heterophylla A. manshuriensis A. cucurbitifolia A. heterophylla A. cucurbitifolia A. heterophylla A. indica A. argentina A. indica
201 220 56, 159 293 241 56, 159 293 56, 159 293 179 295 294
895
O
COOCH3
O
COOCHj O—Phylyl
H3COOC
R
200 201
H OH
202 203
R H OH
Fig. (6) The Structures of Chlorophylls
Phenanthrenes Aristolide A-C (207-209) are the examples of novel dihydrophenanthrenelactones known to Aristolochia species. Aristolide A (208) and C (209) were reported from A. cucurbitifolia [56,159] and A. heterophylla [293] have R configuration at C-10, whereas aristolide B (208) found in the same plants as well as in A. manshuriensis [241] possesses S configuration at C-10. Aristolophenanlactone I (206) and 9ethoxyaristolactone (205) occur in A. tubflora [220] and A. mollissima [201], respectively are also aristolactone derivatives with 8,9-oxygenation at 8 and 9 positions. Aristolamide (210) isolated from A. indica [179] contain -CONH 2 group at C-l. Teresa et al. isolated compound 204, as a new companion of aristolochic acid with -CONH-CH(CH3)-COOCH3 group at C-l and its optical rotation suggests the possibility of L-(+)alanine derivative [62]. Aristololide (212), a phenanthroid lactone is the first 10-oxygenated aristolic acid derivative encountered in nature [294].
896 Argentinine (211), isolated from A. argentina is the only -CH2CH2N(CH3)2 containing phenanthroid amine reported so far [295].
OR, 206 204 205
R, H OCH3
R2 CH3 CH3
NH 2
R. 207 CH3 208 CH3 209 CH3
NHCH(CH3)COOH N(CH3)2
NO,
OCH3 210
R2 H OH OCH2CH3
OCH, 211
212
Fig. (7). The Structures of Phenanthrenes
Monoterpenoids Monoterpenoids were widely distributed in essential oils of Aristolochia species being mostly present as acyclic monoterpenoids, menthanes, pinanes and camphanes (Table 16). In addition to these types, thujanes, caranes, fenchanes and tricyclic monoterpenes were also identified as minor components. A total of forty-three compounds were identified in the essential oils from leaves, aerial stems and underground organs of A. argentina, constituting 84-98 % of total essential oils [296]. The essential oils of A. argentina are characterized by the unusual presence of argentilactone (223) and isomers of undecatriene (219-222). Argentilactone (223), a volatile compound was identified as the lactone of the (-)-(5/?)-5-hydroxydodeca-Z,Z-2,6-dienoic acid [297]. Its presence and quantitative prevalence over any other component in the volatile oils is the
897
Table 16. Monoterpenoids Isolated from Aristolochia Species Structure Acyclic monoterpenoids CH, CH3
JL.
H,C
.-
Source
Ref.
Geraniol (213)
A. brevipes A. gigantea A. ovalifolia A. acutifolia
304 301 306 299
A. argentina A. asclepiadifolia A. gigantea A. indica A. longa A. elegans A. debilis A. longa
296 305 301 307 311 308 298 311
;ra«i-Ocimene (217)
A. argentina A. gibertii
296 300
Myrcene(218)
A. argentina A. debilis A. gibertii A. argentina
296 298 300 296
(3E,5Z)1,3,5-Undecatriene (220)
A. argentina
296
(3E,5E)1,3,5-Undecatriene (221)
A. argentina
296
(2Z,4Z,6E)2,4,6Undecatriene (222)
A. argentina
296
Argentilactone (223)
A. argentina
296, 297
Limonene (224)
A. argentina A. asclepiadifolia A. debilis A. elegans A. gibertii A. gigantea A. ringens
296 305 298 308 300 301 309
JL. CH2 OH
^ ^ ^ ^ ^ ^ CH,
CH, CH3
Compound
H ,C
o
pH
Geranyl acetone (214)
(SVLinalool (215)
2 H,C ^W^ ^ - ^/ \^ •^C/CH ^
CH,
CH3
JL
J^ c H
CH 3
^L
ri,L
(3-0cimene (216)
CH 2
^^^ 11 ^ C H 2
^^^^CH,
CH2
r^^cH, /
(3Z,5E)1,3,5-Undecatriene (219)
\
^W^^CH,
1 1 Men thanes r. \
H3c—V \
/
)
CH 2
'/ y,CH
3
898
H( =
v \_/ C H 3
HiC
A. brevipes
304
a-Teqiineol (226)
A. acutifolia A. asclepiadifolia
299 305
a-Terpenyl acetate (227)
A. argentina
296
a-Terpinolene (228)
A. indica
307
y-Terpinene (229)
A. ovalifolia
306
a-Phellandrene (230)
A. argentina A. ringens
296 309
P-Phellandrene(231)
A. argentina A. gibertii
296 300
Perillyl aldehyde (232)
A. brevipes
304
Perillyl acetate (233)
A. brevipes
304
Cuminyl alcohol (234)
A. heterophylla
228
p-Cymen-8-ol (235)
A. brevipes A. ringens
304 309
1,8-Cineole(236)
Carvacrol (237)
A. argentina A. brevipes A. debilis A. elegans A. gibertii A. brevipes
296 304 298 308 300 304
Carvone (238)
A. gibertii
300
\/~foH N
'
H3C—V ^ H
m-/7-Menth-2-en-l-ol (225)
CH3 >
f-OAc CH,
'
3C
/
^
/
CH3
'
CH,
'
H 2 C=^
CHj
>
( CH,
'
^
0—v
/
\
CH, CH3 /
/ H,C HOH2C—(/ y \—' H 3 C—^ \
^ ' H3C
CH3 ( CH, ^OH CH,
^ ^ " 3
0
».^_/H CH] HO O
V
/
CH,
899
6t:
HjC^XH/
(3-Cyclocitral (239)
A. argentina
296
Borneol (240)
A. asclepiadifolia A. brevipes A. debilis A. elegans A. helerophylla A. mollissima A. ovalifolia A. reticulata A. zenkeri
Bornyl acetate (241)
A. longa A .debilis
305 304 298 308 228 203, 204 306 281 310 311 298
Bornanol (242)
A. debilis A. elegans A. longa
298 308 311
Isobornyl formate (243)
A. argentina A. gibertii
296 300
Camphor (244)
A. brevipes A. debilis A. ovalifolia
304 298 306
Camphene (245)
A. argentina A. brevipes A. debilis A. elegans A. gibertii A. zenkeri
296 304 298 308 300 310
ercrfo-Fenchyl acetate (246)
A. brevipes
304
Bicyclic monoterpenoids Camphanes
CHO
H,C^/-
Fenchanes OAc
900 Pinanes CH,
CH3
CH2
CH3
CH2OH
a-Pinene (247)
A. argentina A. debilis A. elegans A. gibertii
296 298 308 300
Pin-2-en-8-ol (248)
A. longa
311
8-Acetoxy-pin-2-ene (249)
A. longa
311
(5-Pinene (250)
Pinocarvone (251)
A. argentina A. brevipes A. debilis A. elegans A. gibertii A. brevipes
296 304 298 308 300 304
Verbenone(252)
A. brevipes
304
Myrtenol (253)
A. brevipes
304
A-2-Carene (254)
A. gibertii
300
A-3-Carene (255)
A. gibertii A. reticulata
300 281
a-Thujene (256)
A. brevipes
304
Sabinene (257)
A. gibertii
300
cw-Sabinol (258)
A. brevipes
304
Caranes
1*^'
1
Thujanes
H3CH(3 ^ 2
\ HO
C
/
CH3
/
CH,
/—\ F^ \^J
CH,
901 Tncyclic monoterpenoids H3C^/CH3
Tricyciene (259)
A. gibertii
300
most relevant chemical characteristic of A. argentina. Four isomers of undecatriene are reported to have very interesting olfactive properties. The strong earthy odor that the essential oils of A. argentina display can be also largely assigned to the presence of E, Z (220) and E, E (221) undecatriene isomers. The essential oils of A. debilis were analyzed by Hayashi et al. [298] and identified eleven monoterpenoids. Myrcene (218) and limonene (224); and camphor (244) and camphene (245) were the major components of aerial parts and roots of A. debilis, respectively. Two monoterpenoids, geranylacetone (214) and a-terpineol (226) were identified as minor constituents in the hexane extract of the aerial parts of A. acutifolia [299]. Nineteen monoterpenes were identified in the essential oils of leaves and stems of A. gibertii [300]. The essential oils isolated from stems were dominated by limonene (224) content. A comparative study of the essential oils of the stems and leaves of A. gigantea also showed a significant increase in the percentage of monoterpenes in the stem oil, although in this case such increase is probably due to a higher content of linalool (215) and a-terpineol (226), rather than limonene (224) [301]. The essential oil of A. brevipes was characterized by the presence of a high percentage of monoterpenes by Nieves et al. and 1,8-cineole (236), camphor (244), and P-pinene (250) were the main constituents [304]. Linalool (215) and borneol (240) were the major and limonene (224) and terpineol (226) were the minor monoterpenoids found in the freshly prepared essential oil of roots of A. asclepiadifolia which has a characteristic pleasant aroma and smells like many absolutes [305]. The essential oil of leaves of^. ovalifolia [306] was reported to contain thirty two compounds corresponding to 80.43 % of the oil. The major components were geraniol (213), ^-terpinene (229), borneol (240) and camphor (244). Four monoterpenes, linalool (215), bornyl acetate (241), pin-2-en-8-ol (248) and its acetyl derivative (249) were isolated from the essential oil of the aerial parts of A. longa [311]. The later compounds are first cases to be described in the literature of pinanes, functionalized at C-
902 Sesquiterpenoids The sesquiterpenoids with various types of CI5 carbon skeletons constitute the largest group of metabolites in Aristolochia. Twenty four types of sesquiterpenoids were reported so far from the genus Aristolochia (Table 17). However, all these types of sesquiterpenoid frame work arise from the common precursor, farnesyl pyrophosphate, by various modes of cyclizations followed, in many cases, by skeletal rearrangement. The major types of sesquiterpenoids that have been found in the Aristolochia species include cadinanes, aristolanes, germacranes and bicyclogermacranes. Palmeira et al. identified fourty-seven components in three nonpolar fractions from the hexane extract of A, acutifolia [299]. In all the fractions, sesquiterpenes, P-bisabolene (264), oc-selinene (319), aeudesmol (325), spathulenol (372) and trans a-bergamotene (386) were the most abundant. Urzua et al. separated sesquiterpenoids, P-farnesene (260), (-)-P-bisabolene (264), (+)- sesquiphyllandrene (266), p-elemene (268), p-caryophyllene (313), l(10)-aristolene (331), y-cadinene (345), guaiol (360), P-aromadendrene (369), a-aromadendrene (370), ochimachalene (380), a-trans- bergamotene (384), and cc-cedrene (384) from a terpenoid mixture with germination inhibiting property obtained from a nonpolar fraction of the roots of A. chilensis [312]. Volatile oils from roots, stems and leaves of A. elegans [308] were found to be rich in sesquiterpenoids. Sesquiterpene hydrocarbons, in particular bicyclogermacrene (288), P-caryophyllene (313), and isocaryophyllene (314) were the predominated components in the oils from the leaves, whereas the oxygenated sesquiterpenes, mainly is-nerolidol (263), were the main constituents of the oils from stems and roots. Extensive research on Aristolochia species by our group has revealed that A. mollissima, A. heterophylla and A. cucurbitifolia were rich sources of sesquiterpenoids [313,314]. Moreover, sesquiterpenoids were widely distributed in the stems and roots of these species rather than in leaves. Seven sesquiterpenoids, madolin A-E (300, 299, 296, 295, 271) as new and aristolactone (278) and manshurolide (307) as known,were isolated from the stems and roots of A. cucurbitifolia, an endemic species of Taiwan [159,315]. The only difference in the structures of madolins A (300), B (299) and C (296), all possessing 1,10-epoxybicyclogermacrane skeleton was the functionality of C-14: an aldehyde group in the former, with carboxylic acid group in the second and with simple hydrogen in the third. Madolin-D (295), with bicyclogermacrane skeleton is an acetyl derivative
903
Table 17. Sesquiterpenoids Isolated from Aristolochia Species Compound Farnesanes CH3
CH3
Name
Occurrence
Ref.
P-Farnesene (260)
A. argentina A. elegan A. macroura
296 308 301
Faraesol (261)
A. elegans A. peltato-delloidea
308 352
Z-(ft)-Nerolidol (262)
A. elegans
308
£-(/?)-Nerolidol (263)
A. brevipes A. elegans A. gibertii A. gigantea A. macroura A. triangularis
304 308 300 301 301 301,30 2, 303
Bisabolanes CH,
P-Bisabolene (264)
A. acutifolia A. chilensis
299 312
CH 3
ot-Bisabolene (265)
A. elegans A. asclepiadifolia A. impudica
308 305 353
CH 3
P-Sesqui- phellandrene (266)
A. acutifolia A. chilensis
299 312
CH,
CH,
CH3 HC
CH2
CH,
CH3 - ^ ^ - ^ ^ i H3C^ JOH
c c c
H c'
J
j
H
H
904 CH,
a-Curcumene (267)
A. elegans
308
(3-Elemene (268)
A. acutifolia A. argentina A. brevipes A. chilensis A. debilis A. elegans A. gibertii A. rodriguesii A. triangularis
Y-Elemene (269)
A. elegans A. giganiea A. macroura A. triangularis
299 296 304 312 298 308 300 301 301, 303 308 301 301 301, 303
8-Elemene (270)
A. argentina A. elegans A. gibertii A. macroura
296 308 300 301
Madolin E (271)
A. cucurbitifolia A. heterophylla
159, 315 228
Madolin R (272)
A. mollissima
204
Madolin S (273)
A. mollissima
204
Elemanes
CH2
CH,
1
CH,
Germacranes
905 Germacrene A (274)
A. brevipes A. birostris A. impudica
304 301 353
Germacrene B (275)
A. elegans
308
Germacrene D (276)
A. argentina A. brevipes A. elegans A. gibertii A. odoarlissima
296 304 308 300 354
Hedycaryoi (277)
A. elegans A. peliato-deltoidea
308 352
Aristolactone (278)
A. conlorta A. cucurbitifolia A. gibertii A. helerophylla A. kaempferi A. kunmingensis A. liukiuensis A. mollissima
230 159, 315 300 228 244 187 328 200, 203, 204, 329 281, 318 281, 318 222
.CH, CH-,
CH,
CH,
CH,
H,C
CH,
CH,
•O
CH3
A. reticulata A. serpentaria A. versicolar
Isoaristolactone (279)
A. versicolar
222
Madolin J (280)
A. heterophylla
228
906 Versicolactone B (281)
O
CH3
A. heterophylla A. mollissima A. versicolar
203,
Madolin X (282)
A. mollissima
204
Madolin U (283)
A. mollissima
204
Madolin I (284)
A. heterophylla
228
Costunolide (285)
4. yunnanensis
333
Mollislactone (286)
A. elegans A. mollissima
308 200
Melompolide (287)
A. yunnanensis
333
Bicyclogermacrene (288)
A. argentina A. elegans A. gibertii
2% 308 300
228 204 322
CH,
O
H,C
Bicyclogermacranes
907
OHC
(+)-Isobicyclogermacrenal (289)
A. heterophylla A. kaempferi A. manshuriensis A. mollissima
232 244 325 203, 204
Madolin P (290)
A. kaempferi
244
Madolin K (291)
A. heterophylla A. mollissima
238 204
Madolin T (292)
A. mollissima
203, 204
Madolin Y (293)
A. mollissima
317
Madolin N (294)
A. heterophylla
232
Madolin D (295)
A. cucurbitifolia
159, 315
Madolin C (296)
A. cucurbitifolia
159, 315
H3C^CH
I^CH3
CH,
C
°°H
A/ OHC
HjC^Y^
0H
H3C
\Jr\\.,
OHC
OHC
\jr\\^,
ri3^-
CH OAc
H3C^^CH H2C
H3C H2C
n3^
OH
^tH OH
CH OAc CH3
908 CH 3
\
Madolin 0 (297)
A. heterophylla A. kaempferi
232 244
Madolin V (298)
A. mollissima
203, 204
CH3
Madolin B (299)
A. cucurbitifolia A. heterophylla A. kaempferi A. mollissima
159, 315 228 244 204
\ H CH3
Madolin A (300)
A. cucurbitifolia A. heterophylla A. kaempferi A. mollissima
(-)-Lepidozenal (301)
A. heterophylla A. kaempferi A. mollissima
159, 315 228, 232 244 203, 204 228 244 204
1,10-Epoxylepidozenal (302)
A. heterophylla A. mollissima
228 204
a-Humulene (Caryphyllene) (303)
A. argentina A. birostris A. elegans A. gibertii A. indica A. macroura A. papillaris
296 301 308 300 307 301 301
"'•£-)
OHC
H3C H3C
\ H
Tj
''',
nnp
C^
3
HOOC
OHC
CH O Ac
H3C
H3C H,C
OHC
OH
H,C
\
H
\ H
3
CH3
OHC
H3C^^CHi Humulanes CH,
H3C-J H,C
1
909 H,C.
Humulene epoxide II (304)
A. acutifolia
299
Madolin M (305)
A. heterophylla A. mollissima
228 204
Madolin L (306)
A. heterophylla
228
Neoaristolactone (manshurolide; versicolactone A) (307)
A. cucurbitifolia A. heterophylla A. manshuriensis A. mollissima A. versicolar
159, 315 228 327 204 320, 321
Versicolactone D (308)
A. heterophylla A. kunmingensis A. versicolar
228 187 324
Madolin W (309)
A. mollissima
204
CH3
H3CO*" CHO
HO
Madolins
v/
CHO
910 AcO,,
Madolin H (310)
A. heterophylla A. mollissima
228, 316 204
MadolinG(311)
A. heterophylla
228, 316
Madolin Z (312)
A. mollissima
317
(3-Caryophyllene(313)
A. acutifolia A. argentina A. brevipes A. chilensis A. debilis A. elegans A. gibertii A. indica A. longa A. melanoglossa A. ringens A. argentina A. elegans
299 296 304 312 298 308 300 84, 307 311 355 309
A. acutifolia A. elegans A. indica A. longa A. melanoglossa A. peltato-delloidea A. chamissonis A. pubescens
299 308 307 311 355 352
CHO
AcO/,,,
CHO
OH
CHO Caryophyllanes H3C
H
Isocaryophyllene (314)
H3C^ ' V HC I
\, % *CH, ^_V
Caryophyllene oxide (315)
8/?,9«-Oxide-pcaryophyllene(316)
H,C
/
296 308
356 357
911
H,C
H ^ )
A. pubescens
357
Caryophyllenol 1(318)
A. elegans
308
a-Selinene (319)
A. acutifolia
299
Selina-3,7(ll)-diene (320)
A. argentina
296
8-Selinene(321)
A. peltato-deltoidea
352
(3-Selinene (322)
A. acutifolia A. brevipes A. gibertii
299 304 300
5PH, 7|3, lOa-Selina4,ll-diene(323)
A. indica
335
Aristolochene (324)
A. indica
334
a-Eudesmol (325)
A. acutifolia A. peltato-deltoidea
299 352
'OH
CH Eudesmanes CH,
c:H,
Kobusone(317)
CH,
CH,
H ( • H, CH,
C
\ C^H, CH,
(:H,
do T
YP
TCH,
L/CH,
Ti CH, ,,lfCH,
i CH, CH, CH,
CH,
CH, CH,
CH,
912 CH
P-Eudesmol (326)
A. brevipes A. odorntissimn
304 354
a-Cyclocostunolide (327)
A. yunnanensis
333
Isoalantolactone (328)
A. debilis
337
Dihydroisoalantolacto ne(329)
A. debilis
337
(5-Gurjunene (330)
A. argentina
296
A1(10)-Aristolene (calarene)(331)
A. brevipes A. arcuata A. chilensis A. debilis A. elegans A. longa A. papillaris A. contorta
304 188 312 344 308 311 301
Aristolanes
i CH3 CH 33
CH3
"K CH3 CH,
I CH3 CH3 A-Aristolene (332)
CH3
I CH3 CH,
CH,
9-Aristolen-la-ol (333)
A. debilis
230 344
. peltato-deltoidea
352
913
i CH3 CH3
^f,,, 1 'CH3 CHO
^ CH, CH3
l(10)-Aristolen-13-al (334)
A. debilis
A'"0)-Aristolone (2oxocalarene) (335)
A arcuata A. clematitis A. debilis
I CH3 CH3
3
346
A -Aristolenone (336)
/(. debilis
188 153 338, 339, 344 311 ~344~
A1(10)'8-Aristolodien-2one (337)
A. debilis
345
A'uu)-Debilone (338)
A. debilis
344
9a-Hydroperoxy1(10)- aristolenone (339)
A. debilis
345
A9-Aristolone(-)aristolone) (340)
A. albida A. debilis
99 344
1 a-Hydroxy-9aristolenone (341)
A.debilis
345
A. longa
I CH3 CH3
CH3
345,
C CH, OH
OOH
= CH3 CH3
I CH3 CH,
CH,
914 CH,
HO* 1 H CH,
Maaliol (342)
A. longa
Madolin F (343)
A. heterophylla
228, 316
5-Cadinene (344)
A. acutifolia A. debilis A. elegans A. odoratissima A. papillaris
299 298 308 354 301
Y-Cadinene (345)
A. acutifolia A. debilis A. elegans A. gibertii A. papillaris
299 298 308 300 301
y-Muurolene (346)
A. acutifolia A. elegans
299 308
T-Muurolol (347)
A. elegans
308
8-Cadinol (348)
A. ovalifolia
306
""K 1 CH, CH3 CH
Cadinanes
915 HO
T-Cadinol (349)
A. elegans
308
a-Cadinol (350)
A. acutifolia A. brevipes A. elegans
299 304 308
Cubeno1(351)
A. elegans
308
e/?/-Cubenol (352)
A. elegans
308
Calamenene (353)
A. brevipes A. debilis A. elegans
304 298 308
CH2
p-Calacorene (354)
A. giberlii
300
CH3
a-Calacorene (355)
A. brevipes A. elegans A. giberlii
304 308 300
CH
HJC^^CHJ
II
1 1
CH3 OH? '
0
H
3
C ^
916 CH3
Cadalene (356)
A. brevipes
304
0
CH3
Mansonone G (357)
A. liukiuensis
328
o
X
Dehydrooxoperezinon e (358)
A. manshuriensis A. liukiuensis
325 328
a-Guaiene (359)
A. brevipes A. debilis
304 298
Guaiol (360)
A. chilensis A. elegans A. asclepiadifolia
312 308 305
8-Guaiene(361)
A. debilis
298
Bubnesol (362)
A. brevipes
304
H,C Guaianes H
VM
H3C
^-CH
3
CH3
H,C^H" 3 H3C
H3C
H
>-CH3 H,C
H3C
C; H3C
H
A~CH3 H3C
OH
917 H3C
3
'
6,9-Guaiadiene (363)
A. elegans
308
Guaiazulene (364)
A. elegans
308
y-Gurjunene (365)
A. aculifolia
299
DehydrocostuslactoneA(366)
A. yunnanensis
333
Madolin Q (367)
A. heterophylla
314
Versicolactone C (368)
A. heterophylla A. versicolar
228 324
P-Aromadendrene (369)
A. chilensis
312
H,C H3C
H3C
^ C H
3
H,C H3C H ':
vC)
H3C
U~CHl
H7C H2C H U
O
CH
H C
n~
2
°
O Aromadendranes H2C J_T
H3C
H
\\
'""f^
918 H2C
H
HP
>L H3C
L
H3^SC H3C H2C
"3 1 "
H
A. chilensis A. debilis A. gibertii
312 298 300
fl//o-Aromadendrene (371)
A. acutifolia A. brevipes A. gibertii
299 304 300
Spathulenol (372)
A. acutifolia A. argentina A. chamissonis A. elegans A. gibertii A. melanoglossa A. mollissima A. peltato-deltoidea A. brevipes A. indica A. ovalifolia A. asclepiadifolia
299 296 356 308 300 355 204 352 304 351 306 305
Globulol (374)
A. elegans
308
Viridiflorol (375)
A. elegans
308
Aromadendrane4p,10p-diol (376)
A. heterophylla
228
"3
LHi
1H HO
^J AN-u H3C C " 3 HQ CH3
C
5
C
"3
7^ H,C C H 3 H3C OH
H^C
H3C
7^ H,C C " 3 HO H
H0
H
(+)-Ledol (373)
^
H,C H3C pU
H3<
a-Aromadendrene (370)
V^
H,C Cubebanes
CH3
919 H3Q
"3C v / H3C
H H,C Patchoulanes H3C
H,C H 3 C Himachalanes CH2 O
a-Cubeben (377)
A. brevipes A. debilis A. elegans
304 298 307
B-Cubeben (378)
A. elegans
307
Cyperene (379)
A. argentina
296
a-Himachalene (380)
A. chilensis A. elegans
312 308
Longifolene(381)
A. elegans
308
Isolongifolene (382)
A. elegans
308
B-Chamigrene (383)
A. debilis
298
CH3
1 1 M
H3C C H . Longifolanes H3C CH3
CH,
Chamigranes CH3
YCL CH , Cedranes
920 CH,
ct-Cedrene (384)
A. chilensis
I 312 i
a-Santalene (385)
A. debilis
298
a-iraHS-Bergamotene (386)
A. acuiifolia A. argentine! A. brevipes A. chilensis A. giganlea
299 296 304 312 301
(-)-oc-Copaene (387)
A. acutifolia A. argentine! A. brevipes A. elegans A. gibertii A. macroura A. papillaris A. rodriguesii A. triangularis
299 296 304 308 300 301 301 301 301, 303
Ishwarane (388)
A. argentina A. indica
296 231, 307
Ishwarol (389)
A. indica
307,
Santalanes
CH,
H,C,
•CH,
Copaanes
H3C
CH3
Ishwaranes
OH
350
(12S)-7,12Secoishwaran-12-ol (390)
A. indica
92, 184, 351
Ishwarone (391)
A. indica
307,
348
921
i CH3 CH, Bourbonanes H CH3
'/
tj
O
HO
A. debilis
344
P-Bourbonene (393)
A. nrgentina A. brevipes A. elegans A. giberlii A. rodriguesii
296 304 308 300 301
Bourbonanol (394)
A. elegans
169
Aristololide (395)
A. elegans
171
V CH
H,C H CH3
3
3-Oxoishwarane (392)
H H
3
y^cHj H,C
O.
H CH3
\\ , L /"0Ac H3C
H H
VcH3 H,C
of madolin-N (294). Madolin-E (271) is an elemane-4,6-y- lactone and the lactone ring is formed by the oxidation of 15-methyl group of elemane followed by lactonization to C-6. Madolins A (300), B (299), E-M (271,343,311,310,284,280,291,306,305) and Q (367), versicolactones AD (307,281,368,308), aromadendrane-4p,10P-diol (376), aristolactone (278) and 1,10-epoxylepidozenal (302) were discovered from the roots and stems of A. heterophylla [228,316]. Madolin F (343) is a tricyclic sesquiterpene with />ara-quinone function belongs to a novel skeleton, normaaliane type [316]. Madolin G (311) and H (310) were bicyclic sesquiterpenes with novel skeleton named as madolin type which consists of a three- and an eleven membered rings [316]. Madolin G (311) is a 10,11-epoxide of madolin H (310). Madolin I (284) and J (280) were very similar to aristolactone (278), but first one having ketone and a terminal methylene groups at C-l and C-10, respectively instead of a double bond, whilst the second contains (3,y-unsaturation in the lactone ring [228]. Madolin-K (291) is a bicyclogermacrane identical with isobicyclogermacrenal (289) and possessing cis geometry at C-4 and -5 and an exo 12-hydroxymethyl group [228]. Madolin L (306) has a C-l2 membered ring with three E- configurated double bonds, an aldehyde at C-l and a
922 methoxyl at C-4 with R stereochemistry. The only difference between madolin M (305) and L (306) is the Z-configuration of A2'3 double bond in the former instead of ^-configuration in the latter [228]. Madolin-Q (367) considered to be an artifact formed in the course of extraction and separation with CHC13. The CC13 radical obtained from CHC13 was substituted on C-l position. Madolin Q (367) has a rearranged guaiane skeleton with a 4,6-y-lactone [314]. The spectral data of aromadendrane4p,10P-diol (376) was found to be similar to aromadendrane-4a,10p-diol, isolated from Brasilia sickii, however, a single crystal X-ray analysis and NOESY experiments indicated that the hydroxyl groups at C-4 and -10 have P- relative stereochemistry [228].137Madolin N (294) and O (297) along with madolin A (300), (-)-lepidozenal (301) and isobicyclogermacrenal (289) have been isolated from the leaves of A. heterophylla [232]. Madolin N (294) has a bicyclogermacrane skeleton with a ketone function at C-l, an aldehyde at C-4 and a terminal methylene group at C-10. Madolin-0 (297) is very close to madolin-N (294), but having an epoxide and a methyl group on C-l and -10 instead of ketone and terminal methylene groups. A bicyclogermacrane type sesquiterpenoid with a C-l4 carboxylic acid, madolin P (290) was isolated from stems and roots of A. kaempferi [244]. It is very similar to madolin B (299) but having a C-l/10 double bond instead of an epoxide. Sesquiterpenoid metabolites, madolin R-Z (272,273,292,283,298,309, 282,293,312) along with several other known sesquiterpenoids have been reported from A. mollissima [203,204,317] growing in Mainland China. Two interesting products, madolin R (272) and S (273) are the only examples, having rearranged elemane frame work, with 2-substituted propyl side chain and an aldehyde group attached to C-6 and C-4, respectively of the elemane skeleton. Madolin S (273) is, essentially, the methyl derivative of C-l 2 hydroxy group of madolin R (272) [204]. Madolin T (292), V (298) and Y (293) are bicyclogermacranes with a conjugated formyl group. Madolin T (292) and V (298) were acetyl derivatives of madolin K (291) and O (297), respectively [203]. Moreover, madolin V (298) is an epoxide of madolin T (292) [203]. Madolin Y (293) is very close to madolin N (294), the only difference is the presence of a hydroxyl group at C-l instead of keto function [317]. Thus, biogenetically, it may obtained by the reduction of C-l keto group ofmadolinN[287].
923 Madolin U (283), W (309), X (282) and Z (312) were discovered as minor sesquiterpenoid metabolites from roots and stems of A. mollissima [204,317]. Madolin W (309) and Z (312) are 4,6-cyclohumulane derivatives belongs to madolin type sesquiterpenoids. The only difference between madolin W (309) and Z (312) were the presence of a C10/11 vinyl methyl group in the former instead of a terminal methylene and an additional hydroxyl group in the second compound. Madolin U (283) and X (282) were 4,6-y-lactones of germacrane type sesquiterpenoids. Madolin U (283) is quite similar to madolin I (284), but with a hyrodxyl group at carbon C-l instead of a carbonyl group. Madolin X (282) was structurally closely related to versicolactone B (281), but having an opposite geometry (Z) at A910 double bond. A sesquiterpenoid lactone, aristolactone (278) has first been isolated from A. reticulata, then from A. serpentaria and was shown to be a germacrene type sesquiterpene with P,y-unsaturated lactone by Williams and co workers [281,318]. Later, Smith et al. [319] revised the structure of aristolactone (278) as a,P-unsaturated lactone. Isoaristolactone (279), isolated from A. versicolar was quite similar to aristolactone (278), but just differ in the position of double bond [222]. Zhang et al. [320-322] have isolated versicolactone A (307), B (281) and C (368) from the roots of A. versicolar collected in China. Versicolactone A (307) is an humulane type sesquiterpenoid with 12-membered ring and an ct,p-unsaturated-y-lactone ring. It was also named as manshurolide and neoaristolactone [320,321]. Versicolactone B (281) is a germacrane type sesquiterpenoid with an a-oriented hydroxyl group at C-l position. Versicolactone C (368) was neither a psuedoguaianolide nor guaianolide. From the X-ray analysis it was found that, it has a novel hydroazulene skeleton with a- and P- oriented hydroxyl groups at C-l and C-5 positions, respectively. The biogenesis of versicolactone C (368) from the versicolactone B (281) was proposed through addition of H2O and cyclization [322,323], Scheme (1). A structurally interesting sesquiterpenoid lactone, versicolactone D (308) was also obtained from the roots of A. versicolar [324], for which the structure was characterized with the aid of X-ray analysis and found to be possessed a 12-membered ring, 10-membered ring with a y-lactone and a 6-membered ring. Finally, it was assumed that this sesquiterpene lactone was formed by the condensation of aristolactone (278) and versicolactone A (307). These two molecules connected at 4, 5 and 6 , 1 5 , respectively, which resulted in the formation of a new 6-membered ring. Rucker et al. [325] isolated
924 OH
rr
OH
Scheme (1). The proposed biogenesis of versicolactone C
(+)-isobicyclogermacrenal (280) from the stems of A. manshuriensis of Korean origin. It was identical in all respects with (-)-isobicyclogermacrenal, isolated from the liverwort, Lepidozia vitrea [326], but having opposite optical rotation. It is a (+)-(6S,7/?)-enantiomer of (-)(6i?,7.S)-isobicylcogermacrenal. A sesquiterpene lactone, manshurolide (307) was reported from the stems of this species [327]. It has a 12membered ring with an a,p-unsaturated-y- lactone ring. This is the first report of the occurrence of a 12-membered ring among the sesquiterpenes. Recently, P. L. Wu et al. [241] reported a tricyclic sesquiterpene, dehydrooxoperezinone (358) from the stems of A. manshuriensis of Chinese origin. Its structure with (9-naphthoquinone basic skeleton is very similar to mansonone G (357), isolated from A. liukiuensis [328], but having an additional five membered ring which is formed by cyclization between C-2 and C-8a through oxygen. Three sesquiterpene lactones [200,201,203,204,329-332], aristolactone (278), neoaristolactone (307) and mollislactone (286) were isolated from radix of A. mollissima. Mollislactone (286) has a novel skeleton with 10- membered ring, it may be formed from the germacrene skeleton via rearrangement [200,201]. Versicolactone D (308) and aristolactone (278) were also separated from the aerial parts of A. kunmingensis [187]. Sesquiterpene lactones, costunolide (285), dehydrocostuslactone A (366), a-cyclocostunolide (327) and melampolide (287) were characterized from the underground parts of A. yunnanensis [333] growing in Yunnan province of China. Among them, costunolide (285) and dehydro- costuslactone A (366) were identified as germacranolide and guaianolide, respectively, whereas occyclocostunolide (327) as eudesmanolide. The last compound, melampolide (287) was characterized as l(10)-czs- costunolide by X-ray analysis and it possessed non-oxygenated melampolide skeleton. Nine eudesmane derived sesquiterpenes were isolated from Aristolochia species. Among them, a- and P- eudesmols (325), (326), a-, p- and 5- selinenes (319), (322), (321) and selina-3,7(ll)-diene (320) were found in the essential oils of various Aristolochia members. Govindachari et al. [334] isolated aristolochene (324), a sesquiterpene
925 hydrocarbon from the roots of A. indica. It has an eremophilane type carbon frame work. Aristolochene (324) represents structurally the simplest member of the biogenetically interesting and steadily growing group of eremophilane type sesquiterpenoids. Another sesquiterpene hydrocarbon belonging to the eudesmane group, 5p//,7p,10a-selina4(14),ll-diene (323) has also been isolated from A. indica by Govindachari et al. [335] Catalytic hydrogenation over PtO2 in ethanol gave two stereoisomeric saturated hydrocarbons one of which was enantiomeric with P-selinene (322). Formation of this product during hydrogenation was due to epimerization at C-7, possibly through the migration of double bond of the isopropenyl group, which is well documented in the sesquiterpenoids bearing an axial isopropenyl group [336]. From the biogenetic point of view, the co-occurrence of aristolochene (324) and 5p//,7p,10a-selma-4(14), 11-diene (323) in the same plant suggests that both are derived from a common precursor. Wall et al. separated two lactones, isoalantolactone (328) and dihydroisoalantolactone (329) from the antimutagenic fractions from A. debilis [337]. Fourteen aristolane derived sesquiterpenes were reported so far from the Aristolochia species and this is one of most abundant sesquiterpenoid groups of Aristolochia. Moreover, aristolanes are particularly abundant in the essential oils obtained from the roots of A. debilis. The aristolone (335), an aristolane type sesquiterpenoid component of the essential oil of A. debilis [338-341] was reported to contain a double bond on one side and cyclopropane ring on the other side, conjugated with the ketone group on the basis of the analyses of its reduction products: dihydroaristolone, deoxoaristolone and aristolol. Aristolone (335) has a hydronaphthalene skeleton with ketone group at C-2 position and a methyl group at C-5 position. The underground parts of A. debilis also gave aristolane sesquiterpenoids, A1(10>-331 and A9- aristolenes (332), A1<10)-335 and A9aristolones (340) and debilone (338) [342-344]. Among these, A1(10)-331 and A9- aristolones (332) are simple aristolane sesquiterpene hydrocarbons and just differed in the position of double bond. The A1(10)-aristolone (335), debilone (338) and A9- aristolone (340) have keto function at C-8 and C-2 positions, respectively. Debilone (338) possessed an additional oxygenation as hydroxyl group on C-9. Further studies by Rucker et al. on underground parts of A. debilis have also afforded oxidized aristolane sesquiterpenoids [345]. The 9oc-hydroperoxy-l(10)- aristolenone (339) has hydroperoxy group on C-9 in a-orientation. This compound is also
926 obtained up on treatment of A1(10)- aristolone (335) with oxygen. Oxidation opposite to the cyclopropane ring is obviously preferred in aristolane sesquiterpenoids. la-Hydroxy-9-aristolenone (341) possesses the aristolane structure as A9-aristolone (340), but additionally bears a hydroxyl group at C-l in the axial orientation of the chair conformation of ring A. The l(10)-aristolen-12-al is an aristolane sesquiterpene aldehyde. Rucker et al. proposed exo position for the aldehyde group and cis configuration for the aldehyde and cyclopropane protons. As the aldehyde group is in a position unusual for sesquiterpenes, l(10)-aristolen-12-al is formed biogenetically by oxidation of the exo methyl group. Later extensive NMR studies, by Rodriguez et al. [346] established that the structure of this substance must be amended to l(10)-aristolene-13-al (334). Exhaustive NOE experiments provided conclusive proof on the endo configuration of the aldehyde group, which must be at C-l3 instead of the C-l2 exo position, consequently methyl has an exo configuration. It is reasonable to assume that l(10)-aristolen-12-al has a preferred rotamer for the ewdo-aldehyde group owing to the electrostatic repulsion between the C-l/C-10 olefinic double bond and the oxygen atom of the aldehyde. This preferred rotamer, with aldehyde hydrogen close to the olefinic bond (ewdo-conformation), precludes the W-coupling of the aldehyde proton with the H-6a and H-7a cyclopropane protons, thus explaining the mistake in the structure previously attributed to this sesquiterpene. Aristolodien-2-one (337) is the only aristolane sesquiterpenoid contained two double bonds in both rings conjugated to keto function.Teresa et al. found that only tricyclic sesquiterpenes, maaliol (342) with maaliane skeleton were present in the roots of A. longa [311], while from the aerial parts it was possible to isolate sesquiterpenes, P-caryophyllene (313) and caryophyllene oxide (315) with caryophyllene skeleton and some monoterpenes. This is the first report of maaliol (342) with the maaliane skeleton in the genus Aristolochia. Rao et al. isolated ishwarone (391) as one of the chief constituents of the roots of A. indica [347]. Subsequent investigations by Ganguly et al. and Govindachari et al. led to the first structural assignment of ishwarone (391) as a novel tetracyclic sesquiterpene ketone based on eremophilane skeleton, the first of its kind to occur in the nature [348,349]. Ishwarane (388), reported from the roots of A. indica by Govindachari et al. is the first tetracyclic sesquiterpene hydrocarbon based on the eremophilane skeleton [334]. A tetracyclic sesquiterpene alcohol, ishwarol (389) is also reported from the roots of A. indica [350]. Pakrashi et al. isolated (125)-
927
390 Scheme (2). The proposed biogenetic sequence for (12S)-7,12-Secoishwaran-12-ol (390)
7,12-secoishwaran-12-ol (390) as one of the active principles [92,184,351] from the petroleum ether extract of the roots of A. indica, with 100% interceptive activity in mice at a single dose of 100 mg/ kg. Pakrashi et al. also proposed that biogenetically, this sesquiterpene is derivable from the common carbonium ion that might be considered as the immediate precursor to ishwarane (388) [351], Scheme (2). 3Oxoishwarane (392) was detected as the sole ishwarane (388) sesquiterpene component from A. debilis collected from Mainland China and it contains carbonyl function at C-3 [344]. It is interesting to note that ishwaranes isolated from Indian species having an oxygen function at C-l whereas at C-3 in the Chinese species. Only three bourbonenes have been isolated from Aristolochia species. One of them, P-bourbonene (393) was found in the essential oils of several species viz. A. argentina [296], A. brevipes [304], A. elegans [308] and A. gibertii [300]. The other two, bourbonanol (394) and aristololide (395) were isolated from A. elegans [171]. Boubonanol (394) is very similar to bourbonene (393), the only difference being the presence of a hydroxyl group and methoxyl groups instead of an exocyclic methylene group on the carbon C-4. The last compound aristololide (395) possessed a structure which was somewhat different from that of other bourbonanes and the differences were the oxidation of C-2, migration of A415 and the presence of an acetyl group at C-8. Diterpenoids Diterpenoids are also a large group of metabolites from Aristolochia, however their distribution is limited to few species. Mainly, three types of diterpenoids, clerodanes, labdanes, and kauranes were encountered so far in the Aristolochia species (Table 18). However, is-phytol (396) a sole acyclic diterpenoid, was found as the essential oil component of A. elegans [308], A. odoratissima [354] and A. peltato-deltoidea [352]. Diterpenoids, (+)-(4 2)-aZ>eo-kolavelool-3-oicacid (397), (-)-13-epr- 2oxokolavelool (399), (-)-2p-hydroxykolavelool (401), (-)-2p-hydroperoxykolavelool (402), (+)-13-epz'-2o>hydroxykolavelool (400), (-)kolavelool (398) and (-)-3a,4P-dihydroxykolavelool (403) were isolated
928
Table 18. Diterpenoids Isolated from Aristolochia Species Structure Phytanes
Compound
Source
Ref.
(£)-Phytol (396)
A. elegans A. odoratissima A. peltato-deltoidea
308 354 352
2)-o6eo-kolavelool3-oic acid (397)
A. chamissonis
356
Kolavelool (398)
A. chamissonis A. cymbifera A. galeata
356 267 363
13-ep/-2-oxokolavelool (399)
A. chamissonis
356
(+)-13-epi-2a-hydroxykolavelool (400)
A. chamissonis
356
(-)-2[5-hydroxykolavelool (401)
A. chamissonis
356
("*H
H C
HOH C^^V^^^^^^T CH, Clerodanes
CH, OH
(+)-(4
J "CH3
HO 2 C—4,
OH
TH? 3
OH
1 CH CH3 3 OH
H | HO"""
] ^
f""CH3 #
|' 'CH 3 C H 2
TH™ 3 OH
CH3
3
929
A. chamissonis
356
(-)-3a,4p-dihydroxykolavelool (403)
A. chamissonis
356
A1314-Kolavenic acid (404)
A. brasiliensis A. galeala
360 363
Kolavenic acid methyl ester (405)
A. esperanzae A. galeata
360 363
(2S, 5R, 8 R, 9S, \0R)-2Hydroperoxy-en/-3cleroden-3,13 -diene-15-oic acid methyl ester (406)
A. esperanzae
360
A1-Kolavenol (407)
A. galeata
363
rel-(5S, &R, 9S, ]0R)-entClerod-3,13- diene-15-oic
A. brasiliensis
360
hydroperoxykolavelool (402) CH,
CH,
CH, rcHCOOCH3
CO,H
CH,
acid (408)
930 CH,
A 1314 -2-Oxokolavenic acid
A. brasiliensis
360
2-Oxokolavenicacid methyl ester (410)
A. esperanzae
360
Populifolic acid (411)
A. brasiliensis A. cymbifera A. galeata
360 267 363
Populifolic acid methyl ester (412)
A. esperanzae A. galeata
360 363
Dihydrokoiavenol (413)
A. galeata
363
(2S,5R$R,9S,\0R)-2Hydroperoxy-enf-3cleroden-15-oic acid methyl ether (414)
. esperanzae
360
2-Oxopopulifolic acid (415)
A. brasiliensis A. cymbifera A. galeata
360 267 363
(409) ,,CHjCO2H
I""CH3COOCH
CO 2 H
-,, C H CO 2 H
931
CH,
2-Oxopopulifolic acid methyl ester (416)
A. esperanzae
360
rel-{5S, %R, 95, 10/?)-2-oxo • ent-3- cleroden-15-oic acid (417)
4. brasiliensis
360
e/?('-Populifolic acid (418)
A. cymbifera
267
Columbin(419)
A. albida
364
e«/-Labd-8P-ol-14-ene (420)
A. cymbifera
363
A13l4-e«r-Labdan-8|3-ol-15oic acid (e«(-labd-13-ene8P-ol-15-oic acid) (421)
A. galeata
363
-,CHCO2CH3
I CH3C°2H
COOH
Labdanes
H,C
CH
.„„ CO22H "OH
1
H,C
CH,
932 Copalic acid (422)
A. cymbifera A. esperanzae A. galeala
363 363 363
en/-Labd-8|3-ol-15-oic acid (423)
A. galeata
363
en/-Labd-6p-ol-8(17),13dien-15-oic acid (424)
A. esperanzae
363
e«r-16P(H)-Kaurane (425)
A. Iriangularis A. elegans
270 366
Kauranal (426)
A. elegans
366
Kauranoic acid (427)
A. elegans
366
(-)-Kaur-16-en(428)
A. acutifolia A. argenlina A. chilensis A. Iriangularis
299
Kauranes
v
AH H,C
CH,
AH H,C
CHO
H,C
t\ H COOH
296 312 302,303
933
H,C
H,C
H,C
H,C
303 366
ent-\ 6(3,17-Epoxykauran (430)
A. triangularis A. elegans
270 169
(-)-Kaur-16-ene-18-ol(431)
A. triangularis
269
ent-3f>, 19-Dihydroxy- kaur16-ene(432)
A. rodrigueisii
365
(-)-Kaur-16-en-18-al(433)
A. triangularis
269
(-)-Kaur-16-en-18-oic acid (434)
A. triangularis A. rodrigueisii
269 365
(-)-Kaur-16a-ol-18-a1(435)
A. triangularis
269
CH?OH •CH
H,C
A. triangularis A. elegans
CH,
- _ C CH,
H,C
16a, 17-Epoxykauran (429)
CH,OH
CHO
COOH
CHO
934
H,C
16o-Hydroxy-(-)-kauran-l 9al (436)
A. rodrigueisii
365
(-)-Kauranol (437)
A. rodrigueisii
365
e«M 60,19Dihydroxykaurane(438)
A. rodrigueisii
365
e«<-Kauran-16p, 17-diol (439)
A. elegans A. pubescens A. triangularis
208,357 270
en/-16(3, 17-Dihydroxy-(-)kauran-19-oic acid (440)
4. rodrigueisii
365
en/-16p, 17-Isopropylidenedioxy-(-)- 19-oic acid (441)
A. rodrigueisii
365
(-)-Kaur-15-en-17-ol (442)
A. elegans A. pubescens A. triangularis
168, 169 208 270
CHO
CH,OH
H,C
168,169
COOH
H,C COOH
H,C
CH,
935
H,C
H,C
17-Hydroxy-en/-kaur-l 5-en19-oic acid (443)
A. rodrigueisii
365
ent-\ 5(3,16(3-Epoxykauran17-ol (444)
A. elegans A. triangularis
168, 169 270
15a, 16a-Epoxy-17hydroxy- enJ-kauran-19-oic acid (445)
A. rodrigueisii
365
enr-16(3(H)-Kauran-17-oic acid (446)
A. triangularis
270
COOH
- VH COOH
HjCCH,
from a Brazilian species A chamissonis [356]. All these diterpenes belong to the same ew^-clerodane series and their ent clerodane absolute stereochemistry was established by application of the reversed Octant rule [358], NMR and X-ray analysis as 5R, SR, 9S, 10R and 13/?. The first compound is an unique example of clerodane derivative isolated from Aristolochia with a rearranged (4->2)-afeeo-clerodane skeleton. It has been suggested that this abeo clerodane diterpene could be formed from kolavenic acid via oxidative cleavage followed by Aldol conden- sation pathway, since the yielded rearranged aldehyde could be oxidized into the corresponding carboxylic acid [359]. It is interesting to notice that (+)-13epz'-2a-hydroxykolavelool (400) is unstable, being rapidly transformed into (-)-13-e/>/-2-oxokolavelool (399) under storage. The diterpene, (-)2p-hydroperoxykolavelool (402) having p-hydroperoxy group at C-2 is also not stable and is easily transformed into its 2-oxo derivative. Twelve clerodane diterpenes 404-406, 408-412, 414-417 have been reported from the stems of A. brasiliensis and roots of A. esperanzae, collected in Brazil
936 [360]. It constitutes the first report of the occurrence of clerodane diterpenes in Aristolochiaceae. Treatment of 412 with m-chloroperbenzoic acid resulted in the oxidation of C-2, the reaction product was identified as 416. The formation of this carbonyl compound was explained by an acid promoted 1,2-nucleophilic rearrangement of the epoxide initially formed in the reaction [361]. The presence of peroxide function on C-2 in 414 and 406 was confirmed by dehydration of 414 with acetic anhydride and pyridine to 416 [362]. The compounds 414 and 406 differ only by the saturation 414 or unsaturation 406 at C-13. These two compounds undergo rapid decomposition. On the basis of rapid conversion of the peroxy derivatives 414 and 406 to their corresponding carbonyl derivatives 416 and 410, it was suggested that the 2-oxo-3- clerodene diterpenoids could be artifacts. Among these diterpenes, 415, 416, 404, 412 and 414 possessed a saturated side chain, whereas 409, 410, 417, 408, 405 and 406 contain a double bond between carbons C-13 and -14. It is of interest to note that diterpenes 415, and 417 are the only diterpenes possessing cis stereochemistry at the junction of A and B rings. Lopes et al. [363] examined three Brazilian Aristolochia species A. cymbifera, A. esperanzae and A. galeata and reported labdane and clerodane diterpenoids. Extraction of the leaves of A. cymbifera gave two labdanes, copalic acid 422 and e«f-labd-8P-ol-14-ene 423. Compound 423 has a hydroxyl group at C-8 in the axial orientation. Two labdane diterpenes were isolated from the leaves of A. esperanzae. The structures of these compounds 422 and 424 were elucidated through their corresponding methyl esters. The diterpene 424 contains a side chain analogous to that of 422 and a secondary alcohol group in the axial orientation on the C-6. The roots of A. galeata contain six clerodane diterpenoids, populifolic acid (411), kolavenic acid (404), kolavenol (407), dihydrokolavenol (413), kolavelool (398) and 2-oxopopulifolic acid (415). Populifolic acid methyl ester (412), kolavenic acid methyl ester (405), acetyl derivatives of 406 and 413 were also identified [370]. The conversions 411 to 415 and acetyl derivative of 413 to corresponding 2-oxo derivative reinforce the previous suggestion that the 2-oxokola- venic compounds are artifacts. The clerodane diterpenes, populifolic acid 411 and its new C-5 epimer, epipopulifolic acid (418) together with other diterpenoids, 2-oxopopulifolic acid (415) and kolavelool (398) were isolated from the roots of A. cymbifera [267]. This result suggested that clerodane diterpenes predominate in the roots whereas labdane diterpenes predominate in the leaves of the species. A furanoid diterpene lactone belongs to clerodane type was isolated from the rhizomes of A. albida [268] and identified as
937 columbin (419). Rucker et al. [269] have reported seven e«^-kaurane diterpenes 428, 430, 431, 433, 434, 435, and 442 from the roots and stems of A. triangularis collected in Rio Grande do sul. Further investigation on A. triangularis [270] collected in Parama by Lopes and co workers led to the isolation of seven en?-kaurane type diterpenes 425, 428, 430, 439, 442, 444, and 446. Their studies provide evidence that the main difference in the chemical composition between A. triangularis collected in Rio Grande do sul and in Parama is the occurrence in the species from the first region of considerable amounts of kaurane diterpenes oxidized at C-19. These two reports revealed that A. triangularis is a rich source of e«Z-kaurane diterpenoids. A diterpene (-)-kaur-16-ene (428) was also found in the essential oils of A. acutifolia [299], A. argentina [296], and A. chilensis [312]. Isabele et al. [208,357] reported the e«r-kaurane-16a,17-diol (439) from A. pubescens, which was previously isolated from A. triangularis. Eight e«/-kaurane diterpenoids 425-427, 429, 430, 439, 442, and 444 were discovered by Tsai and co-workers [168], and Luiz et al. [366] in A. elegans. It is of interest to note that the Aristolochia species of Brazilian origin were the only rich sources of diterpenoids. Triterpenoids The triterpenoids are apparently rare in Aristolochia. The foliar epicuticular waxes of leaves of A. esperanzae from Cerrado was analyzed by Oloveira et al. and triterpenoids lupeol (447), P-amyrin (448), epifriedelinol (449), and ursolic acid (451) were identified as major constituents [367]. These triterpenoids clearly predominate over alkanes in the waxes from the Cerrado species. Another triterpenoid friedelin (450) was found in A. indica [368] and A cucurbitifolia [166]. Tetraterpenoids Loliolide (249), an apocarotinoid was isolated as the sole tetraterpenoid from Aristolochia species, A. gehrtii [287]. Apocarotenoids are carotenoids in which the carbon skeleton has been shortened by the formal removal of fragments from one or both ends. Lignans Lignans were another important class of metabolites found in several species of Aristolochia. There are six types of neolignans and lignans with
938 H,C
,CH
r
CH 3
452
Fig. (8). Structures of Tri- and Tetraterpenoids
structural diversity reported to date from Aristolochia genus (Table 19). Compounds 453 and 454 from A. manshuriensis (374) and 455-458 from A. birostris [370] were acyclic neolignans found in Aristolochia. Ligans 459-468 were examples of 2-aryl-3-methyl-2,3-dihydrobenzofuran type neolignans of Aristolochia. They are also termed as eupomatenoids. Eupomatenoids are 3-methyl-2-phenyl-5i?-propenylbenzofuran derivatives and they owe their name to the family of Eupomatiaceae, which is a rich source of these compounds. Among the ten lignans of this type from Aristolochia, 461, 462, and 464-468 were reported from A. pubescens [357]. Licarinediol A (465) and B (467), and O-mehyllicarinediol A (466) and B (468) related to lacarin A (464) were separated as two lildiastereoisomeric mixtures, in which A represents the (25r,35r,85,9/?)
939
Table 19. Lignans Isolated from Aristolochia species Compound &ypropen-3-ol)-phenoxy)-propan-l,3-diol (453) Erythro-1 -(4-hydroxy-3-methoxyphenyl)-2-(2,6-dimethoxy4-(l-(£)-propen-3-ol)-phenoxy)-propan-l,3-diol (454) re/-(8fl)-A-3,4-Methylenedioxy-3',5'-dimethoxy- 8.0.4'neolignan (455) re/-(8/?)-A8'-3,4-Methylenedioxy-5,3',5'-trimethoxy-8.0.4'neolignan (456) re/-(7«,8i?)-A-3,4-Methylenedioxy-3' ,5,5 '-trimethoxy-7hydroxy-8.0.4'-neolignan (457) re/-(8R)-A-3,3',4,5,5'-pentamethoxy-8.0.4'-neolignan (458) Eupomatenoid-1 (Eupomatene) (459) Eupomatenoid-7 (460)
(2R,3«)-2,3-Dihydro-2-(4-hydroxy-3-methoxyphenyl)-7methoxy-3-methylbenzofuran-5-aldehyde (461) (2«,3ft)-2,3-Dihydro-2-(4-hydroxy-3-methoxyphenyl)-7methoxy-3-methylbenzofuran-5-carboxylic acid (462) Eupomatenoid-8 (licarin-B) (463) (+)-(ra«s-Dehydrodiisoeugenol (licarin-A) (464) Licarinediol A (465) O-Methyllicarinediol A (466) Licarinediol B (467) O-Methyllicarinediol B (468) Zuihonin-B (469) 3-e/j/-Austrobailignan-7 (fragransin Ei) (470) Nectandrin-B (471) re/-(7S>,8S,7'«,8'«)-3,3\4,4',5'5'-Hexamethoxy7.0.7',8.8'-lignan (472) (+)-Austrobailignan-7 (473) (+)-Calopptin (474) (+)-Aristolignan (475) (-)-Galbacin (476) Zuonin-A (477) re/-(8R,8'fi)-3,4;3',4'-Dimethylenedioxy-9(3-hydroxy8.0.4'-ligan (478) (-)-Cubebin (479)
Source A. manshuriensis
Ref. 369
A. manshuriensis
369
A. birostris
370
A. birostris
370
A. birostris
370
A. birostris A. laliscana A. arcuatn A. peltato-deltoidea A. taliscana A. tubflosa A. pubescens
370 122,371 277 352 122,371 288 357
A. pubescens
357
A. taliscana
122,371
A. pubescens A. laliscana A. pubescens A. pubescens A. pubescens A. pubescens A. arcuata A. chilensis A. taliscana A. chilensis A. ponticum A. birostris
357 122,371,372 357 357 357 357 277 373 122 374 207 370
A. chilensis A. taliscana A. chilensis A. chilensis A. arcuata A. triangularis A. chilensis A. birostris
374 122 374 374 277 303 259, 374 370
A. birostris A. chamissonis A. cymbifera A. elegans A. esperanzae A. galeala
370 356 267 169 363 363
940
3',4'-Dimethoxy-3,4-desmethylenedioxycubebin (480) 3,4-Dimethoxy-3',4'-desmethylenedioxycubebin (481) p-Methylcubebin (482) a-Methylcubebin (483) rel-(%R,%'S,9R)-3,4-Dimethoxy-3',4'-methylenedioxy- 9aethoxy-8.8',9.O.9'-ligan (484) re/-(8«,8'5,95)-3,4-Dimethoxy-3',4'-methylenedioxy-9pethoxy-8.8',9.O.9'-ligan (485) Aristelegin-C (486) Aristelegin-B (487) (-)-Hinokinin (488)
Kusunokinin (489)
Pluviatolid (490) Bursehernin (491) (-)-5"-Methylhinokinin (492) Aristelegin-A (493) /•e/-(8/?,8'/?)-3,4-Dimethoxy-3',4'-methylenedioxy-9-oxo8.8',9.O.9'-ligan (494) Savinin (495) (-)-Dihydro cubebin (496) Piperitol (497) (+)-Methylpiperitol (498) (-)-Eudesmin (499) Asarinin (500) Fargesin (501)
Sesamin (502) (+)-Eudesmin (503) (+)-Medioresinol (504) (-)-Kobusin (505) (-)-Pinoresinol (506) (-)-Aristotetralol (507) (-)-Aristotetralone (508) (-)-2-Hydroxyaristotetralone(509) (-)-2-Acetoxyaristotetralone(510) (-)-Aristochilone(511)
A. gehrtii A. indica A. pubescens A. triangularis A. triangularis A. triangularis A. elegans A. elegans A. peltato-deltoidea
287 294 208, 357 302,303, 375 303, 375 303,375 169 169 352
A. peltato-deltoidea
352
A. elegans A. elegans A. birostris A. chamissonis A. cucurbitifolia A. cymbifera A. elegans A. gehrtii A. indica A. pubescens A. triangularis A. galeata A. pubescens A. triangularis A. triangularis A. peltato-deltoidea A. pubescens A. elegans A. elegans A. peltato-deltoidea
169,206 169,206 370 356 159 267 169 287 294 208 302 363 208 302 302 352 208 169 169,206 352
A. indica A. pubescens A. gehrtii A. gehrtii A. gehrtii A. galeata A. albida A. cymbifera A. galeata A. pubescens A. tagala A. pubescens A. elegans A. elegans A. elegans A. chilensis A. chilensis A. chilensis A. chilensis A. chilensis
92 208 287 287 287 363 376 267 363 357 158 357 169 169 168 377 377,378 377 377 377
941 A. chilensis A. chilensis
(-)-Aristoligone (512) Aristosynone (513)
311 377
HjCO
R 453 H 454 OCH,
455 456 457 458
-CH,-CH,-CH,CH 3 CH 3
H OCH3 OCH 3 OCH3
R, R, 459 -CH,460 H CH,
H H OH OCH,
OR, 4
469 470 471 472
-CH,H -CH,H CH 3 H H CH 3 CH 3 CHj
*^-s
-CH,CH 3 H CH 3 CH 3 CH3 CH,
*^fi
H H H CH 3
473 474 475
R, R, -CH,CH,CH, H
Rj CH 3 CH 3 CH 3
R, H CH 3 CH,
isomer and B the (2S,3S,8R,9R) isomer. Lignans 461 and 462 were the corresponding bisnor-neolignan aldehyde and acid of licarin A (464). Considering that the configurations of C-2 and C-3 do not change during transformation one could suggest that the enantiomer of licarinediol A (465) might be the key biosynthetic intermediate from licarin A to its bisnorneolignan aldehyde. Thus this later one by oxidative processes could yield bisnorneolignan acid. Eupomatenoid-1 (459), -7 (460), -8 (463), and dehydrodiisoeugenol (464) were isolated from A. taliscana [122,371,372]. The lignans, 469-477 were representatives of 2,5-diaryl3,4-dimethyltetrahydrofuranoid lignans, in Aristolochia species. Zuihonin B (469) from A. arcuata [277], 3-e/?/-austrobailignan-7 (470), zuonin A
942 OH l
R,0
H,CO.
HjCO'
'OR,
478 479 480 481 482 483
OH H H H OCHj H
H -CH,OH -CH," OH -CH,OH CH, CH, H -CH2OCH, - C H r
-CH,-CH;CH, CH3 -CH,-CHr -CH2-
484
H
485
0 C H
,CH
H,CO
R,0
R 4 0'
Rr,
R, R, R4 R5 Ri -CH H -CH,CH, CH, H -CH,-CH ,H CH, H -CH H CH, CH, -CH H -CH,OCH , -CH,-CH
488 489 490 491 492 493
xx
<° Y O
495
Rf, H H H H OCH, H ,OR,
A./
OH OCH3
^,OH
V V-o 496
Hi" \
\ R,0
R2O
T 497 498 499
^0 R, R, -CH,-CH,CH3 CH3
R, H CH, CH,
0
/..IIH
0 500
(477) from A. chilensis [373,374], nectandrin-B (471) from A. chilensis [374] and A ponticum [207], and other lignan 472 from A. birostris [370] contained cis relationship between 3,4-methyl groups whereas, (+)austrobailignan-7 (473), (+)-calopiptin (474) and (+)-aristolignan (475) from A chilensis [374] and (-)-galbacin (476) from A arcuata [277] have trans relation. Compounds 478-496 were dibenzylbutyrolactone type lignans known to Aristolochia. Occurrence of this type lignans is common
943 in Aristolochia species. Among them (-)-cubebin (479) and (-)-hinokinin (488) were identified in several species of Aristolochia. Three new lignans of this type, aristelegin A (493), B (487), C (486) along with (-)-cubebin (479), a-methylcubebin (483), P-methylcubebin (482), (-)-hinokinin (488) and 5"-methylhinokinin (492) were isolated from A. elegans by our group [175,216]. Aristelegin A (493) is a dibenzylbutyrolactone derivative with an unusual methoxyl substituent at C-9', whereas aristelegin B (487) and C (486) have hydroxyl group at C-7 which is also rare [206]. o> Methylcubebin (483), p-methylcubebin (482), were epimeric dibenzylbutyrolactol lignans. Lignans 484 and 485 reported from A. peltato-deltoidea [352] were also epimeric dibenzylbutyrolactol lignans with a rare ethoxy substituent on C-9. The other two lignans 491 and 494 isolated from the same plant were diastereomeric dibenzylbutyrolactone lignans. (-)-Dihydrocubebin (496) reported from A. pubescens [208] has been obtained by the cleavage of butyrolactol ring of cubebin. The other type of lignans frequently encountered in Aristolochia species were furofuran lignans which were exemplified by compounds 497-506. Among these lignans, 488 and 495, and 492 and 494 were diastereoisomers. The 4-aryltetralones are a small group of lignans characterized from Aristolochia species. It is noteworthy that all the 4aryltetralone lignans reported so far from the genus, Aristolochia were only from A. chilensis [377,378]. Urzua et al. separated six 4-aryltetralones, (-)-aristotetralone (508), (-)-aristochilone (511), (-)-aristoligone (512), (-)-aristosynone (513), (-)-2-hydroxyaristotetralone (509) and (-)-2acetoxyaristotetralone (510) and a 4-aryltetralol, (-)-aristotetralol (507) from A chilensis [377]. Compound 513 is epimeric with compounds 508, 511 and 512 at C-2. Urzua suggested that the both analogues, (-)-2hydroxyaristotetralone (509) and (-)-2-acetoxyaristo- tetralone (510) found in much smaller amounts may probably formed in nature through in vivo oxidation of 508. Compounds 513, 409 and 410 have a C-2 a pseudo-axia\ substituent. Based on the reported data, a generalization that may be safely drawn is that the negative specific rotation for a 4aryltetralone lignan is to be associated with the a orientation of the 4-aryl substituent, regardless of the configuration at C-2 and C-3 [379,380]. The purification process of lignans is usually made difficult by the fact that several of these closely related species may co-occur within the same botanical source, while their chromatographic properties may be nearly identical. So, several lignans were isolated as diastereoisomeric mixtures from their sources.
944
502 503 504
505 506
R, R, R, R4 R5 -CH r -CH,H CH, CH, CH, CH, H CH, H CH, H OCH, O
R, R, -CHr CH, H
OR, OR 4 508 509 510 511 512 513
R, CH, CH, CH, CH, CH, H
R, H OH OAc H H CH,
R, R4 -CH : -CHr -CHr CH, CH, CH, CH, CH, CH,
Fig. (9). Structures of lignans
Flavonoids Flavonoids are widely distributed in species of Aristolochia, being mainly present as flavonol glycosides (Table 20). The flavones are very rare in Aristolochia, with apigenin (514) being the only representative found in A. cucurbitifolia [56]. Only three flavonol aglycones, kaempferol (515), kaempferol 3-methyl ether (516) and quercetin 3'-methyl ether (528) (isorhamnetin) have been reported to date in Aristolochia Kaempferol (515) itself was present in A. cucurbitifolia [229] and A. kaempferi [313], while kaempferol 3-methyl ether (516) was reported only in A. acutifolia [210]. Isorhamnetin (528) was found in A. heterophylla [232], A. kaempferi [244], and A. reticulate [281]. From the Table 20 it is evidant that the flavonol O-glycosides are apparently common in Aristolochia species of Taiwan origin and most of them are flavonol 3-O-glycosides and few are flavonol 7-O-glycosides. There are seven kaempferol glycosides known, but they cover a range of mono-, di-, and acylated glycosides which were exemplified by compounds 517, 518;
945 Table 20. Flavonoids Isolated from Aristolochia Species Compound Apigenin (514) Kaempferol (515) Kaempferol-3-methylether (516) Kaempferol-3-O-glucoside (517)
Kaempferol-7-O-glucoside (518)
Kaempferol-3-O-p-D- (6"-p-coumaroyl) glucoside (519) Kaempferol-7-O-(3-D- (6"-p-coumaroyl) glucoside (520) Kaetnpferol-3-O-rutinoside (521) Kaempferol-3-p-D-robinobioside (522) Kaempferol-3,7-O-diglucoside (523) Quercetin-3-O-glucoside (524) Quercetin-7-O-glucoside (525) Quercetin-3-O-rutinoside (526)
Quercetin-3,7-O-diglucoside (527) Isorhamnetin (528)
Isorhamnetin 3-O-glucoside (529) Isorhamnetin 3-O-(3-D- (6"-/j-coumaroyl) glucoside (530) Isorhamnetin 3-O-rutinoside (531)
Isorhamnetin 3-O-robinobioside (532)
4"',5",7,7"-Tetrahydroxy-3"\4',7trimethoxy- 3,6"-biflavone (533) 4"\5,5",7"-Tetrahydroxy-3"\4',7trimethoxy- 3,6"-biflavone (534) 4"\5",7,7"-Tetrahydroxy-3",3"',4'trimethoxy-6-O-a,7-p-flavone-chalcone (535) 3"',5,5",7"-Tetrahydroxy-3",4 > ,4'"-
Occurrence A. cucurbitifolin A. cucurbitifolia A. kaempferi A. acutifolia A. cucurbitifolia A. heterophylla A. kaempferi A. cucurbitifolia A. foveolata A. kaempferi A. heterophylla A. kaempferi A. heterophylla
Ref. 56 229 313 210 56 232 313 56 246 229 232 229 232
A. cucurbitifolia A. kaempferi A. rigida A. cucurbitifolia A. cucurbitifolia A. sipho A. cucurbitifolia A. cucurbitifolia A. indica A. kaempferi A. shipo A. foveolata A. heterophylla A. kaempferi A. reticulata A. kaempferi A. zollingeriana A. heterophylla A. kaempferi A. cucurbitifolia A. foveolata A. heterophylla A. kaempferi A. mollissima A. zollingeriana A. cucurbitifolia A. heterophylla A. kaempferi A. ridicula
56 233 238 56 56 381 56 56 368 233 381 246 232 244 281 244 223 232 229 314 246 314 244 204 223 159 228 61, 158 382
A. ridicula
382
A. ridicula
382
A. ridicula
382
946 trimethoxy-6-0-a,7-P-flavone-chalcone (536) 4',5,5",7"-Tetrahydroxy-3',3",4'"trimethoxy-6-0-a,7-P-flavone-chalcone (537) 4',5,5"J"-Tetrahydroxy-3',3",4'"trimethoxy-6-O-p,7-a-flavone-chalcone (538) (4"',5",7-Trihydroxy-4',5,7"-trimethoxy3,6"-biflavone)-3"'-O-4'"-(5,5">7"trihydroxy- 3\3",4'-trimethoxy-6-Op,7a-flavone- chalcone (539)
A. ridicula
382
A. ridicula
382
A. ridicula
382
521, 522, 523; and 519, 520, respectively. Four quercetin glycosides, known to Aristolochia species include quercetin 3-O-glucoside (524) and the 7-O-glucoside (525), rutin (526), and quercetin 3,7-di-6>-glucoside (527) [56, 245]. The four isorhamnetin glycosides, 529, 531, 532, and 530 found in Aristolochia are exclusively 3-0-glycosides, which include mono-, di-, and acylated glycosides. However, recently Carneiro et al. described two biflavones, four unusual chalcone-flavone dimers and one tetraflavonoid from A. ridicula [382]. It is the first report of bi- and tetraflavonoids in the family Aristolochiaceae. In the biflavones, 533 and 534, two flavone units were linked by a bond involving C-3 and C-6" and they differ by the position of substituents at C-5 and C-7 in the A-ring. The dimers, 535-537 with unusual oxygenation pattern in the A-ring were formed due to the linkages C-P-C-7 and C-a O C-6 between the chalcone and flavone moieties. Compound 535 differed from 536 only by substituent positions at C-3'" and C-4'" whereas 537 differed from 535 by aromatic substituents at C-2 (B' ring) and C-P (B ring), which were reversed. The dimer 538 contained an inverse mode of linkage between the flavone and chalcone moieties, i.e., through bonds C-a-C-7 and C-P-O-C-6. All these six dimers exhibited optical activity. The rotation around the carbon-carbon intraflavonyl bond between C-3 and C-6" could be sterically hindered for compounds 534 and 533, leading to atropisomerism [383]. The same effect could explain the optical activity of 535 and 536, since the rotations of the substituents at C-a, C-P were not free, as indicated by strong NOESY interactions of MeO-3" with H6 ' " and of H-8 with H-2'". Compound 539 is a tetraflavonoid composed of two units, flavone-chalcone moiety (unit I) and biflavone moiety (unit II). These two were linked through C-I-4'"-O-C-II-3'". The biosynthesis of compound 534 and 533 could involve an oxidative coupling of two flavone units, leading to linkage of these units through C-3 and C-6"
947 bonds. Compounds 535-538 may arise from oxidative coupling between 6-hydroxyflavone and chalcone derivatives, followed by further elaboration, which could lead to rearranged benzofuran derivatives. Compound 539, in turn, could be formed by oxidative coupling of a biflavone and a benzofuran derivative through a C-I-4'" O C-II-3'" linkage.
515 516 517 518 519 520 521 522 523
Rl H CH3
R2 H H H
524 525 526
(6"-coumaiyl)-glc
glc H
527 528
H
(6"-coumaryl)-glc
rha(|--6)glc rha(l— 6)gal
H H
529 530
glc
glc
glc H
.OCH3
HjCO OH 533 534
R, CHj H
O R, H CH3
Fig. (10). Structures of Flavonoids
531 532
Rl glc H
rha(l— 6)glc glc
R2 H glc H glc H H
H glc (6"-coumaryl)-glc H H rha(l— 6)glc H rha(l— 6)gal
R3 II H H H
CHj
CH, CHj
CH, CH,
948 Biphenyl Ethers
The seven biphenyl ethers that have been described to date include aristogin A (541), B (542), C (545), D (540), E (544), and F (543), and 4methoxy-3,4-oxydibenzoic acid (546) [169,252,292]. All these compounds have only been reported from A. elegans. Biphenyl ethers are considered as one of the end products in the catabolic process of bisbenzylisoquinoline alkaloids.
R2 H COOCH3 CH 3 CHO CH 3 COOCH3 CH 3 COOH CH 3 COOCH3 CH 3 COOCH3 CH 3 COOH ethers Ri
540 541 542 543 544 545 546 Fig. (11). The structures of Biphenyl
R3
COOCH3 COOCH3 CHO CH2OH CH2OH COOCH3 COOH
Tetralones
Among seven tetralones known to Aristolochia so far, four tetralones, anstelegone A (547), B (548), C (549) and D (550) have been described in A. elegans collected in Taiwan [168,252]. The other three, 4-hydroxy-4,7dimethyl-1-tetralone (551), 4,7-dimethyl-6-methoxy-l-tetralone (553) and aristolindiquinone (552) were reported from A. brevipes [273], A. tagala [384], and A. indica [92,385], respectively. All the tetralones except aristolindiquinone possess a keto function at C-l. Aristolindiquinone (552) is a fully aromatic tetralone with a/?-quinone unit.
o 547
R,
R,
H
H
548 OH CH 3 HO. CH 3
Fig. (12). The Structures of Tetralones
949 Benzenoids A number of benzenoid derivatives were isolated from different Aristolochia species, which include phenylmethanoids, phenylethanoids and phenylpropanoids (Table 21). Our group has identified seven new benzenoids along with several known ones from Aristolochia species collected in Taiwan. Sodium 3,4-dihydroxybenzoate (560) [159,228] and sodium (2i?)-(p-hydroxyphenyl)lactate (581) [56,229,244] were sodium salts. The presence of sodium was determined by the acidification of compounds with HC1 followed by the atomic absorption spectroscopy. The latter compound contained P-hydroxypropanoyl moiety on phydroxyphenyl unit. The absolute configuration of P-hydroxy group was identified as R. Ariscucurbin A, B, and C (588-590) were hydroxyl- ethyl esters of phenyl propanoids, ferulic, trans- and cis- p-coumaric acids [56]. Compound 558 is also a hydroxyethyl ester of p-hydroxy- benzoic acid [56]. co-Hydroxypropioguaiacone (567) has 3-hydroxy- propanone moiety on 4-hydroxy-3-methoxyphenyl unit [169]. Similarly, ficusol (582) and aristogin G (569) have interesting side chain substituents on substituted phenyl basic unit [169]. Six phenethyl derivatives, tyrosol (575), icariside D2 (576), salidroside (577), 3,4-dihydroxyphenethyl alcohol (578), thalictoside (580) and its aglycone (579) were isolated from A. gehrtii [287]. Thalictoside (580) and its aglycone (579) have an aliphatic nitro group which is unusual in nature. The occurrence of phenethyl derivatives is significant in the species A. gehrtii. Three propenylphenols, methyleugenol (607), myristicin (608), and elemicin (609) and 3methoxy-4,5-methylenedioxybenzaldehyde (570) were described from the nonpolar fraction of A. acutifolia [299] and it is first occurrence of these compounds in the genus Aristolochia. Compounds 583-606 are phenylpropanoid derivatives found in the various Aristolochia species, which include cinnamic, p-coumaric, ferulic and caffeic acids and their alkyl esters and glycosides. Steroids Steroids are widespread in nature, and those present in Aristolochia species are mostly derivatives of p-sitosterol and stigmasterol. PSitosterol (610) and its glucoside (611) were frequently encountered in several Aristolochia species (Table 22). P-Sitosterone (612), 3-one derivative of P-sitosterol was found in A. zollingeriana only [245]. Stigmasterol (613) and its derivatives stigmast-4-en-3-one (614), 3p-
950 Table 21. Bezenoids Isolated from Aristolochia Species Compound Benzoic acid (554)
p-Hydroxybenzaldehyde (555)
p-Hydroxybenzoic acid (556)
Methylparaben (557)
4-Hydroxybenzoic acid 2-hydroxyethyl ester (558) Methyl 3,4-dihydroxybenzoate (559) Sodium 3,4-dihydroxybenzoate (560) Vanillin (561)
Vanillic acid (562)
Isovanillic acid (563) 4-O-(3-D-Glucosyl-3-methoxybenzoic acid (Picrorhizin) (564) Methyl vanillate (565)
Methyl isovanillate (566) a)-Hydroxypropioguaiacone (567) 3,4,5-Trimethoxytoluene (568)
Occurrence A. foveolata A. heterophylia A. kaempferi A. cucurbitifolia A. elegans A. kaempferi A. zollingeriana A. cucurbitifolia A. elegans A. gehrtii A. heterophylia A. kaempferi A. manshuriensis A. cucurbitifolia A. elegans A. heterophylia A. kaempferi A. zollingeriana A. cucurbitifolia
Ref. 174 232 229 56, 159 168, 169 229, 244 171,245 56 168, 171,292 287 232 229, 244 241 56 168, 169,292 228 229, 244 171,223 56
A. heterophylia A. cucurbitifolia A. heterophylia A. asclepiadifolia A. elegans A. heterophylia A. kaempferi A. pubescens A. argentina A. asclepiadifolia A. cucurbitifolia A. elegans A. heterophylia A. kaempferi A. manshuriensis A. mollissima A. pubescens A. tubflora A. gehrtii A. heterophylia A. kaempferi A. elegans A. foveolata A. heterophylia A. kaempferi A. manshuriensis A. zollingeriana A. heterophylia A. elegans A. debilis
228 159 228 386 168, 169 228 244 357 130 386 56, 159 171 228,232 229, 244 241,265 202 357 288 287 228,232 244 168, 169,292 246 228 229, 244 241 223 228 168, 169 298
951 Aristogin F (569)) 3-Methoxy-4, 5methylenedioxybenzaldehyde (570) Syringicacid (571) Methyl 3,5-dimethoxy-4-hydroxybenzoate (572) Glucosyringic acid (573) Phenyl ethylalcohol (574) Tyrosol (575) lcariside D2 (576) Salidroside (577) 3,4-Dihydroxyphenethyl alcohol (578) Thalictoside aglycone (579) Thalictoside (580) Sodium (2#)-(p-hydroxylphenyl) lactate (581) Ficusol (582) Cinnamic acid (583) c;s-/?-Coumaric acid (584) trans-p-Coumar'\c acid (p-Hydroxycinnamic acid ) (585)
Methyl p-cw-coumarate (586) Methyl p-
Ariscucurbin C (588) Ariscucurbin B (589) Ariscucurbin A (590) p-Coumaroyl (3-D-glucoside (591) 3-O-(£)-p-Coumaroyl-glucoside(592) 6-O-(Z)-p-Coumaroyl-ethyl-glucoside (593) 6-O-(£)-p-Coumaroyl-ethyl-glucoside (594) Caffeic acid (595) Methyl caffeate (596) Ferulic acid (597)
A. elegans A. aculifolia
168 299
A. heterophylla A. kaempferi
228,232 244
A. cucurbitifolia A. brevipes A. gehrtii A. gehrtii A. cucurbitifolia A. gehrtii A. gehrtii A. gehrtii A. gehrtii A. cucurbitifolia A. kaempferi A. elegans A. elegans A. indica A. zollingeriana A. argentina A. clematitis A. cucurbitifolia A. elegans A. heterophylla A. indica A. kaempferi A. manshuriensis A. moupinensis A. zollingeriana A. pubescens A. zollingeriana A. clematitis A. cucurbitifolia A. foveolata A. kaempferi A. pubescens A. zollingeriana A. cucurbitifolia A. cucurbitifolia A. cucurbitifolia A. cinnabarina A. cucurbitifolia A. manshuriensis A. manshuriensis A. manshuriensis A. manshuriensis
159 304 287 287 159 287 287 287 287 56 229, 244 169 169 92 223 130 153 56, 159 168 232 92 229, 244 241,265 205 223 208 171 153 56 174,246 229,244 208 171,223,245 56 56 56 143 56 369 369 369 369
A. heterophylla A. kaempferi A. argentina A. clematitis A. cucurbitifolia
232 229 130 153 159
952
Methyl c/s-ferulate (598) Methyl (rans-ferulate (599)
4-O-p-D-Glucosylferulic acid (600) 6-O-(£)-feruloyl-p-glucoside (601) Methyl 22-feruloyloxy-docosanoate (602) Octadecyl ferulate (603) Eicosyl ferulate (604) Alkyl ra-ferulate (605) Alkyl (rans-ferulate (606)
Methyl eugenol (607) Myristicin (608) Elemicin (609)
A. heterophylla A. kaempferi A. manshuriensis A. cucurbilifolia A. cucurbitifolia A. elegans A. foveolata A. kaempferi A. zollingeriana A. foveolata A. kaempferi A. cucurbitifolia A. manshuriensis A. elegans A. heterophylla A. zollingeriana A. zollingeriana A. heterophylla A. mollissima A. heterophylla A. mollissima A. zollingeriana A. acutifolia A. acutifolia A. acutifolia
HOH,C
228 229, 244 241 159 159 168,169 174 244 171,213 246 244 159 369 168 228 223 223 228 204 228 204 223 299 299 299
COOCH, 569
.COOCH,CH,OH
.OCH,
COOCH,CH,OH 589
Fig. (13). The Structures of Benzenoids
COOCH,CH,OH
CH, R, R, 606 H OCH, 607 -0CH,O6O8OCH3 OCH,
953 hydroxystigmast-5-en-7-one (615), 6P-hydroxystigmast-4-en-3-one (616), 5a-stigmastane-3,6-dione (617) and stigmast-4-en-3,6-dione (618) were isolated from various Aristolochia species [180,294,368], In addition, campesterol (612) and cycloeucalenol (619) were also found in A. albida [268] and A. indica [368], respectively.
CHj
CH,
Fig. (14). The Structures of Steroids
954
Table 22. The Steroids isolated from Aristolochia species Compound P-Sitosterol (610)
P-Sitosterol-p-D-glucoside (611)
p-Sitosterone(612) Campesterol (613) Stigmasterol (614)
Source A. albida A. arcuata A. austrozechuanica A. birostris A. bracteata A. chamissonis A. championii A. chilensis A. clematilis A. contorta A. cucurbitifolia A. elegans A.fangchi A. galeata A. gehrtii A. grandiflora A. heterophylla A. indica A. kaempferi A. kwangsiensis A. longa A. manshuriensis A. mollissima A. moupinensis A. peltato-deltoidea A. pubescens A. rotunda A. serpentaria A. tagala A. triangularis A. lubflora A. versicolar A. zollingeriana A. chilensis A. clematitis A. contorta A. cucurbitifolia A. elegans A. gehrtii A. heterophylla A. indica A. kaempferi A. manshuriensis A. mollissima A. reticulata A. serpentaria A. versicolar A. zollingeriana A. albida A. albida A. bracteata
Ref. 268 277 133 370 387 356 138 374 153 154, 155 156, 159 169 172 363 287 289 176,228,232 92, 180 229,233,388 188,190 389 195,232,241 204 176 352 187,208 212 390 158 302 288 221 169,245 259 153 154,155 56, 159 169 287 176,228,232 92, 180 158,388 241,265 202 209 390 222 245 268 268 387
955
Stigmast-4-en-3-one (615)
3P-Hydroxystigmast-5-en-7-one (616) 6p-Hydroxystigmast-4-en-3-one (617)
5a-Stigmastane-3,6-dione (618)
Stigmast-4-en-3,6-dione (619)
Cycloeucalenol (620)
A. cucubitifolia A. heterophylla A. manshuriensis A. mollissima A. peltato-deltoidea A. zollingeriana A. indica A. kunmingensis A. mollissima A. triangularis A. zollingeriana A. chamissonis A. indica A. cucurbiiifolia A. indica A. manshuriensis A. zollingeriana A. chilensis A. indica A. manshuriensis A. triangularis A. tubflora A. versicolar A. cucurbitifolia A. heterophylla A. triangularis A. tubflora A. versicolar A. indica
159 228,232 241 204 352 171,245 368 187 204 303 245 356 180 180 56 195 245 374 294 195 303 288 221 159 228 303 288 221 368
Miscellaneous Various compounds isolated from Aristolochia species, which are not otherwise classified in the text, are listed in Table 23. Allantoin (624) was the most abundant and significant nitrogenous compound found in several Aristolochia species. Compounds, 9-methoxytariacuripyrone (629) and 7, 9-dimethoxytariacuripyrone (630) were also significant physiological active compounds containing nitro group found in A. brevipes [52]. Compounds, 5-hydroxymethylfuran 2-carbaldehyde (633) and its hemiacetal derivatives were interesting furfural derivatives isolated from A. gehrtii [287]. a-Tocopherylquinone (631) found in A. peltato-deltoidea [352] and morindaparvin (632), isolated from^. heterophylla [228], were the quinone derivatives. It is interesting to note that two 2-deoxyribonolactones, 2-deoxy-D-ribono-l,4-lactone (634) and 2-deoxy-D-ribono- 3,5bis(tripolyphosphate)-l,4-lactone (635) with the two tripolyphosphate groups in the latter compound were identified in A. arcuata [285]. Biologically important aromatic amine, dopamine (686) also isolated from A. manshuriensis [265]. Sugars including glucose (636), fructose (637),
956 Table 23. Species
Miscellaneous compounds Isolated from Aristolochia
Compound Pyridine-3-carboxylic acid (621) Indole-3-carboxylic acid (622) Indole-3-carboxylic acid methyl ester (623) Uracil (624) Allantoin (625)
Adenine (626) 6,7-Dihyroxycoumarin (627) Isoscopoietin (628) 9-Methoxytariacuripyrone (629) 7,9-Dimethoxytariacuripyrone (630) a-Tocopherylquinone (631) Morindaparvin (632) 5-Hydroxymetliylfuran-2-carbaldehyde (633) 2-Deoxy-D-ribono-l ,4-lactone (634) 2-Deoxy-D-ribono-3,5bis(tripolyphosphate)-l ,4-lactone (635) Glucose (636) Fructose (637) Sequoyitol (638)
D-(+)-Pinitol (639)
Sucrose (640)
Occurrence A. kaempferi A. heterophylla A. cucurbitifolia A. kaempferi A. heterophylla A. austrozechuanica A. championii A. contorta A. cucurbitifolia A. cymbifera A. debilis A. elegans A.fangchi A. gigantea A. heterophylla A. indica A. kaempferi A. kwangsiensis A. mollissima A. moupinensis A. pubescens A. reticulata A. tagala A. triangularis A. versicolar A. zollingeriana A. cucurbitifolia A. kaempferi A. sipho A. cucurbitifolia A. brevipes A. brevipes A. peltato-deltoidea A. heterophylla A. gehrtii A. kaempferi A. arcuata A. arcuata
Ref. 229 232 56 244 232 133 138 154,155 159 267 160, 161, 165 170 173 59 176,228,236 184 244 188, 190 200,201,203,204 176,205 208 209 216 302 221,222 223 159 244 381 159 52 52 352 228 287 244 285 285
A. pubescens A. rotunda A. arcuata A. baetica A. arcuata A. debilis A. gigantea A. arcuata A. gigantea A. macrophylla A. arcuata
208 212 285 49 285 391 278 285 278 392 285
957
Saccharose (641) Glycerol (642) 1-Glyceryl stearate (643) Glyceryl-a-linocerate (644) 1-Octacosanoyl glyceride (645) l,2-[di(9Z,12Z,15Z)-octadeca-9,12,15trienoyl]-3-galactosyl-Sn-glycerol (646) a-Carotene (647) (3-Carotene (648) 3-Octanol (649) Dodecane (650) Pentadecane(651) Hexadecane (652) n-Heptadecane (653) Tetradecanol (654) H-Triacontane (655) 2-Hexenal (656) 3-Hydroxy-l-octene (657) 3-Acetoxy-l-octene (658) Octadecene (659) Stearic acid (660) Okie acid (661)
Linoleic acid (662) Methyl linoleate (663) Plamitic acid (664)
Ethyl acetate (665) Allyl decanoate (666) Ethyl octanoate (667) Methyl nonanoate (668) Ethyl nonanoate (669) Ethyl dodecanoate (670) Methyl tetradecanoate (671) Ethyl tetradecanoate (672) Methyl pentadecanoate (673) Ethyl pentadecanoate (674) Methyl hexadecanate (675) Ethylhexadecanoate (676)
A. rotunda A. zenkeri A. baelica A. arcunta A. cucurbitifolia A. manshuriensis A. manshuriensis A. macrophylla
212 311 49 285 56 241 195 514
A. zenkeri A. zenkeri A. elegans A. brevipes A. acutifolia A. acutifolia A. brevipes A. acutifolia A. indica A. acutifolia A. indica A. debitis A. elegans A. elegans A. elegans A. acutifolia A. acutifolia A. acutifolia A. argentina A. bracteata A. bracteata A. acutifolia A. acutifolia A. argentina A. bracteata A. indica A. mollissima A. moupinensis A. zenkeri A. brevipes A. brevipes A. argentina A. peltalo-deltoidea A. peltato-deltoidea A. acutifolia A. acutifolia A. acutifolia A. grandiflora A. acutifolia A. acutifolia A. peltato-deltoidea A. acutifolia A. grandiflora A. peltato-deltoidea
311 311 308 304 299 299 304 299 368 299 368 298 308 308 308 299 299 299 296 136,387 136,387 299 299 296 136,387 368 202 205 311 304 304 296 352 352 299 299 299 289 299 299 352 299 289 352
958 Ethyl 9-hexadecenoate (677) Heptadecanoic acid (678) Methyl 16-methyl-9-heptadecenoate (679) Methyl octadecanoate (680) Ethyl octadecanoate (681) Ethyl 9-octadecenoate (682) Methyl 14-methyl-9-octadecenoate (683) Ethyl 9, 12-octadecadienoate (684) Hexacosanoic acid (685) Methyl 12-nanacosenoate (686) Dopamine (687) O-Methylanisole (688)
A. acutifolia A. acutifolia A. grandiflora
299 299 289
A. acutifolia A. acutifolia A. acutifolia A. grandiflora A. grandiflora A. acutifolia A. indica A. indica A. manshuriensis A. argentina
299 299 299 289 289 299 368 368 265 296
sucrose (640) and saccharose (641), important polyhydroxy compounds, sequoyitol (638) and D-(+)-pinitol (639), glycerol (642) and its derivatives (643-645) and several long chain fatty acids and their alkyl esters were also described from Aristolochia species. o ^? NN
H
NH
H 3 CO
H3CO
H
625
OCH, 630
H,C
Fig. (15). The Structures of Miscellaneous compounds
BIOSYNTHESIS The striking structural kinship of the aristolochic acid skeleton with that of the aporphine alkaloids suggested a biogenetic relationship. The
959 biosynthesis of aristolochic acids is considered to begin with 1-benzyl tetrahydroisoquinoline precursors and to proceed via aporphine intermediates [393-395]. In radioactive 14C-labelling studies in A. sipho by Spenser and Tiwari, aristolochic acid derived from [3-14C]-DLtyrosine lost more than 60% of its activity on decarboxylation. There was no loss of activity during decarboxylation when aristolochic acid I was derived from [2-14C]-DL-dihydroxyphenylalanine. Decarboxylation of the acid derived from [2-14C]dihydroxyphenylethylamine gave an inactive nitrophenanthrene derivative. From these observations, they postulated the biosynthetic route: norlaudanosoline -» orientaline -> orientalinone -> orientalinol -» stephanine -> aristolochic acid I, which involves a phenol coupling step and a dienol-benzene rearrangement [396]. From [2-14C]DL-noradrenaline incorporation experiments, they speculated that the aristolochic acids are related to 4-hydroxynorlaudanosoline, rather than to norlaudanosoline, and that it is the presence of a benzylic hydroxyl group which predisposes an aporphine intermediate to oxidative conversion into an aristolochic acid. Later, this hypothesis was confirmed by the feeding experiments of Schuette et al. [397] In their experiments, they found that [4-14C]- tetrahydropapavarine-HCl feeding to A. sipho gave no radioactive aristolochic acid I, whereas feeding of [4-14C]- norlaudanosolineHCl yielded acid with the carboxylic group containing 69% of the radioactivity. Further labeling studies in A. sipho by Comer et al. proved that tyrosine, dopa, dopamine and noradrenaline can be served as specific precursors in the biosythesis of aristolochic acid. On the basis of the feeding experiment with doubly labeled [p-14C,15N] tyrosine they predicted that the nitro group of aristolochic acid originated from the amino group of tyrosine [398]. The incorporation of tyrosine, (3,4-dihydroxyphenyl)alanine, nororientaline, prestephanine, and stephanine into aristolochic acid in A. bracteata by Sharma et al. demostrated specific utilization of nororientaline [399]. This strongly supported the hypothesis that the oxidative coupling of orientaline gives prestephanine, which is converted to stephanine; oxidative cleavage of stephanine then furnished aristolochic acid. An experiment with doubly labeled nororientaline showed its incorporation intact into the product and confirmed the view that the methylenedioxy group in aristolochic acid originates from an Omethoxyphenol precursor. Parallel feedings of (-)- and (+)-orientaline confirmed that the stereospecificity is maintained in the biosynthesis of aristolochic acid from the 1-benzyltetrahydroquinoline precursors. These feeding experiments strongly supported the following sequence for the
960 biosynthesis of aristolochic acid: norlaudanosoline -» nororientaline -> orientaline -> prestephanine -> stephanine -> aristolochic acid, Scheme (3).
H,CO.
Scheme (3). Biosynthetic pathway of aristolochic acid (5)
Concurrent isolation of the aristolochic acids, aphorphines, 4,5-dioxoaporphines, 7-oxoaporphines, aristolactams and aristolactam Af-glycosides in Aristolochia species is of interesting point in view of biogenesis [260]. Several aristolactams showed a similar substitution pattern to that of the accompanying aristolochic acids [257]. The structural relationship between both these two groups suggested that aristolochic acids are derived from aristolactams rather than directly from quarternery aporphine alkaloids as proposed [400]. Also, Castedo et al. suggested that aporphine alkaloids be postulated as precursors of aristolactams in plants. Thus, the biosynthetic pathway can be enlarged with the introduction of the oxoapophinoids and aristolactams as possible intermediates of aristolochic acids [401]. The 4,5-dioxoaporphine generated from the oxidation of aporphines can function as intermediate for the biosynthesis of aristolactam, while aristolochic acids were derived from aristolactam, Scheme (4). Indeed, conversion of pontevedrine, a 4,5-dioxoaporphine, into an aristolactam takes place in vitro and can be regarded as a benzilic acid rearrangement followed by loss of carbon.
961
HO
Scheme (4). Biosynthetic pathway of aristolactam
Aristolactones, the 10-oxygenated denirtoaristolochic acid derivatives can be viewed as a by-product of the biosynthesis of aristolactam from aristo- lochic acid [294]. Thus the intermediate amino acid might tautomerize to the corresponding imine which could then be hydrolyzed to the lactone via hydroxyacid, Scheme (5).
'OCH 3
'OCH,
Scheme (5). Biosynthesis of aristolactones
OCHj
OCHj •OCH,
962 9-Methoxytariacuripyrone and 7, 9-dimethoxytariacuripyrone with 5nitro-2//-benzo[h]chromen-2-one skeleton, for which Achenbach et al. suggested the trivial name, tariacuripyrone, might originate from a corresponding aristolochic acid: oxidative cleavage of ring A between C-l and C-2 and subsequent decarboxylation and oxidative decarboxylation steps directly produce the tariacuripyrones [52], Scheme (6). This hypothetical biosynthetic pathway is corroborated by the identical substitution pattern observed in aristolochic acid III (4) and 9-methoxytariacuripyrone (629) as well as in aristolochic acid IV (14) and 7, 9-dimethoxytariacuripyrone (630). Lou et al. proposed the biogenetic path way for the neoaristolactone (manshurolide) a typical sesquiterpenoid of Aristolochia with C-l2 membered ring skeleton from the farnesylpyrophosphate [167], Scheme (7). This compound was formed by the end to end coupling of three isoprene molecules. The isolation of a series of manshurolide type sesquiterpenoids with slight structural variations allowed us to propose possible biosynthetic conversions of these sesquiterpenoids from each other, Scheme (8) [314].
Scheme (6). Hypothetical biosynthetic pathway to tariacuripyrones.
Bisbenzylisoquinoline alkaloids can be biogenetically formed by the dimerization of two enantiomeric benzylisoquinoline units through phenolic oxidative pathway. In contrast, oxidative cleavage of bisbenzyltetrahydroisoquinolines through TV-oxide to produce tetrahydroisoquinolone-benzyltetrahydroisoquinoline dimers as well as that of a simple monomeric benzyltetrahydroquinoline, which in turn lead to tetrahydroisoquinolones and biphenyl ethers is an intrinsic part of the general alkaloid catabolic process. It has also been adumbrated that the tetrahydroisoquinolone alkaloids originate in plants from the oxidation of simple benzyltetrahydroisoquinolines [402,403]. These assumptions were corroborated by the co-occurrence of bisbenzyltetrahydroisoquinolines, TVoxide benzoyl benzyltetrahydroisoquinolines, tetrahydroisoquinolines and biphenyl ethers in A. elegans. The structural kinship of these metabolites allowed us to consider a definite possibility that these metabolites were derived biogenetically from bisbenzyltetrahydro- isoquinolines in general alkaloid catabolic process [169]. Thus we proposed a possible biogenetic transformation pathway of these metabolites, Scheme (9).
963
Double bond tranformation OPP
Farnesylpyrophosphate
Cyclization Oxidation Hydroxylation
-H.O COOH
Cyclization
Scheme (7). Biosynthetic pathway for neo-aristolactone
SPECTRAL PROPERTIES Spectroscopy methods are widely used for the structural determination of aristolochic acids. These compounds all show characteristic UV absorption bands of a substituted phenanthrene chromophore at 223, 250, 318 and 390 nm whereas aristolactams absorb at 241, 250, 259, 291 and 300 nm [125,404]. The phenolic derivatives of this series generally display considerable bathochromic shifts on addition of alkali [129,140]. The IR spectrum is useful for detecting functional groups of aristolochic acids [405]. These compounds show two characteristic bands at 1550 and 1350 cm"1 due to the nitro group, and the carboxyl OH group appears at 3000-2500 cm"1 as a broad continuous absorption. Hydroxy derivatives of aristolochic acids or aristolactams show OH and NH absorptions at 33003500 and 3200-3400 cm"1. The carboxylic or lactamic carbonyl is present at 1710-1690 cm"1, whereas the carboxylic group absorption of the sodium salts of aristolochic acids appears at 1540-1580 cm"1 [159,171, 174,246]. In general, the aromatic ring system shows stretches at 1625-1575 and
964 ,CH,
An A
41 R= Anstolochic acid I 42 R= Anstolochic acid II
312
CHO
SAM- S-Adenosyl-L-methionine H COO~
CHO
CHO 311
NH 3 + Ad
Scheme (8). Biogenetic sequences of madolin type sesquiterpenoids
1525-1475 cm"1 as usual, and observation of the bands in the range of 900700 cm"1 is based on the substitutions in the aromatic ring. 1 H-NMR is the most commonly used technique in the structural elucidations of aristolochic acids. The aristolochic acids and aristolactams display a C-2 aromatic proton at 5 7.50-7.90 and C-3, C-4 hydroxy at 8 10.00, or methoxy at 8 3.90-4.10, or methylenedioxy at 8 6.35-6.55 of
965
H3C
R, = H, R, = b-H, R, = CH3, R4 = a-H, (•)-(R, R)-7'-0-metliylcuspidaline (147) R, = CH3, R, = b-H, R3 = H, R4 = b-H, (-)-temuconine (146)
(143) R,= -CH3, R, = -H,R, = C (145) R, - -CH3, R, « -H, R3 = (144) R,= -CHj, R2= -H,R3 =
(162) R, =H,R 5 = R,, = OH
'oxidative cleavage
(166) R, = CH3, R5 = OH, R(, = OCH3 (163) R, = H, R, = OH, R6 = OCH3 (167) R, = CH3, R5 = R6 - OCH3 R7
(543) R6 = CH,OH, R7 = COOH, R8 = OCH3 (546) R,, = R7 = COOH, R, = OCH3 HjCN
A'-methyl-6, 7-dimethoxyisoquinolone (168)
(541) R6 = COOCH 3 , R7 - CHO, R, = OCH 3
(542) R6 = CHO, R7 = COOCHj, R, = OCH3 (545) R6 = R, = COOCH3, R, = OCH, (540) R6 = R7 = COOCH3, R, = OH (544) R6 = CH,OH, R7 = COOCH3, R, = OCH3
Scheme (9). Possible biotransformation pathway from bisbenzyltetrahydroisoquinolines
ring A. The presence of a strongly deshielding nitro group results in a downfield shift about 0.9 ppm of the H-9 of ring B to form a chemical shift at 5 8.40-8.70 in aristolochic acids, but in aristolactams H-9 appears
966 rather upfield at 8 7.05-7.40. The H-9 of the sodium aristolochates shifts upfield and resonate usually from 8 8.15-8.35. The chemical shift of H-5 of ring C can be recognized by its downfield position at 5 8.10-9.12. Most of the aristolochic acids and aristolactams have one or two substitutions on the ring C; therefore, interpretation of coupling constants is useful to determine the ortho or meta H-H splitting and thus the positions of substituents [62,65,127,178,257]. The 13C NMR spectrum is an elegant method to distinguish aristolactams and sodium salts of aristolochic acids from aristolochic acids. The carbonyl carbon of aristolochic acids usually resonates around 8 170-175, whereas it shows an upfield shift of 5-10 ppm in aristolactams and downfield shift of 10-15 ppm in sodium salts of aristolochic acids [246,262]. The mass spectra of aristolochic acids were first reported by Pailer et al. [146], who found that the nitro radical is very easily split off from the molecular ion, giving the base peak [M-46]+, and then the CH3, CO, etc. were removed. Eckhardt et al. [406] found that primary cleavage of aristolochic acid occurs in the condensed aromatic system having a carboxy and a nitro functions in the peri positions, with elimination of NO2, and then loss of small units such as H, CH3, CO, and CHO. In 1987, Priestap reported the mass fragmentation pattern of aristolochic acids [130], Scheme (10). Rucker et al. described the mass spectral fragmentation of methyl ester of debilic acid. It displayed strong molecular ion peak followed by fragments [M-31]+, [M-46]+, [M-46-15]"1" and [M-46-15-15]+ [193]. Priestap explained the principal cleavages observed in the mass spectrum of aristolactam CII and DII [266], Scheme (11, 12). The mass spectrum typical of aristolactams exhibits very strong [M]+ peaks, usually the base peak, and the principal ions were associated with loss of methyl and carbonyl derived from initial cleavages around the methoxy functions. Aristolactam showed a preferential loss of a hydrogen atom to give the base peak followed by the elimination of a carboxy group. Expulsion of a hydrogen atom from the CH2OH group is more pronounced in aristolactam CII, presumably because the resultant ion was stabilized by the two adjacent methoxy groups. Initial expulsion of methyl may represent an equally likely process. The degradation path [M-2Me4CO-HCN]+ can be formulated in which fission of the CH2OH group proceeds with migration of hydrogen atoms on the aromatic ring system. In contrast to typical aristolactams, the [M]+ peak of aristolactam CII is not recognizable, possibly because of preferred stabilization through loss of one hydrogen atom and also by a more facile methyl elimination.
967
Scheme (10). Mass spectral fragmentation of aristolochic acid OCH,
Scheme (11). Mass spectral fragmentation of aristolactam CII (93)
Cleavage of one methoxy group with expulsion of a methyl radical may be favoured by a hydride transfer from the adjacent CH2OH group to the ether oxygen. In mass fragmentation pattern aristolactam DII behaves like typical O-hydroxybenzoic acids. It showed a preferential loss of water to give the most abundant fragment species. The primary cleavage of the
968
Scheme (12). Mass spectral fragmentation of aristolactam DII (92)
methoxy group with loss of methyl observed in aristolactams, is noted only to a very small extent in aristolactam DII. An alternative mode of breakdown in aristolactam DII occurs through dehydration between the carboxyl and methoxy groups with cyclization to a new ring. The resulting fragment then suffers the successive losses of hydrogen, three carbonyls, and hydrogencyanide. The mass spectra of 4,5-dioxoaporphine alkaloids were characterized by a direct loss of CO ([M-28]+) from the molecular ion leading to a prominent peak and subsequent loss of a methyl group accounted for other significant peak [181]. Teresa et al. have explained the mass spectral fragmentation of 2-(phenanthro[3,4-GT]-l,3-dioxole-6-nitro-5- carboxamido)propanoic acid methyl ester isolated from A. longa [62], Scheme (13). It has amino acid side chain joined to the phenanthrene structure by means of an amido linkage. The main fragmentation is due to the loss of the nitro group to give the base peak, and/or cleavage of the amino acid side chain. Priestap et al. reported the mass fragmentation of argentilactone, an active constituent of the rhizomes of A. argentina [297], Scheme (14). We have applied the circular dichroic (CD) exciton chirality method to determine the absolute configuration of sesquiterpene esters of aristolochic acids [407,408], Scheme (15,16). A positive Cotton effect at 221 nm was indicative of the ^-configuration at C-5' of aristophyllide A (27) and C (29) which is due to a clockwise configuration between the double bond and unsaturated aldehyde of the sesquiterpene, aristophyllene. A negative Cotton effect at 262 nm caused by the exciton interaction between the 3,4-methylenedioxybenzoate and the ring double bond inferred the ^-configuration at C-12' of 27 and 29 [251]. The opposite Cotton effects, i.e., a negaive Cotton effect at 227 nm and a positive Cotton effect at 261 nm concluded C-5'5 and C-12'/? stereochemistry for
969
o ,
-NO,
Scheme (13). Mass spectral fragmentation of compound (211)
m/z 150 /-C 2 H 3
C7H13 m/z 97 \ C7H,, mh 95
^\~^ 0 m/z 65 m/z 117
nk 91
Scheme (14). Mass spectral fragmentation of argentilactone (223)
970
JOOOO-
Scheme (15). CD spectra of 39-42
Scheme (16). CD spectra of 27-30
971 aristophyllide B (46) and D (45) [251]. A negative Cotton effect at 250 nm due to the arylcarboxylate chromophore proved the ^-configuration at C-4' for aristoloterpenate I (40), II (39), III (42) and IV (41) [249,409]. TOTAL SYNTHESIS Kupchan et al. achieved the first total synthesis of aristolochic acid involving photocyclization of substituted 2-iodostilbenes [410]. Piperonal (689) provided a suitable skeleton to build ring A of aristolochic acid. It was reduced to the piperonoyl alcohol (690) with lithium aluminium hydride. Bromination of 690 afforded 6-bromopiperonoyl bromide (691). It was then hydrogenated to the corresponding 2-bromo-4,5methylenedioxytoluene by «-butyllithium carbonation method. The toluic acid (693) was converted to acid chloride by oxalyl chloride, and bromination of acid chloride and by radiation with a 200-W tungsten lamp followed by methanolysis afforded the ester 694, which on treatment with silver nitrate produced methyl ester (695). In the synthetic sequence of ring C of aristolochic acid, 2-nitro-6-methoxytoluene (696) was oxidized to the nitroaldehyde (697), by Kronhke reaction. It was converted to the oxime 698 and then to iodaldehyde 700 via Sandmeyer reaction. The synthetic precursor of ring A on condensation with the Schiff s base of 695 in glacial acetic acid gave the 2-carbomethoxy-4,5-methylenedioxy-2'-iodomethoxy-a-nitro-c/5-stilbene (702). Photolysis of this product afforded aristolochic acid I methyl ester. Aristolochic acid (5) was prepared by hydrolysis of its methyl ester using Pailer and Schleppnik method [411], Scheme (17). PHARMACOLOGY To provide scientific support for the wide use of Aristolochia species in folk/traditional medicines, number of scientific groups world wide studied the pharmacological properties of both crude extracts and constituents of Aristolochia species. Many worthy achievements in the pharmacology of Aristolochia have been published. The aristolochic acids have been considered to be the most potent fraction of the Aristolochia constituents. Aristolochic acid I, the most active constituent of Aristolochia has been used for medicinal purposes since the GraecoRoman period [412]. The pharmacopeia of the People's Republic of China indicated that aristolo- chic acid can be used to relieve pain by subdueing hyperactivity of the liver, counteract toxicity, and cause subsidence of
972
HO Ac 68%
THF ] 00%
])n-BuLi CO, 2)H' AgNOj 3)Me0H 46%
ccc
CH, COOH
Scheme (17). Total synthesis of Aristolochic Acid I (5)
swelling [413]. On top of that it was also reported to relieve pain and induce diuresis. Following the observations that the compound was mutagenic and carcinogenic, it was removed from pharmaceutical products since a decade [412]. Antitumor Activity/Cytotoxicity Kupchan and Doskotsch found that an alcoholic extract of A. indica possessed reproducible activity against the adenocarcinoma 755 test system and isolated aristolochic acid I (5) as an active principle [414].
973
5 A A 1 R=H I AA-II R=OCH,
66 AA-I R=H 62 AA-II R=OCH,
+ DNA
dG-AA I dG-AA II
Scheme (18). Metabolic activation and DNA adduct formation of AAI and AAII
Kamatsh and co-workers reported that growth of mouse sarcoma-37 cells incubated with aristolochic acid (5) at concentration of 100-200 u.g/ml for 3 hours was completely inhibited. Treatment of mice with aristolochic acid (5) (1.25-5 mg/kg ip per day) for 3 days, after subcutaneous implantation of sarcoma-37 cells inhibited tumor growth by 40-50 %. The cytotoxicity on HeLa cells in culture was observed at concentration of 25 ug/ml [415]. The acute toxic effects of aristolochic acid I (5) were observed in rats and mice, oral or intravenous administration of high doses was followed by death from acute renal failure within 15 days [416]. The biological activities of aristolochic acid I (5), aristolactam I
974 (66), and aristolactam-/V-P-D-glucoside (71) have been evaluated towards Erlich ascities carcinoma cells in Swiss albino mice [417]. These studies showed that the cytotoxic activity is in the order aristolochic acid I aristolactam-A^-P-D-glucoside aristolactam I. Aristolactam III (65) has been reported to exhibit cytotoxic activity against three kinds of human cancer cell lines (A-549, SK-ME L2 and SK-OV-3) [418]. It is interesting that the cytotoxicity of aristolochic acid I (5) was not only obsereved in animal cells, has also been confirmed for plant cells [419]. Thus Aristolochia plants seem to have developed the aristolochic acid I (5) as chemical barriers against herbivores. Aristoloside (16), a glucoside, was also reported to possess antitumor activity [420]. Aristoloside (16) was given in the drinking water for 30 days to 4 months old SHN mice, a strain with a high incidence of mammary tumors. It inhibited preneoplastic mammary gland growth and elongated the estrous cycle. Aristoloside (16) had little effect on normal mammary gland growth. Since these findings were consistent with the antitumor effect of Guanmu-tong (Gmt, radix of A. manshuriensis), aristoloside was considered to be one of the active component of Gmt [421]. It was developed by the Otsuka Pharmaceutical Co. group as an antitumor agent [420]. In cytostatic potential screening of aristolochic acids and aristolactams against cultured KB and P388 cells by Pezzuto et al, aristolochic acid I (5) showed to be most potential with ED50 value 0.58 uM and aristolactam I (66) and aristolactam iV-(3-D-glucopyranoside (71) also demonstrated appreciable activity with ED50 vlaues 4.2 uM and 6.0 uM, respectively against P-388 cells [413]. In our cytotoxicity screening program on the isolates from Formosan Aristolochia species, most of the compounds exhibited some degree of cytotoxicity against KB, P-388, A-549, HT-29 and HL-60 cell lines with significant ED 50 values [159,223,244], Table 24. Aristolochic acid I (5) displayed excellent inhibition on the growth of HT-29 cell line with the ED50 value of 8.3xlO"4 ug/ml. Incidentally, ariskaninB (35) and cepharanone A (62) are equally potent towards P-388, A-549 and HT-29 cell lines. Ariskanin A (34), aristolochic acid All methyl ester (33) and aristolactam III (65) were cytotoxic against only P388 cell line, whereas ariskanin C (36) was active against only A-549 cell line. Interestingly, aristolactam I (66) and isorhamnetin-3-O-rutinoside (531) well inhibited the growth of all cell lines tested, KB, P-388, A-549, HT-29 and HL-60 cell lines, however the latter compound showed greater extent of inhibition with ED50 values of 1.3, 1.9, 0.8, 1.2 and 0.5 u.g/ml, respectively. Ariskanin E (38), aristolochic acid II methyl ester (25) and
975 Table 24. Cytotoxicity of compounds Isolated from Aristolochia ED50 Cone (u.g/ml) Compound ariskanin A (34) ariskanin B (35) ariskanin C (36) ariskanin D (37) ariskanin E (38) aristolochic acid A II methyl ester (33) aristolochic acid 11 methyl ester (25) aristolochic acid C (2) aristolochic acid I (5) aristolochic acid II (1) aristolactam (66) aristolactam III (65) aristolactam All (83) cepharanone A (62) aristolactam A III (89) aristolactam-A'-P-D-glucoside (71) aristolactam-C-jV-P-D-glucoside(69) isorhamnetin-3-O-rutinoside (531) KB : human epidermoid carcinoma P-388 : mouse lymphocytic leukemia A-549 : lung adenocarcinoma HT-29 : colon adenocarcinoma HL-60 : human leukemia
KB >50 4.1 6.2 11.4 >50 >50 >50 >50 4.0 >50 3.3 13.5 >50 4.1 15.8 >50 >50 1.3
P-388 1.5 2.3 5.1 0.5 0.5 1.5 0.5 8.8 0.7 10.9 1.0 3.7 1.3 2.3 0.8 N 2.4 1.9
Cell lines A-549 >40 1.7 2.3 3.8 1.2 17.4 1.2 >20 5.0 >50 3.2 19.1 2.7 1.7 1.7 N 4.6 0.8
HT-29 8.0 3.3 7.3 2.2 4.9 14.8 4.9 17.1 0.00083 8.4 2.6 18.6 13.2 3.3 4.5 N 12.9 1.2
HL-60 9.3 3.9 5.8 1.3 >50 "1 11.9 >50 40 3.4 >50 2.4 6.8 12.9 3.9 1.8 N 11.4 0.5
aristolactam All (83) were active against P-388, and A-549 cell lines, whereas ariskanin D (37) and aristolactam AIII (89) were active against P388, HT-29 and HL-60 cell lines and P-388, A-549, HL-60 cell lines, respectively. In addition, the novel constituents from our research, sesquiterpene esters of aristolochic acids (39-46) have also showed moderate cytotoxicity against hepatoma G2,2,2,15 cell line [249,251]. Aristolactam All (83) snowed cytotoxicity against PS and KB cells in culture at ED50 3.2, and 2.1 g/ml, respectively [422]. In vitro cytotoxicity of aristolactam la (64) and aristolochic acid I (5) isolated from the roots of A. longa against P-388 lymphocytic leukaemia and NSC LCN6 (brainchial epidermoid carcinoma of human origin) was observed [191]. Zhang et al. reported that the versicolactone A (307) showed growth inhibition against human liver cancer cell line QQy-7703 [320]. An amide, aurantiamide acetate (172) found in A. tubflora showed cytotoxicity against A-549, MCF-7, and HT-29 cells in culture with ED50 values of 2.15x10"', 7.73xlO"2 and 2.60 |ig/ml, respectively [288]. NCoumaroyl- tyramine (178) suppressed the growth of human tumor cells such as U937 and Jurkat cells. In addition, the suppression of the growth
976 of the cells was strongly associated with an increased percentage of cells in the S phase of the cell cycle progression. Furthermore, Ncoumaroyltyramine (178) was able to inhibit the protein tyrosine kinases including epiderma growth factor receptor (EGFR) [423]. Matsuda et al. demonstrated the antimitotic activity of argentilactone (223) isolated from A. argentina [424]. However, it bears a close structural relation with parasorbic acid. Parasorbic acid and a series of ot,p-unsaturated lactones have been reported to induce cancer in rats [425-427]. Argentilactone has irritative effects on the skin and mucosa and also may cause allergic reactions on the skin. Thus Priestap et al. warned the health risk of ingesting the tinctures of A. argentina as it contain 0.05 mg/ml of argentilactone, a possible carcinogenic agent [296]. Mutagenic Activity Pezzuto et al. found that aristolochic acid I (5) serves as a direct-acting mutagen in Salmonella typhimurium strains TA100, TA102, TA1537 and TM677, but was not active in the nitroreductase defficient strains TA98NR and TA100NR [412]. However, aristolic acid (51) was also found to be a direct-acting mutagen in Salmonella strains TA98, TA100, TA102, TA1537, and TM677 as well as strains TA98NR and TA100NR. Both compounds were active mutagens in cultured Chinese hamster overy cells. Thus nitro group at position 10 is not required to induce a mutagenic response. A series of structural relatives, aristolochic acid I methyl ester (28), aristolic acid methyl ester (55), aristolochic acid D (10), aristolactam I (66), aristolactam All (83) and aristolactam-jV-P-D- glucoside (71) were found to be inactive with S. typhimurium strain TM677. But compound 28 and 55 were found to be active mutagens with strains TA98, TA100, TA102 and TA1537. Pistelli et al. isolated aristolochic acid IV (14) from A. rigida and evaluated the mutagenic activity using the plate incorporation assay for S. typhimurium strain TA100 [238,428]. Aristolochic acid IV (14) showed dose dependent activity on mutation of the TA100 strain. The assay with S9, rat liver enzyme system which is added to stimulate metabolic activation process showed no significant dose response relationship, only variations of revertants per plate with in spontaneous levels were noted. On the contrary, in the absence of S9, a significant increase of revertants per plate took place. These results indicated that aristolochic acid IV (14) is endowed with weak direct mutagenic properties, and this effect seemed to be inhibited at least in part, by metabolic reactions.
977 9-Methoxytariacuripyrone (629), a nitro phenanthrene compound isolated from A. brevipes showed strong mutagenic activity in strain TA98, TA100 and some YG strains of S. typhimurium with and without S9 addition [429]. Incubation with cytosol resulted in a heavy increase in mutagenecity. When incubated with microsomes the activity was dramatically decreased. Schimmer et al. viewed that enzymes may possibly involved in activation and detoxification of the compound. The role of the basic structure on the mutagenecity mediated through the nitro group was also considered. Antifertility Activity Aristolochic acid (5) and its methyl ester (28) were found to possess significant antifertility activity in mice [87-89]. Wang and Zheng reported that aristolochic acid (5) showed significant antiimplantation and early pregnancy-interrupting effects when administered orally to mice at a dose of 3-4 mg/kg [430]. This acid showed neither estrogenic nor antiestrogenic actions. Treatment with exogenous progesterone failed to prevent its pregnancy interrupting action. In addition, intra-amniotic injection of aristolochic acid I (5) in mid-term pregnant dogs and rats led to termination of pregnancy. Later, Che et al. reported that aristolochic acid (5) and its methyl ester (28), aristolic acid (51) and (125)-7,12-secoishwaran-12-ol (390) were ineffective in antifertility tests, when given to mice, hamsters and rats [92]. Anti-Oestrogenic and Anti-Implantation Activity Studies on endocrine and contraceptive property of aristolic acid (51) in the pre-implantation stage of pregnancy in mice by Pakrashi et al. elicited anti-oestrogenic nature as it prevented oestrogen induced weight increase and epithelial growth of mouse uterus and prevented implantation in the early stage of pregnancy in mice [431]. Finding of antioestrogenicity of aristolic acid was corroborated by the depletion of alkaline phosphatase activity, glycogen content and mitotic counts of oestrogen treated uterus. Ganguly et al. reported that aristolic acid disrupted nidation in mice when administered on day 1 of pregnancy [432]. The compound did not affect the tubal transport of eggs, but the uterine blue reaction caused by extravasation of the dye, pontamine blue at future implantation sites was inhibited significantly in treated mice.
978 This acid induced impairment of development and reconciled with decreases found in uterine weight and its total protein contents. It prevented specific uterine alkaline phosphatase activity. On the otherhand, specific uterine acid phosphatase activity remained low on days 4 and 5 and increased significantly thereafter. It indicated that aristolic acid interferes with steroidal conditioning of the uterus and renders it hostile to ovum implantation. A sequiterpene, (12S)-7,12-secoishwaran-12-ol (390) isolated from the root of A. indica which is reputed for its emmenagogic and abortifacient properties was found to exert 100% interceptive activity and 91.7 % antiimplantation activity in adult female mice at a single oral dose of 100 mg/kg body weight on day 1 of pregnancy [89]. Subsequent lower doses showed lower percentages of activity. Laparotomy indicated abortion to occur between day 8 and 10 of pregnancy. No toxic effect was found at the dose levels used. It also showed anti-estrogenic potency when administered to immature female mice along with estrogen [433]. When administered alone, sesquiterpene did not showed any uterotropic activity but when given together with estrogen, it inhibited the uterotropic action of estrogen. Since estrogen is necessary for implantation, and the compound is having anti-implantation and abortifacient activities, this action may be due to inhibition of estrogen by the compound during the pre- and post implantation period. Interceptive Activity A phenolic acid compound, /7-coumaric acid (585) isolated from A. indica has been investigated for its antifertility effect at different stages of pregnancy in mice [434,435]. It showed 100% interceptive activity in mice when administered at 50 mg/kg dose. The therapeutic value of the 100 % active dose is investigated by acute toxicity and teratogenic studies. In acute toxicity study, it had high margin of safety (>1000 mg/kg). The compound did not show teratogenic effect. Abortifacient Activity Methyl ester of aristolic acid (55) isolated from A. indica exerted 100%, 25% and 20 % abortifacient activity in mice when administered at the dose level of 60 mg/kg body weight on days 6 or 7, 10 and 12 post coitum, respectively [88].
979 A sequiterpene, (12S)-7,12-secoishwaran-12-ol (390) exerted 100% abortifacient activity in mice when administered at a single oral dose of 100 mg/kg body weight on 6th or 7th day of pregnancy [89]. Short Term Toxicity Study As methyl ester of aristolic acid (55) showed potent abortifacient activity, Pakrashi et al. designed a short term toxicity study to predict reasonable safety and to identify undesirable drug effects [436]. Chronic adminis- tration of methyl ester of aristolic acid (55) at the dose level of 60 mg/kg/day for 5 days per week over a period of 4 weeks increased liver alkaline phosphatase activity, depleted liver glycogen and decreased kidney alkaline phosphatase activity in the treated group showing damage to liver and kidney. There was also an increase in the treated uterine weight. The treated animals which later became mothers were indifferent towards their letters and exhibited cannibalism. These results provide indications of undesirable drug effects one should look for in human, during clinical trials. Since a possible damage to liver and kidney is indicated here, a special attention should be paid to these organs in experiments with higher dose in same or higher animals. Immunomodulating Activity Experiments performed in rabbits and guinea pigs showed marked stimulation of leukocyte phagocytosis following application of Aristolochia extracts, and aristolochic acid I (5) was characterized as the active principle. It was capable of offsetting chloramphenicol- and prednisolone- impaired phagocytic activity without influencing the number of leukocytes. When phagocytosis was impaired by prednisolone, acid showed the effect depending on the dose of prednisolone: from 2.5 mg/kg prednisolone upwards its lesional activity could not be influenced, but for doses under 1 mg/kg values were clearly normalized by treatment with aristolochic acid I (5). Offsetting of damage by cyclophosphamide to phagocytosis was seen even after higher drug doses. Further investigation proved that 5 has a protection activating effect on the phagocytosis of leukocytes. This effect was also observed in cold blooded animals including carps, Aesculopius snake, and Elaphe longssima [437]. It acts not on the invading pathogens but on the naturally existing endogenous defense of the diseased organism, via vigorous stimulation of the phagocytic activity of host leukocytes [437]. Aristolochic acid I (5)
980 stimulated the reticuloendothelial system (RES) and abolished the RESdepressing effect of chloramphenicol in mice and rats. Treatment with aristolochic acid I (5) increased the oxygen consumption and thus the metabolic activity in mice liver cells and splenocytes [438]. Antibacterial Activity Aristolochic acid I (5) was also reported to exhibit antibacterial action against Staphylococcus aureus, Diphococcus pneumoniae and Streptococcus pyogenes in infected mice at 50 ug/kg ip [415]. When, rats with wounds infected with S. aureus were treated intraperitoneally or orally with aristolochic acid I (5), they recovered much faster than control. In mice with Pneumococci infections were influenced very well by aristolochic acid I (5). Rabits after intravenous application of aristolochic acid I (5) showed an increased antibacterial action of serum. Aristolactam la (64) and aristolochic acid I (5) showed antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, S. faecalis, S. aureus and S. epidermides [191]. Neurological disorders, especially Parkinson's diseases have been treated by the administration of the aristolactam taliscanine (91) to the affected patient [439]. Cepharadione A (107) exhibited antimicrobial activities [440]. An aristolactam, aristolactam ./V-p-D-glucopyranoside (71) characterized from the roots of A. contorta showed significant antibacterial activity against Gram-positive bacteria [260]. The essential oil of A. indica containing P-caryophyllene (313), a-humulene (303), caryophyllene oxide (315), and linalool (215) as major constituents was found to be moderately active against Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, B. shaericus, and Salmonella typhimurium [84]. A sequiterpenoid, l(10)-aristolen-13-al (334) isolated from A. debilis showed moderate antimicrobial activity against Staphylococcus aureus [346]. Anti HIV Activity In anti HIV evaluation by Wu et al, dehydrooxoperezinone (358) isolated from A. manshuriensis showed moderate anti HIV activity in acutely infected H-9 lymphocyte cells with IC50 and EC50 values of 25.1 and 17.5 |^g/ml, respectively and therapeutic index is 1.43 [241].
981 Antiplatelet Aggregation Xu and Sun reported that moupinamide (179), an amide found in several Aristolochia species, inhibited rat platelet aggregation and MDA formation in platelets in vitro [205]. In our antiplatelet aggregation activity evaluation studies, aristolochic acid All methyl ester (33), ariskanin A-E (34-38), aristolactam All (83), aristolactam AIII (89), piperolactam A (84), aristolide A (208), aristolactone (278) and manshurolide (307) showed different degree of inhibition of rabbit platelet aggregation based on inducers, arachidonic acid (AA), collagen (Col), platelet activation factor (PAF) and thrombin (Thr) [159,223,244], Table 25. Among screened, no compound inhibited significantly the rabbit platelet aggregation induced by thrombin (Thr) at even high concentrations (100 u.g/ml). Aristolochic acid All methyl ester (33), ariskanin A-E (34-38), aristolide A (208), manshurolide (307), aristolactam All (83), and piperolactam A (84) displayed 100% inhibition of platelet aggregation induced by AA at 100 ug/ml and however, aristolactam AIII (89) and aristolactone (278) also showed little less inhibition at the same concentration. Among these inhibitors, aristolochic acid All methyl ester (33), piperolactam A (84), aristolactam All (83), and ariskanin-E (38) exhibited 100% even at 50 ug/ml whereas ariskanin B-D (35-37), aristolide A (208), and manshurolide (307) showed little less inhibition at this concentration. Aristolochic acid All methyl ester (33), ariskanin B-E (35-38) and piperolactam A (84) were most potent inhibitors (100%) of platelet aggregation induced by Col and ariskanin A (34) and aristolactam AIII (89) also inhibited significantly. Only ariskanin C (36) showed 100% inhibition of platelet aggregation induced by PAF at 100 |ug/ml, but ariskanin A (34), B (35), aristolactam AIII (89) and aristolactone (278) showed significant inhibition. Wang et al. also reported that aristolactone (278) showed inhibition [187] of platelet aggregation induced by PAP with an IC50 = 2.63 x 10"5 M. Smooth Muscle Relaxant Activity Lemos et al. studied the nonspecific and reversible smooth muscle relaxant activities of the ethanol extract of A. papillaris (EE), a fraction containing tertiary alkaloids (TAF) and three alkaloids isolated from it [120]. In non pregnant rat uterus, EE and TAF inhibited both the oxytocin-induced contractions and the amplitude of rhythmic spontaneous contractions with the IC50 values of 0.91 and 0.22 (ig/ml in the first experiment and 1.0 and 0.17 u.g/ml in the second, respectively. The rythmic contractions of the uterus obtained from 21-day pregnant rats
982 Table 25. Antiplatelet aggregation activity of compounds Isolated from Aristolochia Inducer Compounds Cone, (nig/ml Inhibition (%) Compounds
Tlir(0.1U/ml)
Col(10nig/ii]l)
AA(100nM)
PAF(2 g/ml)
100
50
100
50
20
100
50
20
100
50
iclliyl plicophorbidc-ii (21
A
A
A
A
0.9 ± 1.1
N
N
0.1 ± 4.1
N
N
45.2 ±15.1
15.5 ±6.4
0.5 ±3.8
22.5 ±4.8+
N
13.5 ±4.5*
N
N
48.3 ±21.7*
N
arisiohde-A (208)
-0.6 ± 1.8
N
100.0 iO.Of. 89.6 ±8.6*
aristolactonc(278)
-1.5 ±0.8
N
9I.7±6.8+.
11.2 ±7.3
N
maiishurolidc (307)
-3.2 ± 2.6
N
100.0 ±0.0$ 77.3±11.5f
18.2 ±6.7* 21.9 ±5.9+
N
N
51 1±12.3+
N
arsto!aciam-All (83)
0.8 ±3.9
N
100.0 ±0.0J 100.0 ±O.0J 95.3 ± 3.7J J6.8± IO.2t
N
N
10.6 ± 4 1 +
N
pipcroliiciiini-A (84)
4.0 ± 3.4
N
!00.0±0.0j 100 0±0.0f
38.8 ± 13.8+
N
anskaiiin A (34)
7.5 ± 1.7
N
100.0± 1 3
N
N
76.2 ± 5 8
N
N
61.2 ±9.0
N
iiriskiiniii B (35)
4.7 ± 1.2
N
100.0 ± 1.3
80.4 ± 4.1
3.1 ± 2.5
100 0±0.6
36.8 ±6.8
12.6 ± 1.4
60 6 ±8.9
ariskanmC(36)
3I.9±7.3
N
100.0 ± 1.3
51.5 ± 8.0
5.2 ±2.7
100 0 ±0.6
80.9 ± 7.5
13.5 ± I.I
100.0 ± 1.2
19.0 ± 3 1
ariskanin D (37)
4.1 ± 1.9
N
100.0 ± 1.3
93.4±3.8
47.8 ±9.3
100,0 ±0.6
IOO.O±O.6
8.1 ±0.7
90.0 ± 4.9
15.3±2.9
anskanin E (38}
6.9 ± 1.9
N
100.0 ±0.4
100.0±0.4
4.0 ± 1.7
100.0 ±0.5
100.0 ±0.5
12.6 ±4.5
45.3 ± 8.9
N
10.3 ± 1.6J 100.0 ±0 0; 60.6 ± 2 ) 1 * 22 3 ± 2.4
N " "
N
100.0 ±0.4
100.0 ±0.4
6.6 ± 1.3
100.0 ±0.5
I7.8±4.2
2.5± t.6
100.0 ± 1.2
14.2±4.0
arsiolaciam-AH] (89)
3.7 ± 1.2
N
77.9 ±9 1
N
N
67.9 ±5.1
N
N
58.9 ±5.5
N
aspirin
2.5 ± 1.2
N
!00.0± 1.4 51.0 ± 12.5
3.6 ± 1.3
7.3 ±3.9
N
N
2.4 ± 1 2
N
rislolocliic acid A II metli 16.3 ± 10.6 esler (33)
N = not tested. A = platelet aggregation promoted. Thr = thrombin, AA = arachitonic acid, Col = collagen, PAF = platelet activation factor.
were also reduced by EE and TAP with IC50 values of 25.5 and 11.2 )ag/ml, respectively. The relaxation of isolated guinea pig trachea produced by EE and TAP were also observed with the compounds isolated from TAF, moupinamide (179), coclaurine (140) and isoboldine (100) with IC50 values of 1.58xlO"4 M, 3.98xlO"4 M and 7.10xl(T4 M, respectively. Propranolol significantly antagonized the effects of coclaurine (140) and isoboldine (100) but failed to inhibit the responses to moupinamide (179) which suggests that these compounds produce muscle relaxation by P-adrenoceptor-dependent and -independent mechanisms. Antispasmodic Activity The methanol extract of the aerial parts of A. constricta actively inhibited the electrical induced contractions of the isolated guinea-pig ileum with IC50 value of 196.3 ug/ml, Table 26. Five protopine alkaloids,
983 Table 26. IC50 Values and Confidence Limits of Extracts, Fraction VI, and Pure compounds from A. constricta on ECI and Ach- and Histinduced Contractions of Guinea-pig Ileum IC50 values
CHCl 3 -MeOH extract MeOH extract fraction VI
137 139 138 135 136 137 139 138 135 136 137 139 138 135 136
confidence limits (lower-upper) effect on ECI 515.7 711.3 M 681.7 ng/mL 160.3 272.1 M 196.3 ng/mL 180.3 265.4 M 220.3 u.g/mL 6.6 x 1 0 ' M 4.8 x 10"' M 8.9 x 10"'M 1.9 x 10"'M 1.4 x 1 0 ' M 2.5 x 10"'M 1.6 x 10"'M 2.6 x 10"'M 2.1 x 1O"5M 4.6 x 10"' M 9.4 x 10"'M 6.6 x 10"'M 8.6 x 10"'M 5.8 x 10-'M 1.3 x 10"'M effect on Ach-Induced Contractions 4.3 x 10"'M 7.8 x 10-'M 5.8 x 10"' M 2.0 x 10"'M 1.5 x 10"'M 2.6 x 10-'M 2.6 x 10"'M 1.4 x 10"'M 2.8 x 10"'M 5.1 x 1 0 ' M 1.1 x 10"'M 7.5 x 10"'M 8.6 x 10"'M 6.0 x 10"'M 1.2 x 10""'M effect on Hist-Induced Contractions 4.9 x 1 0 ' M 9.3 x 1 0 ' M 6.8 x 10"'M 2.2 x 10"'M 1.5 x 1 0 ' M 3.1 x 10"'M 1.8 x 10"'M 3.9 x 10"'M 2.6 x 10"'M 3.7 x 10"'M 6.9 x 10"'M 5.1 x 10-'M 5.9 x 10"' M 4.2 x 10"'M 8.2 x 10"'M
3,5-di-O-methylconstrictosine (137), 5,6-dihydro-3,5-di-O- methylconstrictosine (139), 5,6-dihydroconstrictosine (138), constrictosine (135) and 3-0-methylconstrictosine (136) were isolated as compounds responsible for the observed antispasmodic activity of the extract. These compounds exerted a significant activity on the ECI, at concentrations of 2.5xlO"5, 5*10~5, 10"4 M. The relative order of potency was: 137<136<135<139 and 138 as indicated by their IC50. These alkaloids also reduced significantly both acetylcholine (Ach-) and histamine (Hist-) induced contractions of isolated guinea-pig ileum [280]. Herman et al. also reported both anticholinergic and antihistaminic properties of protopine type alkaloids [441]. Effect on Morphine Withdrawal The protopine alkaloids, constrictosine (135), 3-0-methylconstrictosine (136), 5,6-dihydroconstrictosine (138), 3,5-di-Omethylconstrictosine (137), and 5,6-dihydro-3,5-di-O-methylconstnctosine (139) purified from the aerial parts of A. constricta were able to reduce dose-dependently the morphine withdrawal when injected 10 min.
984 before morphine. After washout, both Ach response electrical stimulation and the final opiate withdrawal were still reduced. The ability of these alkaloids to reduce morphine withdrawal may be related to their anticholinergic activity since acetylcholine is primarly involved in opioid withdrawal. This hypothesis was strongly supported, because both acetylcholine and electrical stimulation resulted in further reduction after washout. Also, the ability of these alkaloids to block both post- and presynaptic acetylcholine receptors further supported this possibility. Finally, whatever the mechanism may be, this activity results provides evidence that protopine type alkaloids are effective in opiate withdrawal and could be potential anti-addictive agents [442]. Anti Snakevenom Activity Aristolochic acid I (5) inactivated snake venoms including those of Naja atra and Bungarus multicinctus but did not inactivate Trimeresurus muorosquamatus, Agkistroden acutus or T. gramineus venoms [443]. A furanoid diterpene, columbin (419) isolated from rhizomes of A. albida was found to significantly reduce the toxic symptoms and protect the mice against the lethal doses of venoms of snakes, Naja nigricollis (spitting cobra) and Bitis arietans (puff-adder), commonly found in northern Nigeria [444], Table 27. However, the compound is more effective against the venom of N. nigricollis (ED50 = 45 mg/kg) than that of B. arietans (ED50 = 74 mg/kg). The whole animal in vivo studies were conducted using the mortality of male Swiss albino mice after intra-peritoneal injection of lethal doses (LD1Oo),.8.75 mg/kg and 4.20 mg/kg of venoms of Table 27. Anti snakevenom activity of columbin (419) isolated form A. albida Group number
1 2 3 4 5 6
Dose of columbin (419) (nig/Kg) administered
5.0 10.0 20.0 40.0 80.0 Saline
Envenomation with LDioo of N. nigricollis
Envenomation with LDioo of B. arietans
Number of survival out of 15(n=15) 0 1 3 7 11 0
Number of survival out of 15(n=15) 0 2 3 6 9 0
% of survial
0.0 6.7 20.0 46.7 73.3 0.0
% of survial
0.0 13.3 20.0 40.0 60.0 0.0
985 these two species, respectively. It was observed that the mortality of mice was reduced progressively with increasing doses of the compound and significant protection (p<0.05) was observed at 80 mg/kg dose. Death from N. nigricollis resulted mainly due to neurotoxicity by curare-like action of cobrotoxin on the respiratory muscles causing flaccid paralysis leading to respiratory depression. The diterpene was found to interfere with acetylcholine receptors and there, it might mediate its actions by antagonizing the actions of the neurotoxic substances in the venom at the acetylcholine receptor sites. This might be the reason for its higher activity against N. nigricollis venom compared to that of B. arietans which interferes primarily with haemostasis and causes cardiotoxicity. Phospholipase A2 (PLA2) Inhibition Activity Vishwanath et al. found that aristolochic acid I (5) interact with the major basic phospholipase A2 from Vipera russelli venom [445]. It is a non- competitive inhibitor with a Ki of 9.9><10"4 M when phosphatidylcholine is used as substrate. The inhibition of direct and indirect hemolysis is higher compared to the inhibition of phosphatidylcholine hydrolysis. Edema- inducing activity of Vipera russelli phospholipase A2 is inhibited by aristolochic acid I (5) when injected either as a mixture or separately. Both i.m. and i.p. administration of aristolochic acid I (5) following phospholipase injection are equally effective in inhibiting edema. But aristolochic acid I (5) failed to inhibit other pathological activities of the enzyme. The interaction of aristolochic acid I (5) with phospholipase A2 from Vipera russelli venom was also studied by Vishwanath et al. by circular dichroism measurements [446]. The binding of aristolochic acid I (5) to PLA2 induces an extrinsic CD band at 320 nm. Aristolochic acid I (5) forms 1:1 complex with PLA2, with an association constant R, of 5.4xlO3 M~' and a Gibb's free energy change of-5.1 kcal/mol. These values suggest that the interaction is weak. Recently, Chandra et al studied the structural basis of a complex formed between Vipera russelli phospholipase A2 and aristolochic acid at 1.7 A resolution [447]. The structure contains two crystallographically independent molecules of phospholipase A2 in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. They also found that aristolochic acid I (5) is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic
986 acid (5) are provided by its OH group which forms two hydrogen bonds, one each with His and Asp. A basic phospholipase enzyme, TFV Pl-X (pi 9.2) and two acidic PLA2 enzymes, TFV PL-la (pi 4.9) and TFV PL-lb (pi 4.5) purified from the Trimeresurus flavoviridis venom induced edema when injected into the mouse foot pad [448]. Aristolochic acid I (5) interacts with these PLA2 enzymes. It is a competitive inhibitor with varying affinity when PC is used as substrate. Aristolochic acid I (5) inhibits direct and indirect hemolytic activity, as well as edema-inducing activity, of TFV PL-X, but fails to neutralize the lethal potency of the enzyme. On the other hand, it inhibits direct and indirect hemolytic activity of TFV PL-la and TFV PLIb only at 10 fold higher concentrations and it enhances the edemainducing activity of these enzymes. Such effects of aristolochic acid I (5) indicates that different mechanisms may be involved in the edemainducing activity of PLA2 enzymes and catalytic and pharmacological sites are separate on the PLA2 molecule. A neutral-active, Ca2+-dependent phospholipase A2 (PLA2) purified from human synovial fluid (HSF) induced edema when injected into the mouse foot pad. Aristolochic acid I (5) produced a dose dependent inhibition of in vitro phospholipids hydrolysis by HSF-PLA2, porcine pancreatic PLA2, snake venom (Naja naja) PLA2, and PLA2 isolated from human platelet [449]. The sensitivity of these PLA2s to inhibition by aristolochic acid varied makedly: HSF-PLA2 >N. naja PLA2 > Human platelet PLA2 > porcine pancreatic PLA2. The inhibition of HSF-PLA2 by aristolochic acid I (5) was independent of substrate concentration (8-144 uM) and Ca2+ concentration (0.1-4.0 mM). These indicate that inhibition of HSF-PLA2 by aristolochic acid I (5) may result from direct interaction with enzyme. When aristolochic acid I (5) was mixed with HSF-PLA2 and then injected into the mouse foot pad, edema was inhibited in dose dependent manner and was positively correlated with in vitro inhibition of PLA2 activity. It has been reported by Denson et al. that general anaesthetics inhibit Ca2+ activated potassium (BK) channels at clinically relevant concentrations. These anaesthetics produce their effect on BK channels by disrupting the phospholipase A2 (PLA2)-arachidonic acid signal transduction pathway. The PLA2 inhibitor, aristolochic acid I (5) (250 uM) inhibited BK channels by 47 % [450]. Antifeedant Activity
Lajide et al. examined the antifeedeant activity of the metabolites of A. albida root extracts, aristolic acid (51), aristolochic acid (5), 6-hydroxy-
987 aristolochic acid (10), aristolactam (66) and aristolone (335) against tobacco cut worm larvae, Spodoptera litura [99], Table 28. The antifeedant activity is greatest with aristolochic acid I (5), whereas the naturally occurring derivative, possessing a hydroxyl group in the 6position, was less active. The decrease in activity of 10 is consistent with observations reported in some phenanthrene-based alkaloids that an undissociated phenolic group leads to a marked decrease in activity. Furthermore, the esterificiation of the two acids to afford methyl aristolochate (28), methyl 6-hydroxyaristo- lochiate (29) resulted in a very significant loss of antifeedant activity against the larvae of S. litura. This observation might suggest that the free carboxyl (COOH), in close proximity to the nitro (NO2) group, is necessary for antifeedant activity. It is possible that the bonding arrangement between the two functional groups contributes a great deal to the volatility of aristolochic acid and its bitter taste. Aristolactam (66), on the other hand, had no antifeedant activity when compared to aristolochic acid I (5); the free COOH and NO2 groups in latter compound have been transformed into a lactam-type ring in 66. Aristolic acid (51), lacking the NO2 group, and aristolone (335) did not show any antifeedant activity at all. The results obtained showed that decarboxylation of 5 to 8-methoxy-3,4- (methylenedioxy)-lOnitrophenanthrene (5a) or reduction to 8-methoxy-3,4-(methylenedioxy)l-(hydroxymethyl)-10-nitrophenanthrene (5b) and 8-methoxy-3,4(methylenedioxy)-10-nitrophenanthrene-9-carboxaldehyde (5c) significantly decreased the antifeedant activity even at a very high concentration. It is possible that an electrostatic interaction occurs between the carboxylate ion and a positively charged group of the receptor; this interaction may be enhanced by the close proximity of the nitro group in 5. The very high activity of 5 observed is consistent with previous observation on the antifeedant activity of aristolochic acid I (5), in which 0.00001 % of 5 reduced feeding by 50 % against the fifth instar larvae of the locusts L. migratoria and S. gregaria. The potency of 5 as an antifeedant could make this compound a potentially interesting lead for the development of new plant protection chemicals. In a comparative experiment, aristolochic acid demonstrated a level of activity comparable to azadirachtin, one of the most potent feeding ditterent compounds isolated so far from plants. These results clearly demonstrate both the importance of aristolochic acid as a potential plant deffentive agent and its potential for the development of agronomically useful substances. Aristolochic acid I (5) showed herbicidal activity against Chenopodium album, Amaranthus repraflexus, Lindemia pyxidaria and Setaria viridis
988 Table 28. Antifeedant Acitivity of A. albida Metabolites and Synthetic Derivatives against Third Instar Larvae of S. litura Antifeedant index (mean ± SE) Dose,
5T
5
10
28
29
5a
5b
5c
45.61 ±
36.20 ±
33.68 ±
789
9.51
10.93
66
335
AZA
0.00 ±
ppm 10000
1000
68.09 ±
0.00 ±
0.00 ±
30.86 ±
49.70 ±
48.05 ±
49.92 ±
1007
000
0.00
2.35
0.30
5.47
324
500
0.00 ±
2I.42±
41.12±
0.00
664
258
0.00
100
3.73 ±
53.67 ±
51.04 ±
0.00 ±
1.86
17 37
1.86
50
19 96 ± 093
0.00
10
48.56 ±
4 28 ±
000 0.00 ±
0 00 0 00±
4.27
12.52 5
18.59 ±
1
29.54 ±
05
40.25 ±
0.1
68.57 ±
1.80
10.86
2 76
10.00 0
54.25 ±
54.25 ±
54.25 ±
54.25 ±
54.25 ±
41 88 ±
41.88 ±
41.88 ±
54.25 ±
54.25 ±
45.22 *
(control)
1.80
1.80
1.80
1.80
1.80
6.86
686
6.86
1.80
1.80
2.75
but it was not phytotoxic to corn and soy beans [451]. Cepharadione A (107) exhibits insect antifeedant activity [452]. Germination-Inhibiting Activity
Urzua et al. reported that a terpenoid mixture from a nonpolar fraction of the roots of A. chilensis completely inhibited the germination of lettuce and wheat seeds, partially the germination of onion and carrot seeds and is inactive with raddish and beans [312]. The principal components of the terpenoid mixture, (-)-P-bisabolene (264) and (+)-P-sesquiphyllandrene (266), showed different inhibitory properties from those of the mixture. Trypanocidal Activity
Abe et al. reported that the methanolic extract of the roots of A. taliscana, locally known as "Guaco" immobilized all the epimastigotes of Trypanosoma cruzi, in vitro, which is the etiological agent for Chagas' disease one of the most serious protozoan diseases in Latin America, even at lower concentration of 0.5 mg/ml [122]. Four neolignans, eupomatenoid-1 (459), -7 (460), licarin A (464) and B (463), and two
989 lignans austrobailignan-7 (473) and fragransin Ej (470) were isolated from the active fraction. Compounds 459,460,463,464 immobilized all the epimastigotes of T. cruzi even at minimum concentration (MCioo) of 25 ug/ml, 40 ug/ml, 75 ug/ml, and 150 (J.g/ml, respectively, after incubation for 48h, while compounds 473 and 470 were inactive. Corresponding concentration of gossypol, berberine chloride and harmine was 280 ug/ml, 300 ug/ml, and >500 ug/ml, respectively. Thus compounds exhibited higher activity than trypanocidal natural compounds. Comparisons of 459 with 473, and 460 with 470 suggest that loss of hydroxyl group reduces activity. The differences of activity between 459 and 460, and 464 and 463 suggestes that steric structures might have influence on the activity. From the high yield and MC^o value it was concluded that trypanocidal activity of Guaco is mainly due to eupomatenoid-7 (460). Inhibition ofAcetylcholine Esterase
Bisbenzylisoquinoline alkaloids, (-)-obamegine (151) and fangchinoline (152) isolated from A. fangchi inhibited acetylcholine esterase of rat brain with IC50 of 3.2xlO"s and 4.0xl0"6 M, respectively. Acetylcholine esterase inhibitors are useful for treatment of muscular dystrophy, dementia, Alzheimer's disease, digestive tract disease etc. Tablets (200 mg/each) were formulated containing (-)-obamegine 10, corn starch 44, crystalline cellulose 40, CMC ca 5, silica 0.5 and Mg stearate 0.5 g [453]. Radical Scavenging Activity
Phenylpropanoid glycosides, 6-(9-p-hydroxycinnamoyl glucose (591), 6-O- (£)-feruloyl glucose (601), 6-0-(£')-/?-hydroxycinnamoyl ethyl glucoside (594), 6-0-(Z)-/?-hydroxycinnamoyl ethyl glucoside (593), and 3-0-(is)-/>-hydroxycinnamoyl glucose (592) isolated from the stems of A. manshuriensis showed higher scavenging activity of OH free radical [369]. Antimalarial Activity
Cepharanone B (75), taliscanine (91), and aristolactam All (83) showed in vitro antimalarial activity with IC50 values of 7.51-11.01 ug/ml [449].
990 Inhibition ofMPP+ Induced Apoptosis
Fall et ah investigated the role of the mitochondrial transition pore (MTP) in apoptosis induced by the Parkinsonism producing toxin Nmethyl pyridinium ion (MPP+) by using cyclosporin A (CSA) alone or in combination with the phospholipase A2 (PLA2) inhibitor aristolochic acid (5) (APvA). In this experiment, aristolochic acid alone inhibited MPP+ induced apoptosis at 24 hours but did not alter the mitochondrial effects of MPP+, suggesting that ARA inhibits MPP+ induced apoptosis downstream of the initiation event. Thus, ARA may prove to be another useful tool for understanding apoptosis [455,456]. Chinese-Herbs Nephropathy
Chinese herbs nephropathy (CHN) is a rapidly progressive interstitial nephropathy reported after the introduction of Chinese herbs in a slimming regimen followed by young Belgian women [457,458]. Because of manufacturing error, there were several reports on the adverse effects of this slimming regimen. Firstly, in 1992, some cases of women presenting with a rapidly progressive renal failure after having followed a slimming regimen including powdered extracts of Chinese herbs, one of them being Stephania tetrandra were recorded. This outbreak of renal failure eventually resulted in about 100 cases in 1998, 70 % of them being in end-stage renal disease (ESRD) [459]. Chinese herbs nephropathy is characterized by early, severe anemia, mild tubular proteinuria and initially normal arterial blood pressure in half of the patients [224]. Renal histology shows unusually extensive, virtually hypocellular cortical interstitial fibrosis associated with tubular atrophy and global sclerosis of glomeruli decreasing from the outer to the inner cortex. Urothehal malignancy of the upper urinary tract develops subsequently in almost half of the patients. The hallmark of the disease is an extensive paucicellular interstitial fibrosis with atrophy and loss of the tubules, the glomeruli being relatively spared. A suspicion that the disease was due to the inadvertent replacement of Stephania tetrandra one of the herbs of slimming regimen by a carcinogenic and nephrotoxic plant, Aristolochia fangchi because of their very similar Chinese names, fangji and fangchi, respectively was reinforced by identification of the nephrotoxic and carcinogenic aristolochic acid (AA) the principle constituent of Aristolochia in the slimming pills. This hypothesis was substantiated by the identification of premutagenic AA-DNA adducts in the kidney and
991 ureteric tissues of CHN patients. High-dose AA induced glucosuria, proteinuria, and elevated serum creatinine levels and reduced leucine aminopeptidase enzymuria [460]. Tubular necrosis associated with lymphocytic infiltrates and tubular atrophy surrounded by interstitial fibrosis also found in high-dose AA treated rats. Finally, induction of the clinical features (interstitial fibrosis and upper urothehal malignancy) typical of CHN in rodents given AA alone removed any doubt on, the causal role of this phytotoxin in CHN, now better called aristolochic acid nephropathy (AAN). AAN is not restricted to the Belgian cases. Similar cases have been observed throughout the world, but AA is sometimes incriminated on the basis of the known content of AA in the herbs. It has provided a fascinating opportunity to understand the link between a fibrosing interstitial nephropathy and urothelial carcinoma. There is clear evidence that the aristolochic acid I (AAI) and aristolochic acid II (AAII) are genotoxic mutagens forming DNA adducts after metabolic activation through simple reduction of the nitro group [461], Scheme (17). Several mammalian enzymes have been shown to be capable of activating both AAI and AAII in vitro and in cells. The activating metabolism is consistent with the formation of a cyclic nitrenium ion with delocalized charge leading to the preferential formation of purine adducts bound to the exocyclic amino groups of deoxyadenosine and deoxyguanosine. The predominant DNA adduct in vivo, 7-(deoxyadenosm-M>-yl)anstolactam I (dA-AAI), which is the most persistent of the adducts in target tissue, is a mutagenic lesion leading to AT-TA transversions in vitro. This transversion mutation is found at high frequency in codon 61 of the H-ras oncogene in tumors of rodents induced by AAI, suggesting that dA-AAI might be the critical lesion in the carcinogenic process in rodents. DNAbinding studies confirmed that both AAs bind to the adenines of codon 61 in the H-ras mouse gene and preferentially to purines in the human p53 gene. DNA damage by AA is not only responsible for the tumor development but also for the destructive fibrotic process in the kidney. It is concluded that AA is a powerful nephrotoxic and carcinogenic substance with an extremely short latency period, not only in animals but also in humans. In particular, the highly similar metabolic pathway of activation and resultant DNA adducts of AA allows the extrapolation of carcinogenesis data from laboratory animals to the human situation. Therefore, all products containing botanicals known to or suspected of containing AA should be banned from the market world wide. Finally, it has led to the withdrawal in several countries of a previously unsuspected carcinogenic and nephrotoxic substance. Recently, Arlt and co-workers
992 reported that in addition to ochratoxin A, aristolochic acid I (5) should be considered as an additional potential rise factor for Balkan Endemic Nephropathy [462,463]. Martinez et al. conducted a pilot study and follow-up study on the investigation of corticosteroid therapy's role in prevention of Chinese-herb nephropathy (CHN) progression [464]. Corticosteroid therapy was able to slow the progression of renal failure in some CHN patients. The renal protective effect of taurine (TU) in the rats with Aristolochia (ATL) nephropathy was studied by Bo et al. [465] Taurine exerts protective effect on the tubulointerstitial injuries caused by Aristolochia nephropathy in rats with Aristolochia nephropathy, by a mechanism in which the antioxidation effect of taurine might be involved. One of the earliest sign of CHN is the urinary excretion of low molecular weight proteins (LMWP), suggesting that AA is toxic to proximal tubules (PT). The effects of AA on PT functions including reabsorption of LMWP were investigated on the well-established opossum kidney (OK) cell line, a model for PT, and compared with those of the classical PT toxin CdCl2. AA significantly decreased megalin expression and formed specific DNA adducts in OK cells, similar to those found in kidneys from CHN patients. This support the involvement of AA in the early PT dysfunction found in CHN; furthermore, it suggested a causal relationship between DNA adduct formation, decreased megalin expression, and inhibition of receptormediated endocytosis of LMWP [466]. Stiborova et al. confirmed the participation of human enzymes, P450 1A1/2 and NADPH: P450 reductase in the activation of aristolochic acid to metabolited forming AA-DNA adducts [467]. The ability of prostaglandm H synthase (PHS) to activate AA to metabolites forming DNA adducts was examined by Stiborova et al. [468] PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-./V6-yl)aristolactam I, 7-(deoxyguanosin-7V2-yl)aristolactam I and 7-(deoxyadenosin-M>-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-Ar2-yl)aristolactam II and 7-(deoxyadenosin-M>-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. This demonstrated a key role of PHS in the reductive activation pathway of AAI and AAII in the RSV microsomal system. Stibovora et al. [469] also found that aristolactam I. a Drinciral detoxication metabolite of
993 aristolochic acid I, upon activation forms adducts found in DNA of patients with CHN. Aristolacatms are activated by cytochrome P450 (P450) and peroxidase to form DNA adducts. Aristolactam I activated by peroxidase led to the formation of several adducts. Two major adducts were identical to adducts previously observed in vivo. 7-(Deoxyguanosin./V-yl)aristolactam I (dG-AAI) and 7-(deoxyadenosin- A/'-yl)aristolactam I (dA-AAI) were formed in DNA during the peroxidase-mediated oneelectron oxidation of aristolactam I. Aristo- lactam I activated by P450 led to one major adduct and four minor ones. These results indicate a potential carcinogenic effect of aristolactam I in humans. Ray et al. reported that aristolactarri-iV-P-D- glucoside (57) (ADG) binds strongly to B-form duplex DNA by the mechanism of intercalation with considerable sequence specificity toward GC-rich DNA, especially alternating GC polymer. Recently it has also been shown that aristolactam A/-P-Dglucoside (ADG) interact with a protonation-induced structures [H(L)form] of poly(dG-dC)-poly(dG-dC) and poly[dG- m(5)dC]-poly[dGm(5)dC] in a nonlinear noncooperative manner. Protonated poly[dGm(5)dC]-poly[dG-m(5)dC] is more favorable for ADG binding than the corresponding nonmethylated analogue by the mechanism of intercalation [470]. DETECTION As a consequence of the toxicity related to the presence of aristolochic acid in plant preparations, several health institutions, such as the US Food and Drug Administration, Therapeutic Goods Administration have recently published safety information to prevent further cases of intoxication (information available at web address: http://www.cfsan.fda. gov/~dms/ds-bot.html) [471]. So detecting aristolochic acids in plant species that could be used in herbal remedies, and also in herbal preparations of uncertain composition, has attracted great priority in recent years to help prevent future adverse reactions. Aristolochic acids present in medicinal plants or herbs are analyzed by soxhlet extraction followed by TLC in the Chinese pharmacopoeia [412]. Another report used multiple ultrasonic extraction followed by HPLC analysis [472]. Ong's laboratory reported a method using a home made pressurized liquid extraction (PLE) system in dynamic mode to extract aristolochic acid in medicinal plants, followed by gradient elution HPLC [473]. Several scientific communities described various analytical methods for
994 qualitative and quantitative analysis of aristolochic acids after Chinese herb nephropathy out break. Rao et al. described a convenient and sensitive fluorometric assay for the analysis aristolochic acid in A. clematitis based on reduction to the lactam and measurement of the intensity of fluorescence using a characteristic emission spectrum with a maximum at 425 nm. The limit of sensitivity was 0.05 ug/ml. With this method, concentrations of 0.02-0.05 fig/ml of aristolochic acid were determined after reduction to the lactam with sodium hydrosulfite, which gave fastest reaction in a neutral medium [474]. A GLC assay was also described based on flash methylation of aristolochic acid and its lactam using trimethylanilmium hydroxide with a sensitivity limit of 1-5 u.g/ml. The minimum detectable concentration was 0.01 mg/ml [474]. Makuch et al. developed a preparative liquid chromatography method for isolation of aristolochic acid I (5) and II (1) from a crude extract of the A. clematitis using methanol-water-acetic acid (53:47:1, v/v/v) as mobile phase [475]. Makuch et al. also carried out the separation of a mixture of aristolochic acids obtained by extraction with methylene chloride from roots of A. durior using reversed phase HPLC method with RP-18 column and mixture of A+B at 1:1 ratio as mobile phase, where A containing 63.5 % of methanol in 0.08 mol/1 acetic acid, and B containing 37 % of tetrahydrofuran in 0.08 mol/1 acetic acid [476]. Cateni et al. reported a rapid, reproducible, sensitive and suitable HPLC analysis for quantitative determination of aristolochic acids in different organs of A. clematitis [477]. Ratios of methanol-water-acetic acid (80:20:1) used as mobile phase and flufenamic acid used as internal standard. The aristolochic acid I (5) and II (1) were well resolved in the chromatogram with retention times of 3.16 and 3.87 min, respectively. Results obtained showed that in the aerial parts the total amounts of acids were very lower than in the roots. Later, Zhang et al. determined aristolochic acid I in drugs of Aristolochia by reversed phase HPLC using C18 column and methanol-acetic acid-water (60:1:35) as mobile phase [478]. The average recovery of aristolochic acid I (5) and variation coefficient were 100.5%, 1.9%, respectively. Hashimoto et al. determined the amounts of aristolochic acid I (5) and II (1) in four groups of medicinal plants from the Aristolochiaceae, classified by their vernacular name by HPLC using C18 column, and 1% acetic acid-methanol (1:1) as mobile phase. Aristolochic acid I (5) and II (1) (AAI and AAII) were detected in all the plants that originated from the genus Aristolochia (Aristolochiaceae) and in some of the plants from the genus Asarum (Aristolochiaceae) [479]. These points suggest that these medicinal plants
995 usage for remedial purposes should be prohibited due to the harmful effects attributed to aristolochic acids. It was also found that the content of both AA-I and AA-II in A. manshuriensis was quite high. The 0.2 mg/kg, a non toxic effect of AA corresponded to approximately 20 mg/kg of A. manshuriensis. Ong and Woo developed a method for the determination of aristolochic acids in medicinal plants and Chinese prepared medicine (CPM) using capillary zone electrophoresis (CZE) [480]. The buffer used was 30 raM sodium tetraborate at pH 9.5, detection was at 254 nm, applied voltage at 18 kV and the temperature was set at 25°C. The effect of ionic strength, pH, and applied voltage on the seperation was investigated. The precision values (R.SD, %) for the relative migration time and peak area or peak height for aristolochic acids I (5) and II (1) were found to be less than 0.3% and between 2.6 to 4.0%, respectively. The limit of detection for aristolochic acids I (5) and II (1) was found to be 1.2 and 0.9 mg/L, respectively. The method using pressurized liquid extraction (PLE) followed by CZE without further clean up was found to be simple, rapid and accurate for quantitative analysis of aristolochic acids in medicinal plants or CPM samples with complex matrix. Use of internal standard, overcome the reproducible problem in CZE. Using an aqueous buffer system, organic solvent consumption was minimized. This method was found to complement the results obtained by HPLC. Lee et al. reported that a HPLC system using a mobile phase of 0.3% ammonium carbonate solution-acetonitrile (75:25, v/v) with pH 7.5 in combination with a RP-18 reversed-phase column was useful for the determination of aristolochic acids I, and II in medicinal plants and slimming products [481]. With these conditions aristolochic acids I (5), II (1) were separated within 20 minitues with a good resolution. The recovery of aristolochic acids I (5) and II (5) in medicinal plants and slimming products was better than 90% by extraction with methanol and purifying through a PHP-LH-20 column. Aristolochic acid I (5) was the major component in A. fangchi and the level ranged from 437 to 668 ppm. Aristolochic acid II (1) was the major component for A. contorta and its range was <1-115 ppm. The profile of aristolochic acid was quite different depending on the plant species. Twelve out of 16 samples of slimming pills and powders analyzed contained aristolochic acids I (5) and/or II (1). The major component in most slimming products was aristolochic acid II (1) and the level ranged from <1 to 148 ppm. It may indicate that slimming products were not mainly made of A. fangchi. According to Kite et al., targeted liquid chromatography/serial mass spectrometry (LC/MS/MS) analysis using a quadrupole ion-trap mass spectrometer,
996 permitted the detection of aristolochic acids I (5) and II (1) in crude 70 % methanol extracts of multi-component herbal remedies without any cleanup or concentration stages [482]. The best ionization characteristics were obtained using atmospheric pressure chemical ionization (APCI) and by including ammonium ions in the mobile phase. When 200 mg of herbal remedy are extracted in 1 niL, direct LC/MS/MS or LC/MS3 analyses detected aristolochic acid I (5) included at above 0.125-1.25 ppm dry weight depending on the sample matrix. This sensitivity was sufficient to detect the aristolochic acids extracted from 0.1% dry weight of A. manshuriensis in an herbal preparation producing a problematic matrix. These detection limits are below those required to detect aristolochic acids in a quantity of herbal preparation that could feasibly be taken each day to give a dose of 0.1 mg/kg body weight, the long term dosage producing renal lesions in rabbits. A method using direct on-line coupling between HPLC and UV-DAD/MS was developed by Ioset et al. for the rapid detection of aristolochic acid I (5) in plant preparations [483]. The separation was performed on a C-18 column with a linear methanol (0.5 % acetic acid)-water (0.5 % acetic acid) gradient (60:40 to 100:0) and 254 and 224 nm DAD-UV detection in 21 minutes. The retention times of aristolochic acid I (5) and II (1) were 13.9 and 12.6 minutes, respectively. The use of both UV-DAD and MS detectors enables this method to be very selective. The detection limits of 2 ng of aristolochic acid I (5) was determined on column for both UV and SIM-MS measurements. The recoveries of 90.2±3.0 for UV and 92.6±6.3 % for MS of aristolochic acid I (5) were determined after control was performed using the optimized LC/DAD-UV/MS analysis. A quantitative thin layer chromatographic analysis associated with a specific detection of aristolochic acid I (5) in complex mixtures due to the presence of its strongly oxidative NO2 group after spraying with diphenylamine using chloroform:methanol:acetic acid (65:20:2) was also developed. A densitometric method also reported to quantify aristolochic acid on thin layer chromatography with a detection limit of 0.02 mg per gram of sample [484]. The presence of aristolochic acid I (5) in several commercial dietary supplements, teas and Chinese phytomedicines used as slimming regimens was confirmed by Ioset et al. using a HPLC/UV-DAD/MS analysis [485]. A quantitative determination of aristolochic acid I (5) was also achieved in the incriminated preparations using both UV and MS detection. Out of 42 analyzed preparations, four, Fang ji huang qi tang, Han fang ji, Ba zheng san and Han fang ji (Sinomeniutri) were found to contain aristolochic acid I (5) and two, Asari herba and Xi xin Asari herba were suspected to contain
997 aristolochic acid derivatives. Five of the six incriminated products were provided by the same Chinese herb importer. Immediate removal of the products from the Swiss market was called for. Quantitation of aristolochic acid A. indica samples using rapid, sensitive and reproducible HPLC method with photodiode array detection was performed by Singh et al. [486,487] The procedure involves the extraction of aristolochic acid with methanol and chromatographic separation with a mobile phase of acetonitrile-water-trifluoroacetic acid-THF (50:50:1:1) on a Spherisorb S10 ODS2 column. The average recovery of aristolochic acid is 97.8 % and minimum detectable concen- tration is 0.10 mg/injection of 5 mL injection vol. Twenty samples of the stems of A. manshuriensis from the market were analyzed by UV spectro- photometry and content of total aristolochic acid I (5) was determined at a wave length of 310 nm by Shang et al. [488] Their total aristolochic acid contents varied from 0.63%-2.84%. Commercial samples of A. manshu- riensis used in different localities varied markedly in their total aristolo- chic acid contents. Zhao et al. determined aristolochic acid A (5) in A. debilis by RP-HPLC on a C18 column with a mobile phase methanol-1.28 % tetrabutylammonium bromide-pH 3.6 NaOAc buffer solution (2:1:1) and UV detection at 316 nm [489]. The calibration curve was linear within the range of 20.8-104.0 mg/mL for aristolochic acid A (5). The mean recovery was 97.0 % and the precision (RSD) was 2.4 %. The roots of A. bracteata collected from the Sudan and roots and rhizomes of A. contorta, A. debilis, A. heterophylla, and A. mollissima collected from different places in China were analyzed by Mohamed et al. using HPLC on LichroCART column with methanol-acetic acid-water (72:28:1) as mobile phase at detection wave length of 310 nm [490]. The method was very useful to distinguish different species from each other based on the typical peaks. The content of aristolochic acid in A. bracteata is slightly higher. The average recovery of aristolochic acid A (5) and variation coefficients were 100.8 % and 6.35 %, respectively. In a HPLC determination, Zhang et al. found that some A. tuberosa callus cultures on MS medium contained higher aristolochic acid content (0.27% of dry callus wt.) than their original plant organs (0.15/1000 of dry wt.) [491]. Seto et al. analyzed AAI (5) and AAII (1) in the Kampo medicine, Toki-shigyakuka-goshuyu-shokyo-to by detection methods, thin layer chromatography and a high performance liquid chromatography equipped with a photodiode array detector (LC-PDA) and an electric ionization mass spectrometry detector and also found that LC-PDA detection method was suitable for quick detection of AAI (5) and AAII (1) in the ten kinds of
998 Kampo medicines in which A. fangchi root or A. manshuriensis stem may be misused [492]. Moreover, LC-PDA and LC-EIMS detection methods both were effective in distinguishing AAI (5) and AAII (5) peaks by their spectra. Very recently, we have reported the extensive analysis of aristolochic acids and aristolactams in the various Chinese medicinal plants and crude drugs using different techniques. A method has been developed using reversed phase liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry under the positive ion detection mode [LC/(+)APCI/MS/MS] to determine the amount of AA-1(5) in Xinxin, a traditional Chinese medicine that originate from nine Asarum species [493]. By applying this method to the determination of AAI (5) and AAII (1) in nine species of Asarum collected in China, {A. crispulatum, A. debils, A. forbesii, A. fukienense, A. heterotropoides, A. himalaicum, A. ichangense, A. maximum, and A. sieboldii) AAI (5) was found to range from a low of 3.3 ng/mg in A. sieboldii to 3376.9 ng/mg in A. crispulatum. AAII (1) was not detected in any of the analysed species. Better chromatographic shapes and sensitivities were obtained by using LC/MS/MS rather than the traditional LC/UV system. It was found that a great enhancement occurred (about four times) in the ion count of [M+HH2O]+ when 0.005 % TFA was used in the aqueous mobile phase instead of 1 % acetic acid. The detection limit of AA I (5) was as low as 10 ng/ml in this [LC/(+)APCI/ MS/MS] method. A facile reversed phase HPLC method for analysis of aristolochic acids (AA) and arsitolactams (ALs) simultaneously was developed and applied for the quantitative determination and quality control of the traditional Chinese medicine [494]. Hymonymic Chinese crude drugs such as Stephania tetrandra and A. fangchi, Clematis armandii and A manshuriensis were easily identified with the chromatograms obtained in this method. Resolution and quantitative determination of 17 analogues of AAs and ALs in 12 species of Aristolochia {A. contorta, A. cucurbitifolia, A. debilis, A. elegans, A. foveolata, A. heterophylla, A. kaempferi, A. mollis, A. odoratissima, A. shimadii, A. trilobata and A. zollingeriana) under gradient elution with a solvent mixture of sodium acetate and acetonitrile are also demonstrated by applying this method. The chromatograms obtained can serve as fingerprints to identify plant species, thus avoiding inadvertent replacement of herbs in the manufacturing herbal preparations. A similar HPLC method was described to evaluate the effect of pH on retention scale of 17 analogues of AAs and ALs in the simultaneous quantitation under the same chromatographic conditions [495]. The retention scale of
999 analytes was found to be dependent on the pH of mobile phase and highly relevant to the presence of functional groups on analytes as well. Analytes with high polarity elutes early. Increasing the pH of mobile phase decreases the retention time of AA analogues, however it increases that of AL analogues slightly. At higher pH (>5.0) the elution pattern was reversed between AA and AL and it was believed to result from the ionization of a carboxyl group on AA. High correlation between concentration and peak area in quantitative analysis of Chinses herbs such as A. heterophylla and A.fangchi was also noticed in this method. Chen et al. determined aristolochic acid I (5) in A. fangchi, A. heterophylla, A. moupinensis and A. austoszechuanica by TLC scanning and dye calorimetry [496]. ECOLOGICAL ADAPTATION Plant secondary metabolites are thought to pose both as toxicological and behavioral barriers to evolutionary changes in host use by phytophagous insects [497-499]. Arguing that secondary metabolites represent principally behavioral barriers, Dethier and Jermy proposed that colonization of a novel plant by an insect species will be more probable if that plant contains compound similar to those that the insects already use as cues in host recognition [500,501]. Recent years have witnessed tremendous strides in the establishment of the influence of natural compounds on the behavior of the anthropods [502-506]. A large number of swallowtail butterfly species (Papilionidae) feed on the plant family Aristolochiaceae. Rothschild et al. reported that the host adaptation in a large number of swallowtail butterfly species is associated with the hostoriginated aristolochic acid derivatives (AAs) [507]. A Japanese pipevine swallowtail, Atrophaneura alcinous appeared to sequester AAs in body tissue as in other troidine species. Not only adults but also eggs, larvae, and pupae of A. alcinous have been found to store AAs in high concentration demonstrated that AAs act as deterrent allomones against birds [508-510]. Adult females of A. alcinous have been shown to be stimulated to oviposition by AAs, in addition to other components in their host plant A. debilis. Furthermore, AAs were found to initiate feeding activity by A. alcinous larvae [511].. Nishida and Fukama isolated seven analogues of aristolochic acids (I (5), 11(1), 111(4), B(9), C(2), D(10), and E(12)) from the leaves of A. debilis and characterized as the larval feeding stimulants of an Aristolochiaceae-feeding swallowtail butterfly, A. alcinous [166]. Aristolochic acids showed synergistic activity in
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combination with the water-soluble components in the leaf extract of A. debilis. Aristolochic acids were detected in the body tissues and specialized organs throughout all life stages of A. alcinous. Larval osmeterial fluid also contained AAs as high as 2 % of secretions, exhibiting HPLC profiles similar to those of fresh leaf extracts of A. debilis. In contrast, aristolochic acids I (5) and II (5) were present both in the egg yolk and egg coating material as well as in the collateral glands of the adult females. Although six AA analogs were detected from both male and female adults of A. alcinous, the total AA content was much higher in the female specimens. A large portion of AAs in the female butterflies is thus concentrated in the reservoir and transferred efficiently to the next generation. The larvae of A. alcinous often display cannibalistic behaviors toward eggs and pupae and thus suggesting a possible adaptive mechanism in this species. AAs have also been characterized as synergistic factors for oviposition stimulants of A. alcinous. Sequestration of AAs in the adult bodies has also been demonstrated among several troidine species, namely Pachliopta aristolochiae, and Battus archidamas, and B. polydamas, and also in an Aristolochia-feeding Parnassiinae, Zerynthia polyxena. Aristolochic acid I (5) deterred feeding of tree sparrows, which suggested a defensive role against vertebrate predators. Nishida and Fukama isolated oviposition stimulants of A. alcinous, from the leaves of A. debilis and characterized as a mixture of aristolochic acids and sequoyitol [391]. An artificial blend of these components applied to filter paper induced a significant oviposition response by the female butterflies, identical to that elicited by intact leaves of the Aristolochia host plant. Four analogues of aristolochic acids [AA-I (5), II (1), B (9), and C (2)] and sequoyitol have been characterized as oviposition stimulants of A alcinous. Each individual component was inactive or only weakly active alone, but distinct oviposition activity was generated when either one of the AA analogs was mixed with sequoyitol (638). The simultaneous occurrence of AAs and sequoyitol in the plant seems to ensure an extremely high specificity for A. alcinous oviposition. Ecological adaptation of A. alcinous seems to be strongly associated with a sensory mechanism specially developed for perception of AAs. The oviposition stimulant, sequoyitol (638), is a cyclitol closely related to chiro-inositol that has been characterized as an oviposition stimulant component for Papilio xuthus [512,513]. Paraj et al. found that the ethanolic extracts of both young and mature A. macrophylla leaves were active in eliciting oviposition responses by females of the pipevine swallowtail butterfly, Battus philenor [391]. D-(+)-Pinitol (639) was
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isolated and identified from leaf material of A. macrophylla. In combination with chloroform-soluble components of host leaf material, this compound was comparable to the parent extract in stimulating oviposition. D-Pinitol (639) also serves a significant defensive role against herbivores. Gupta et al. reported that the swallowtail butterfly B. philenor in the eastern United States use a synergistic mixture of inositols, acids and a lipid as oviposition cues in recognizing the host plant A. macrophylla on contact. The acids, inositols and lipids were identified as aristolochic acid I, II, D-(+)-pinitol (639), sequoyitol (638) and l,2-[di(9Z, 12Z, 15Z)-octadeca-9,12,15- trienoyl]-3-galactosyl-Sn-glycerol (646), respectively. This indicates substantial evolutionary conservation in chemical oviposition cues within the tribe Troidim [514]. Our group has also investigated the chemical constituents of every stage of the Taiwanese swallowtail butterfly, Pachliopta aristolochiae interpositus and compared with the constituents of the exclusive insect feeding plants, cucurbitifolia [515]. The presence of AA-I (5) in all life stages of the insect was noticed, however high concentration in the larval osmeterial fluid supported the earlier reports that AAs act as deterrent allomones against birds. It is interested to note that aristolactams were detected only from the larvae and larval excrement, but not in the larval osmeterial fluids, pupal shells or adult bodies. It demonstrated that aristolochic acids might be metabolized to aristolactams at the larval stage and then, aristolactams may be further metabolized and excreted when insects are matured in the adult stage. The presence of AAI (5) in all life stages of the insect, especially in the larval osmeterial fluids and pupal shells, suggests a possible adaptive mechanism in this species. Sime et al. found the sequestration of aristolochic acids by B. philenor from Vergenia and east Texas [516]. A comparison of the aristolochic acid profiles of the Vergenia butterflies and their A. macrophylla food plants revealed that aristolochic acid I (5) and II (1) dominated in foliage whereas insects contained much lower proportion of aristolochic acid II (1) and in addition substantial amounts of aristolochic acids la (3) and IVa (10). The presence of aristolochic acids in eggs, larval integument, osmeterial glands, pupal cuticle and adults indicating that both immature and adult B. philenor are unpalatable and protected from natural enemies. Fordyce reported that the California subspecies pipevine swallowtail, B. philenor hirsute and its host plant, A. californica, both contained aristolochic acid [517]. Aristolochic acid I (5) and II (1) were the primary aristolochic acids found in A. californica. The highest concentration of aristolochic acids was found in the flowers, which bloom before B. philenor emerges.
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Concentrations of aristolochic acids decreased in the leaves but not in stem tissue over the course of the season. Butterflies contained primarily aristolochic acid I (5). Aristolochic acid content of individuals from Arizona, which are involved in mimicry complexes, did not differ from California populations. Klitzke et al. demonstrated the presence of aristolochic acids in varying concentrations in the individuals of 17 neotropical troidine species butterflies [518]. Interestingly, aristolochic acids do not occur in A. galeata leaves, but they were present in B. polydamas larvae reared on these leaves, and thus may be synthesized by the larvae from chemical precursors in the plant. In 2000, Tsuchihara et al. found a unique protein of 23 kDa (Jf 23) in the tarsus of the female swallowtail butterfly, A. alcinous and characterized as a putative binding protein for lipophilic substances related to butterfly oviposition [519,520]. The protein, Jf 23 has 38 % identity with a bilin-binding protein found in the cabbage butterfly, Pieris brassicae, which has 2 consensus sequences in common with the members of the lipocalin family, suggesting that it is a binding protein for lipophilic ligands. The Jf 23 was expressed only in the female, and not in the male. Electrophysiological response of the female tarsi was stimulated by methanolic extract of their host plant, Dutchman's pipe (A. debilis) and the stimulated response was depressed by the presence of Jf 23 antiserum suggesting that Jf 23 is one of the chemosensory signaling proteins, which plays one or more roles in female butterfly oviposition. Finally, there has been a concern in India that since A. indica has become endangered, the swallowtail butterfly Tros aristolochae may also be endangered. CHEMOTAXONOMY In 1991, Mizuno et al. suggested chemotaxonomically that the subgenera, and some times their sections of Aristolochia are restricted by the relative content of aristolactam derivatives [158]. The genus Aristolochia is composed of two subgenera, Siphisia and Aristolochia. The aristolactam derivatives with methoxyl group on the A-ring were found to occur abundantly in the subgenus Siphisia, such as A. shimadai, A. manshuriensis and A. kaempferi. On the contrary, the plants of subgenus Aristolochia, A. debilis and A. tagala were rich in derivatives with methylenedioxy group. The plants of Siphisia section, A. shimadai, A. manshuriensis, A. cucurbitifolia and A. westlandii in the subgenus Siphisia were richer in aristolactam 17V-P-D-glucoside (71) than the plants
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in the section Hexodon, A. onoei, A. kaempferi and A. liukiuensis. The stems of A. debilis were characterized by an abundant quantity of aristolactam derivatives within the methylenedioxy group. Thus Mizuno et al. concluded that the aristolactam derivatives were good specificmarker for distinguishing the Aristolochia species. Aristolochia species are characterized by the outlandish shape of the flower [4-7]. From this fascinating genus come many of the most unusual flowers in the world, most of them difficult to describe to the uninitiated. From the side, the flowers often look like birds or "Dutchman's Pipes". Head on, many look like large maroon or green pouches. For all the intricacies of their flowers, Aristolochias have no flower petals, just one fancy calyx. Based on the morphology of perianth-tube or corolla, Aristolochia species can be sub divided in to three groups. They are, firstly Aristolochia species with funnel shape corolla, secondly, with labiate type corolla and thirdly, with tubular corolla. This classification was also correlated with their chemical constituents. Aristolochia species with funnel shape corolla were examplified by species like A. elegans, A. brasiliensis, A. gigantea, A. grandiflora, A. odoratissima, A. westlandii etc. These species were particularly characterized by the accumulation of diterpenoids and lignans, as major constituents with little or no aristolochic acids and aristolactams. However, from A. elegans wide variety of constituents were reported including bisbenzylisoquinoline and isoquinoline alkaloids [169]. A. gigantea was reported to contain number of 8-benzylberbine alkaloids and few bisbenzylisoquinoline alkaloids, but no reports of aristolochic acids and aristolactams so far [279,283]. As far as we know, A. brasiliensis is known for clerodane diterpenoids whereas A. elegans for e«;-kauranes [169,360,366]. The second type Aristolochia species with labiate type corolla were represented by A. labiata, A. chilensis, A. cucurbitifolia, A. foveolata, A. heterophylla, A. mollissima, A. maxima, A. debilis, A. fimbriata, A. longa, A. gehrtii, A. galeata, A. fangchi, A. baetica, A. arcuata, A. watsonii etc. Chemically they were characterized by sesquiterpenoids, aristolochic acids and aristolactams as major constituents. However, A. cucurbitifolia, A. heterophylla, A. mollissima, A. debilis were reported to contain sequiterpenoids as minor constituents. A. chilensis, a unique species chemically is the only species in the genus Aristolochia noted for 4-aryltetralone lignans [377,378]. Lignans and protopine alkaloids were reported from A. arcuata, but no reports of aristolochic acids and aristolactams so far [277,280]. The final subgroup of Aristolochia species with tubular corolla were examplified by several species such as, A. kaempferi, A. zollingeriana, A. rotunda, A.
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pallida, A. contorta, A. ringens, A. ovalifolia, A. trilobata, ect. These species were characterized by the accumulation of aristolochic acids and aristolactams, but no sesquiterpenoids. Chemosystematic conclusions within Aristolochia however, are strongly affected by the frequently occurring chemical polymhorphism. Especially different collections of A. elegans displayed remarkable intraspecific variations. For example, A. elegans of Brazilian origin yielded bisbenzylisoquinoline alkaloids whereas the material collected from Taiwan afforded isoquinolones, biphenyl ethers, tetralones [167,169,282]. On the other hand, A. triangularis collected in Rio Grande do sul differed with the considerable amounts of kaurane diterpenes oxidized at C-19 position with A. triangularis collected in Panama [269,270]. In some Aristolochia species, not only between different habitates, but also between individuals of the same population significant chemical variations were noted. For instance, sesquiterpenoids were widely distributed in the stems and roots of A. cucurbitifolia, A. heterophylla, and A. mollissima rather than leaves [314]. According to Lopes et al., clerodane diterpenoids predominate in the roots whereas labdane diterpenoids in the leaves of A. cymbifera, A. galeata and A. esperanzae [267,360,363]. Despite the surprising individuality of chemical compositions with in the Aristolochia species, the formation of aristolochic acids represents an excellent chemical marker for the genus. This chemical trend was observed in almost all species. CONCLUSION This review of literature covering phytochemical and pharmacological investigations on Aristolochia species have resulted in the compilation of six hundred and eighty eight compounds belonging to the classes of aristolochic acids and their derivatives, aporphines, benzylisoquinolines, isoquinolines, protoberberines, protopines, amides, chlorophylls, terpenoids, lignans, biphenyl ethers, flavonoids, tetralones, benzenoids, and steroids, etc. with broad spectrum of physiological activities. Biosythetic studies, spectral properties, synthesis and detection of aristolochic acids have also been discussed. The last few years have witnessed a revolution in the pharmacology of the Aristolochia species, mainly due to the discovery of the nephrotoxic aristolochic acids responsible for a tragic phenomenon of Chinese herb nephropathy recognized in 1992. Recently, health food supplements containing botanicals known to or suspected of containing aristolochic acids were prohibited for use with full scientific support. It may thus be of more than academic interest to examine the
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remaining Aristolochia plants for their aristolochic acid presence to prevent the issues like Chinese herb nephropathy and Balkan endemic nephropathy. Ecological adaptation of Aristolochiaceae feeding swallowtail butterflies to aristolochic acids, pmitol and sequoyitol metabolites of host plant has also been presented. This review will help fellow scientists in locating the detailed information in a single source on Aristolochia species. ACKNOWLEDGEMENTS We are grateful for the enthusiastic support of all our co-workers of the past fourteen years. Work in the author laboratories was also kindly supported by program project grants from the National Science Council, Taiwan, R. O. C. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
1019
CHEMISTRY AND BIOACTIVITY OF WITHANOLIDES FROM SOUTH AMERICAN
SOLANACEAE ADRIANA S. VELEIRO,1 JUAN C. OBERTI2 AND GERARDO BURTON1 1
Departamento de Quimica Orgdnica and UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2 Ciudad Universitaria C1428EGA Buenos Aires, Argentina. 2 Departamento de Quimica Orgdnica and IMBIV, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, 5000 Cordoba, Argentina. ABSTRACT. Since the isolation of withaferin A in 1965 over 300 withanolides have been described, largely from genera belonging to the Solanaceae. Although until the mid eighties most of the withanolides appeared to adhere to the basic structure of withaferin A, nowadays a considerable number of withanolides and withanolide related compounds are known, which present modified skeletons, aromatic rings, additional rings, etc., posing challenging problems of structure elucidation. Many of these structures coexist in the plants with "normal" withanolides and thus allow us to infer the biogenetic relationships among them and the transformations they suffer in the plant. As a further bonus, several species show marked seasonal and geographical variations in the amount and type of withanolides present, thus adding to the structural diversity. The latter is particularly noteworthy in (but not restricted to) species of the Jaborosa and Salpichroa genera. In recent years these genera, both native to South America, have rendered several novel withanolide types. Exodeconus, Dunalia, Deprea and Vassobia are other southamerican genera where unusual structures have also been found. Recently, some of the withanolides isolated from these plants have shown interesting biological activities as cancer chemopreventive agents (inductors of quinone reductase), as feeding deterrants for several insects, and displaying selective phytotoxicity towards monocotiledoneous and dicotiledoneous species. Trypanocidal and leishmanicidal activities have also been reported.
INTRODUCTION The withanolides are steroidal lactones built on an ergostane skeleton of 28 carbons functionalized at carbons 1, 22 and 26, commonly known as the withanolide skeleton. The first withanolide to be isolated and characterized was withaferin A, Fig. (1), isolated in 1965 almost simultaneously by Lavie from Withania somnifera and by Kupchan from
1020
Acnistus arborescens. The extract from W. somnifera known by its Sanskrit name "Aswagandha", has been used since ancient times in India, due to its medicinal properties. Most of its curative properties are currently associated with the presence of withanolides. At present over 300 withanolides have been described, largely but not exclusively, from genera belonging to the Solanaceae. A variety of biological activities are known for these compounds, including antifeedant, antiinflamatory, antitumor, cytotoxic, immunomodulating and cancer chemoprevention activities. In the last decade, several review articles have dealt with structural and other aspects on this group of compounds [1-3]. This review covers recent developments and findings in the chemistry and bioactivity of withanolides from Southamerican Solanaceae.
Fig. (1). Structure of withaferin A and numbering scheme used.
OCCURRENCE OF WITHANOLIDES The Solanaceae family has been divided into 6 subfamilies comprising 92 genera and ca. 2300 species [4]. Surprisingly, the withanolides have been isolated only from 18 genera belonging exclusively to subfam. Solanoideae, tribes Solaneae and Jaboroseae are the most studied to this date within this subfamily. Ten of the Solanaceae genera containing withanolides are native to South America (Table 1). Outside the Solanaceae, withanolides or closely related compounds, have been found in Cassia siamea (Leguminosae) [5], Ajuga parviflora (Labiatae) [6-8], Taccaplantaginea [9-10] and Tacca chantrieri [11].
1021 Table 1: Southamerican genera and species from Solanaceae, containing withanolides Genera
Species
Acnistus
A. arborescens
Datura
D.ferox
Deprea
D. orinocensis ; D. subtriflora
Dunalia
D. brachyacantha; D. solanaceae
Exodeconus
E. maritimus
Iochroma
I. australe2; I.fuchsioides
Jaborosa
J. araucana; J. bergii; J. integhfolia; J. laciniata* J. leucotricha; J. magellanica; J. odonelliana; J. runcinata; J. sativa
Physalis
P. peruviana; P. philadelphica; P. viscosa
Salpichroa
S. origanifolia
Vassobia
V. breviflora4, V. lorentzii
1 subnom. Acnistus ramiflorum; Acnistus breviflorus.
2
subnom. Acnistus australis; 3 subnom. Trechonaetes laciniata; 4 subnom.
CLASSIFICATION OF WITHANOLIDES Withanolides are generally polyoxygenated, a common feature of all of them being oxidation at C-l, C-22 and C-26. They may be classified into two major groups, withanolides with a 5-lactone or 8-lactol side chain (Group A) and those with a y-lactone side chain (Group B). Most of the known withanolides belong to group A, which may be further divided into 11 subgroups: withanolides with an unmodified skeleton, withaphysalins, physalins, acnistins, withajardins, withametelins, sativolides, subtriflora-8-lactones, ring-D aromatic withanolides, ring-A aromatic withanolides and norbornane-type withanolides, Fig. (2). The withanolides with an unmodified skeleton are the most abundant and are regarded as possible precursors of the other withanolides. A further subdivision of the withanolides on the basis of the orientation of the side chain is again possible as compounds with the "normal" 17poriented side chain as well as the less usual 17a-orientated side chain are known, in the latter case a 17(3-hydroxyl is always present.
1022
acmstins
sativolides
ring-A aromatic withanolides
withajardins
subtriflora-5-lactones
withametelins
ring-D aromatic withanolides
norbornane-type withanolides
Fig. (2). General structures of withanolides with a 8-lactone or 8-lactol side chain (group A). Numbering has been added for clarity in some structures. (R,, R2 = O or H,OH)
1023
The presence of an oxygenated function at C-23 allows the formation of a y-lactone side chain (group B), these withanolides may be divided in five subgroups, spiranoid withanolides, trechonolides, subtriflora-ylactones, ixocarpalactones and perulactones, Fig. (3) 27)
H0
spiranoid
S L I \/OH
trechonolide
subtriflora-y-lactone
'27
ixocarpalactone
perulactone
Fig. (3). General structures of withanolides with a y-lactone side chain (group B). Numbering of relevant positions has been added for clarity.
CHEMISTRY OF WITHANOLIDES FROM SOUTHAMERICAN SOLANACEAE. 14-Oxygenated and 15,21-Cyclowithanolides (norbornane-type) Initial studies on a population of Jaborosa bergii Hieron growing in San Luis province (Argentina), yielded five withanolides with the unusual feature of having hydroxyl groups at position C-14 and C-17 both with p1configuration, e.g. jaborosalactone M (1) [12]. Very recently Nicotra et al. reinvestigated J. bergii and isolated another 14(3,17p-diol withanolide, jaborosalactol 23 (2), and five new compounds, jaborosalactols 1 8 - 2 2 (3-7), all of which presented a novel norbornane-type structure in ring D
1024
resulting from a carbon-carbon bond between C-15 and C-21 [13]. Misico et al. isolated another 14p,17J3-dihydroxywithanolide, named jaborosalactone 8 (8), from Jaborosa leucotricha collected in Mendoza province (Argentina) [14].
o
'"OH 'OH
OH
1 5[5,6p-epoxy; 2,3-dihydro 2 5CC-C1, 6P-OH
3 5P,6p-epoxy 7 5a-Cl, 6p-OH
4 5p,6p-epoxy 5 5a-OH, 6P-OH 6 5a-Cl, 6p-OH
Recently, Ahmad et al. isolated two 14-oxygenated withanolides, compounds 9 and 10 from plants of Physalis peruviana collected from District Chitral, Pakistan and a new withanolide glycoside (compound 11) which presented a 14,20-epoxy functionality [15,16]. Dinan et al. had previously reported the isolation of 28-hydroxywithanolide E (12) from calices of this plant [17].
o
OH 10
1025
OH
12
11
C-16, C-18 and C-20 oxygenated norwithanolides (subtrifloralactones)
withanolides
and
18-
Withanolides with a functionalized C-18 at various oxidation levels (alcohol, aldehyde and laetone carbonyl) have been isolated from plants of the genera Dunalia, Vassobia and Physalis [1,2]. In recent years, several new 18,20-dioxygenated withanolides were found, compounds 13-20 were isolated from Vassobia lorentzii Dammer (subnom. Acnistus lorentzii) [18] and compounds 21 and 22 from Dunalia brachyacantha Miers [19], both species collected in Catamarca province (Argentina). Compound 21 has the unusual feature of a saturated laetone side chain. From the latter plant, three other 18-oxygenated withanolides were also found, compounds 23, 24 and 25; two of them also present a 16-hydroxy substituent. Compounds 23 and 24 are the deacetyl derivatives of iochromolide and withacnistin respectively, found previously in Iochroma coccineum [20].
13 \%RIS
14 R=H,OH; ( i m s ) 15R=H, OCH3;(18/?) 16R=H,OCH3;(185) 17R=O
18 \%R 19 185
1026
HO O
OH 21
22 24,25-dehydro
23 R,=H2; R2=OH 24 R,=H,OH; R2=H 25 R,=O; R2=OH
Compounds 13-22 are structurally related to the withaphysalins, isolated from Physalis minima, which have been regarded as intermediates in the formation of the physalins. Recently Tomassini et al. published a review on withanolides isolated from the Physalis genera [21]. 16-Hydroxywithanolides were isolated from plants of Acnistus arborescens, collected in Rio de Janeiro (Brazil) [22] and from Exodeconus maritimus collected in Trujillo Department (Peru) [23]. Compounds 26 and 27 were reported for the former species while exodeconolides A (28), B (29) and C (30) were isolated from E. maritimus. Alfonso has reported the isolation of some 16,18dioxygenated withanolides from Iochroma gesneriodes (Kunth) Miers (also known as Iochroma coccineum Scheidweiler) [24]. The close relation between the functionality patterns found in withanolides isolated from Dunalia, Vassobia, Acnistus and Iochroma is in agreement with the taxonomic affinity exhibited by these genera.
o
OH 26 R,=OH; R2= OAc; R3=OAc 27R,=H; R2=OAC; R3=OAc
28 R,=OH; R2=H 29 R,=H; R2=OH
"6 30
1027 OH 0
31 R,=Ac R2=
'OH
-7^07 HO
I OH
OH
Two 20-hydroxywithanolide glycosides (31 and 32) were isolated from D. brachyacantha collected in La Paz (Bolivia) [25]. Silva et al. studied Physalis viscosa growing in Argentina and isolated four known 20hydroxywithanolides together with a related pregnane identified as 4(3hydroxy-5p,6(3-epoxypregn-2-ene-l,20-dione (33). Compound 33 could arise as a degradation product of a 20-hydroxywithanolide (e.g. withanolide D, a major component of P. viscosa) by oxidative cleavage of the side chain of the between C-20 and C-22 [26]. Ixocarpalactone A (34) is the major withanolide present in fruits of Physalis philadelphica (subnom. P. ixocarpa Brot), a Solanaceous plant cultivated in Mexico and Guatemala, original from Mesoamerica [1,2] Kinghorn and coworkers isolated a group of 20-hydroxywithanolides (compounds 35-42) from this plant, compounds 38-41 possess a y-lactone side chain of the ixocarpalactone type [27-29].
OH
CH 3 O' 33
34 R=H; 2,3-dehydro 39 R=OCH3
35
1028 --. R
OH 36R=H 37 R=OH
38 R=H; 2,3-dehydro 40 R=OCH3 41 R=H
42
A 16,18,20-trioxygenated withanolide, 13P-hydroxymethylsubtrifloralactone E (43) was isolated from plants of Deprea subtriflora (Riz & Pavon) D'Arcy collected in Peru together with a series of highly oxygenated C-18 norwithanolides, subtrifloralactones A-L (44-55). Compounds A, B, C, K and L (44, 45, 46, 54, 55) incorporate a hemiketal bridge like that found in the trechonolide group, while subtrifloralactones H, I and J (51-53) presented a new type of hemiketal bridge between C-20 and C-12 [30,31]. .o
'OR? OH
43
44 R!=R2=H 46 R,=OH; R2=H
47
48
45
49
1029
o o
50
51
.O
.0
53
12-Oxygenated withanolides A common feature of withanolides isolated from Datura ferox is the presence of an oxygenated function at C-12. [1,2]. From plants of this species collected in Cordoba, Argentina, four 12-oxygenated withanolides were isolated in the last decade: 15p-hydroxynicandrin B (56), and the 7hydroxywithanolides daturolactones 5, 6 and 7 (57-59) [32,33]. Carcamo et al. isolated a 12-oxowithanolide, named (-)-jaboromagellonine (60), from the Chilean Jaborosa magellanica [37].
o
OH ""6 56
58
1030
o
o' 60
59
Sativolides Jaborosalactones R (61), S (62) and T (63), present an additional six membered hemiketal (or ketal) ring, involving what must have been originally a C-12 ketone and a C-21 hydroxyl. They were isolated from Jaborosa sativa (Miers) (subnom. Trechonaetes sativa) A. T. Hunziker & G. Barbosa, collected in Tucuman province (Argentina) [35]. Although several other 21-oxygenated withanolides are known, they form additional rings with the side chain [1,2].
62
63
Trechonolides A hemiketal bridge formed by the 22-hydroxyl and a C-12 ketone, resulting in a six-membered ring with a B-oriented 12-OH group, was the unusual characteristic feature of trechonolide A (64), isolated from Jaborosa laciniata (Miers) (subnom. Trechonaetes laciniata), collected in Mendoza province (Argentina) [36]. This compound was later isolated by Parvez et al. from Chilean J. magellanica and renamed jaborosalactone M
1031
[37]. As this name had been already assigned to a structurally different withanolide (compound 1) isolated from J. bergii [12] the name trechonolide A is retained for 64. A group of withanolides structurally related to trechonolide A (64), have been subsequently isolated from different Solanaceae. From J. magellanica (Punta Arenas, Chile) were isolated (-)-jaborotetrol (65), (-)jaborolone (66), (+)-jaborochlorotriol (67) and (-)-jaborochlorodiol (68) [38]. Jaborotetrol (65) has been found in J. leucothricha (Mendoza province, Argentina) [14] and J. araucana (Chubut province, Argentina) [39], together with the 12-methoxy analogue (69) and threchonolide A (64) respectively. Jaborosalactone U (70) isolated from J. sativa (Tucuman province, Argentine), was characterized as the 24,25-epoxy analogue of trechonolide A, this being the only epoxy-y-lactone side chain found so far among the withanolides [35].
Cl 67
68
70
1032
Aromatic ring-A withanolides and 19-hydroxywithanolides The first 19-hydroxylated withanolide, jaborosalactone O (71), was isolated from Jaborosa leucotricha (Speg.) A. T. Hunziker collected in late spring in El Carrizal, Mendoza province (Argentina) [40]. When plants collected in autumn were investigated, three 19hydroxywithanolides, jaborosalactones V (72), W (73) and X (74) and two withanolides with and aromatic A ring, jaborosalactone Q (75) and jaborosalactone 7 (76), were isolated.[14,41] Compound (75) had been previously found in plants of J. leucotricha collected at another location (Cacheuta, Mendoza province, Argentina) [42]. (+)-Jaborol, is the only other known withanolide with an aromatic A ring [1,2]. The presence in the same plant of 19-hydroxywithanolides and A-ring aromatic 19norwithanolides, is indicative of an oxidative degradation pathway for the loss of C-19.
o
71 R,=H; R2=OH; 2,3-dihydro 72 R,=OH; R2=H 73 R,=OH; R2=H; 2,3-dihydro
75R=H 76 R=OH
Spiranoid withanolides The first withanolide with a spiranoid y-lactone side chain, jaborosalactone P (77), was isolated from plants of Jaborosa odonelliana collected in Salta province (Argentina) [43]. Lately six structurally related compounds were found in the same plant, jaborosalactones 10 (78), 14 (79) and 15 (80) in plants collected in summer and jaborosalactones 11 (81), 12 (82), 13 (83) and 14 (79) in plants collected in autumn [44]. Another group of six spiranoid withanolides, jaborosalactones 1 (84), 2 (85), 3 (86), 4 (87), 5 (88) and 6 (89), was isolated from J. runcinata collected in Entre Rios province (Argentina) [39]. Jaborosalactone 2 (85)
1033
was also isolated from J. araucana. These withanolides were structurally related to jaborosalactone P (77), but with a 17(20)-ene-22-keto system, a novel arrangement within the withanolides. o
77
78 R^Cl, R2=OH 79 R!=OCH3> R2=OH 83 R,=R2=OH
80
81R=H 82 R=OH
O
84R=H 87 R=OH
85 R,=OH, R2=H 86 R,=C1, R2=H 89 R,=C1, R2=OH
88
Comparison of the structure of jaborosalactone 2 (85) with that of trechonolide A (64) indicated that both compounds may have closely related biosynthetic precursors. In the former withanolide, ring closure on C-12 has occurred with a C-22 hydroxyl as shown in Fig. (4) pathway a. Oxidation of this intermediate or a related compound to the 22-ketone would allow cyclization between C-23 and the C-12 ketone to give a spiranoid withanolide (Fig. (4) pathway b). The co-occurrence of trechonolide A (64) and jaborosalactone 2 (85) in J. araucana supports this hypothesis.
1034
.0
OH
o
OH
t 77
64
85
Fig. (4). Proposed biosynthetic routes to trechonolides (pathway a) and spiranoid withanolides (pathway b) via a common precursor. The spiranoid intermediate could render both types of spiranoid withanolides either by reduction at C-22 or dehydration of the 20-hydroxyl.
Aromatic ring-D withanolides Withanolides with a six-membered aromatic ring D constitute nowadays an important group. A small number of these compounds, the nicandrenoids, were isolated from the Peruvian "shoofly" plant Nicandra physaloides (e.g. Nic-1, 90) [1,2] and remained as a curiosity within the withanolides, for many years. A family of these type of withanolides and related ergostane derivatives (termed salpichrolides) were isolated from Salpichroa origanifolia (Lam.) Thell in the last decade. The major components, in plants collected in Cordoba and Buenos Aires provinces (Argentina), were salpichrolides A (91) and G (92), salpichrolides B (93) and C (94) being isolated as minor compounds [45-47]. Compound 91 was the first withanolide having a 5,6-epoxide with a-stereochemistry, a feature found afterwards in several other salpichrolides.
o
o
= O
;o OH ""6 90
91R=H 92 R=0H
1035
O
OH
= O
= O
93
From plants collected in Buenos Aires in winter, two ergostane derivatives, salpichrolides E (95) and F (96) were isolated [48]. The stereochemistry of the C-24 methyl could not be determined. It is noteworthy that this type of side chain has been found only in N. physaloides, the other plant known to contain ring D withanolides [1,2].
o
A group of withanolides hydroxylated in the side chain, was isolated as minor components from S. origanifolia collected in Buenos Aires province in winter and in Salta province in summer. They were named salpichrolides H (97), I (98), J (99), K (100) and M (101) [47,49] Salpichrolides H and M correspond to the two possible products of hydrolytic {trans) cleavage of the side chain epoxide. Salpichrolides H (97) and I (98) could be intermediates in a degradative pathway, leading from salpichrolide A (91) to salpichrolide E (95), by oxidative cleavage of the C-25-C-26 bond, Fig. (5). C-26 would give rise to the formyloxy group. Salpichrolide F (96) may be derived analogously from the corresponding 5a,6(3-diol (salpichrolide C), or by hydroytic cleavage of the 5,6-epoxide of 95.
1036
0
97 26S/26S 2.5:1
9S26S/26R 1:3.5
0
o
I 99
100 R,=OH, R2=H 101 R!=H, R2=OH (26S/26R 1.3:1
A common feature of most withanolides, is the oxidation level of C-22 and C-26, C-26 being oxidized in most instances to the carboxylic acid level, thus allowing the formation of a 22,26-lactone. In some withanolides (e.g. in most salpichrolides) it is at the aldehyde level, allowing the formation of a 22,26-lactol. Salpichrolide J (99) and K (100) are the first withanolides with a side chain in which oxidation levels at C22 and C-26 are reversed. Salpichrolide K (100) slowly cyclized to salpichrolide J (99) in solution. A possible biosynthetic pathway for these compounds is presented in Fig. (6).
Fig. (5). Proposed degradative pathway for the formation of the side chain in salpichrolides E (95) and F (96). Starting from salpichrolide A (91) the first two intermediates correspond to compounds 97 and 98.
1037
Fig. (6). Proposed biosynthetic pathway for the formation of the side chain in normal salpichrolides and in salpichrolides J (99) and K (100).
Besides withanolides with an aromatic D ring, three withanolides with a normal (5-membered) D ring were isolated from S. origanifolia, salpichrolides D (102), L (103) and N (104) [46,49]. All of them retained the characteristic 5a,6a-epoxide moiety, unique to S. origanifolia. ,0
OH
102
103
104
The structure of salpichrolide N (104) is particularly interesting regarding the biosynthesis of withanolides with an aromatic D ring. Whiting has proposed as a possible pathway to ring D aromatization, the oxidation of C-18 followed by a 1,2-shift of C-17 to form a new six-
1038
membrered ring via a cyclopropyl fused intermediate; this would lead to salpichrolide A and related compounds upon cleavage of the C-13-C-17 bond as shown in Fig. (7) pathway a. The cleavage of the C-13-C-18 bond of the cyclopropyl intermediate would result in migration of the angular methyl (C-18 to C-17 via a 13,15-diene intermediate) to yield salpichrolide N (104) (Fig. (7) pathway b). Salpichrolide L (103) may be the precursor of a putative 14,16-diene intermediate.
103
Fig. (7). Proposed biosynthetic pathways for the formation of withanolides with an aromatic D ring (e.g. 91, pathway a) and for the rearranged skeleton in salpichrolide N (104, pathway b).
Acnistins and withajardins The acnistins are withanolides with a bicyclic side chain at C-17 similar to that of the withametelins [1-3], but with C-21 directly bonded to the lactone ring (C-24) via a C-C bond instead of an ether bond. Formation of the new 21,24 bond is considered to take place by a SN2-type reaction in withanolides having a good leaving group at C-21 [1]. The first acnistins, acnistin A (105) and E (106) were isolated from plants of Acnistus ramiflorus Miers (one of the synonyms of A. arborescens) collected in Merida (Venezuela) [50,51]. Luis et al. isolated acnistins B (107), C (108), D (109), F (110), G (111) and H (112) from Dunalia solanacea Kunth. collected in Medellin (Colombia) [52-54] This type of bicyclic side chain was also found in the withasteroid glycosides tubocapside A and tubocapside B isolated from Tubocapsicum anomalum Makino [1].
1039
O
o
O
""OAc 107
105 R!=H, R2=H 106 R,=OH, R2=H 108 R,=H, R2=OAc
109
O
110
111
112
Withajardins are closely related to acnistins, in this case C-21 is directly bonded to C-25 giving rise to a bicyclic lactone side chain with a six-membered homocycle. Withajardins A-E (113-117) were isolated from plants of Deprea orinocensis (Kunth) Raf. collected in El Jardin, Colombia. A common precursor has been proposed in the biogenetic routes to acnistins, withajardins and withametelins [55-57].
O
o
113 R=H 116R=Ac
114 R=H 115R=Ac
117
1040
BIOLOGICAL ACTIVITY Antifeedant and insecticidal properties Insecticidal properties of withanolides were first noticed on components isolated from the Peruvian plant Nicandra physaloides. Nicandrenone (Nic-1) (90), the major component isolated from this plant, was known by its bitter taste and its insecticidal properties. [58-61] During an infestation by larvae of the Egyptian cotton leafworm Spodoptera littoralis (Boisd) in the summer of 1978, it was noticed that shrubs of Physalis peruviana L. (cape gooseberry) were not attacked, whereas other Physalis and Nicandra spp. suffered heavy damage. Asher and co-workers demonstrated that withanolide E (118) and 4(3hydroxywithanolide E (119), isolated from P. peruviana, as well as several related steroids, had insect antifeedant properties. Further studies on other withanolides showed antifeedant effects and species-specific activity on three insects, S. littoralis (Boisd.) (Lepidoptera), the Mexican bean beetle, Epilachna varivestis Muls. (Coleoptera) and the red flour beetle, Tribolium castaneum (Herbst) [62]. R,0
,COOR
o
118 R=H 119R=OH
R2O' 120 R|=glc-(l-»2)-glc-6'-Ac, R2=R3=H, R4=OH 121 R,=glc-(l-»4)-glc-(l-»2)-glc-6'-Ac, R2=glc, R3=R4=H 122 R,=glc-(l-»4)-glc-6"-Ac-(l-»2)-glc-6'-Ac, R2=glc, R3=R4=H 123 R,=glc-(l-»4)-glc-(l->2)-glc-6'-Ac, R2=glc, R3=Ac, R ^ H 124 R,=glc-(l-»4)-glc-6"-Ac-(l->2)-glc-6'-Ac, R2=glc, R3=Ac, R4=H 125 R,=R2=glc, R3=R,=H 126 R,=glc-(l->2)-glc, R2=glc, R3=R4=H 127 R,=glc-(l->2)-glc-6'-Ac, R2=glc, R3=R4=H 128 R,=glc, R2=R3=H, R4=OH
Waiss and co-workers examined P. peruviana as a possible source of insect resistance in intergenetic hybridization and found that its foliage is highly inhibitory to growth and development of Helicoverpa zea, an insect that is an economic pest of numerous crops including the solanaceous plants tobacco and tomato. Bioassay directed extraction and
1041
fractionation of leaf material led to the isolation of several steroidal glycoside esters (120-128) that reduced the growth of H. zea. These compounds are structurally related to withanolides, with the 5-lactone side chain open and the carboxyl group esterified by mono- di- or trisacharides [63,64]. Artificial diets containing the test compounds at several levels were presented to larval H. zea, and their growth was determined after a 10-day period. The most active substance was the 11-hydroxy diglucoside ester 120, which reduced the weight of larvae to 50% of control values (ED5o) at a dietary concentration of 5.4 ppm. The triglucoside esters with 3-0glucosyl substitution, 121 and 122, had ED5o's of 15 and 50 ppm respectively. This may be compared to the 35 ppm value for both 123 and 124 that are the corresponding analogues with position 22 acetylated. Monoglucoside ester 125, had an ED5o of 85 ppm, the corresponding diglucoside esters 126 and 127 had ED5o's of 64 and 22 ppm respectively. The least active compound was the 11-hydroxy monoglucoside ester 128, at 110 ppm. By comparison, 4p-hydroxywithanolide E (119), which was found in P. peruviana at concentrations of over 2000 mg/kg (dry basis), had an ED50 of about 250 ppm. No clear structure-activity relationship could be established for compounds 120-128, the most striking difference was that between 128 and 120, that showed a ca. 27-fold change in activity. Their structures differ only by a single acetoxy glucose unit, and their polarities -as estimated by chromatographic partitioning between the stationary and mobile phases- were very similar, thus in this case the differences in insect inhibitory effects appear to be governed by very subtle factors. The above compounds were not lethal over the concentration range studied; for example, 120 was tested at 10 times the ED50, and all animals lived. This was consistent with the behavior of H. zea on fresh P. peruviana leaves where the larvae search and sample without settling down to feed. Moreover, the leaves showed a fine pattern of "shotgun" holes instead of the usual serrated feeding zones on preferred hosts where a large amount of plant material had been ingested. On the basis of these data, the authors suggested that growth inhibition was a consequence of feeding deterrence, leading to semi-starvation of animals. Baumann and co-workers studied the variation in the concentration of withanolide E (118) and 4(3-hydroxywithanolide E (119) in the berry as well as in the surrounding calyx during fruit development in Physalis
1042
peruviana [65]. On a fresh weight basis, they all decreased except for 4phydroxywithanolide E (119) that remained almost unchanged in the calyx. However, when related to the tissue water to obtain a measure for chemical defense, there was a decrease in the berry but a strong increase in the calyx during maturation, for both withanolides. When the withanolides content was compared with the ppm-concentration reported for antifeedant effect of those compounds, data suggested that the berry itself could be protected by intrinsic withanolides only when young. It appears that chemical defense is later taken over by the calyx abundantly equipped with 119. These authors also determined the concentration of 118 and 119 in leaves (related to water), 640 ppm for 118 and 1140 ppm for 119, which is high enough to explain the full protection against predation. The antifeedant effect of several withanolides isolated from Salpichroa origanifolia were investigated on larvae of the sanitary pest Musca domestica [66], the stored grain pest Tribolium castaneum [67] and the Mediterranean fly Ceratitis capitata [68]. The time needed to pupate 50% of the surviving M. domestica larvae (PT50) exposed to salpichrolides A (91), C (94) and G (92) is summarized in Table 2. On the basis of the intermediate dose (500 ppm), compound 91 showed the greatest development delay. The 2000 ppm concentration produced in all cases 100% mortality before pupation occurred, not allowing the calculation of the PT50. The concentration needed to inhibit complete development in 50% of the larvae (EC50) was calculated from the dose response curves in each experiment with the three natural withanolides, salpichrolide G (92) being the most toxic (ED50 203 ppm). With salpichrolides A (91) and G (92), adults failed to enclose from puparia. Development delays similar to those obtained with salpichrolide A (91) were observed when medium and low nutrition diets -without withanolides- were offered as food, supporting the idea that these compounds act as feeding deterrents. In the case of Tribolium castaneum, significant developments delays from larva to adult were also observed in treatments with salpichrolide C (94) at 2000 ppm and with salpichrolides A (91) and G (92) at 500 ppm and higher concentrations (Table 2). The results paralleled those obtained previously with M. domestica larvae, salpichrolide A showing the greatest development delay. On the other hand, no development delay was observed with salpichrolide C (94) in T. castaneum at 500 ppm. The
1043
different responses may be explained by species-specific detoxification mechanisms. Comparison of adult size data in treatments that produced development delays showed that control adults were significantly bigger (3.60 ± 0.10mm) than individuals treated at 500 ppm with compounds 91 (3.22 ±0.10 mm) and 94 (3.27 ±0.15 ppm), suggesting feeding inhibition by these compounds [67]. Table 2. Pupation time in Musca domestica larvae and development time for T. castaneum larvae exposed to natural salpichrolides A (91), G (92) and C (94) [66,67]. Musca domestica
Tribolium castaneum
Treatment
Cone (ppm)
PT50 (days)
DT50 (days)
control
-
7.7 (7.5-7.9)
57.3 (52.5-60.8)
91
500
10.3(10.1-10.6)
70.7 (68.3-73.0)
91
2000
ID
85.9(81.5-90.4)
92
500
8.0 (7.8-8.2)
69.1(61.9-74.6)
92
2000
ID
106.6(101.8-112.6)
94
500
8.1 (7.8-8.4)
54.6(49.1-58.1)
94
2000
ID
87.0 (82.0-92.2)
ID: Incomplete development
A group of synthetic analogues of natural salpichrolides was assayed on M. domestica and T. castaneum to assess structure-activity relationships. Results indicated that oxidation of the hemiacetal side chain to the lactone (compound 129) eliminated the biological activity on both species. Acetylation of the hemiketal on the side chain (compound 130) resulted in a nonsignificant decrease of the activity in M. domestica and drastically reduced the observed effect in T. castaneum. Reduction of the 2,3-double bond (compound 131) had a small negative effect on the feeding deterrent activity compared to salpichrolide A (91) [66,67]. These results prompted a study of the influence of modifications in rings A and B of the steroid nucleus on the antifeedant activity. Lethal and sublethal effects of natural salpichrolides and synthetic analogues were evaluated on the Mediterranean fly Ceratitis capitata [68]. The analogues selected for testing involved two major modifications of the A and B ring functionalities. On one hand three analogues with varying degrees of
1044
reduction of the ring A enone system were synthesized (compounds 93, 131 and 132). Although salpichrolide B (93) occurs naturally in S. origanifolia, it is a very minor component and cannot be isolated in sufficient amounts for biological testing; it was prepared from salpichrolide A (91). The second modification involved cleavage of the 5,6-epoxide, followed by oxidation (compound 133) or dehydration (compound 134). Significant development delays from larvae to puparia were observed in treatments with the three natural salpichrolides, A (91), C (94) and G (92); these results were similar to those previously obtained with M. domestica and T. castaneum larvae in which salpichrolide A (91) showed the greatest development delay (Table 3).
o =o
E
129 R=O 130 R=a-H, p-OAc
O
131 R=O 132 R=a-0H, p-H (X
JDH
O
= O
-- o OH 134
Table 3. Pupation time (PT50) and mortality of Ceratitis capitata larvae exposed to natural and synthetic salpichrolides (500 ppm). Treatment
a
PT50 (days)
Mortality (%)
91
10.54(10.18-10.83)
47.5
92
8.47 (8.00-9.00)
37.5
94
8.44(8.1-8.75)
5.0
131
6.84(6.53-7.12)
20.0
93
_•
95.0
132
11.64(10.42-15.02)
77.5
133
5.79(5.61-5.97)
7.5
134
6.15(5.87-6.41)
22.5
Control
5.36(5.14-5.58)
10.0
The high mortality produced by salpichrolide B (93) did not allow PT50 calculation
1045
Oxidation of the 6-hydroxy group in salpichrolide C (compound 133) or cleavage of the 5,6-epoxide in salpichrolide A followed by dehydration (compound 134), resulted in loss of the inhibitory effect. Although reduction of the 2,3-double bond (compound 131) had a smaller effect, the ring A reduced analogue 132 showed the greatest delay among synthetic analogues. Salpichrolide B (93) produced a high mortality before pupation, not allowing the PT50 calculation. The resulting EC50 of salpichrolide B (93) was 83 ppm, being this value lower than those informed for salpichrolide A (91) and G (92) against M. domestica. Exposure of adults of Ceratitis capitata to drinking water containing natural salpichrolides A (91), G (92), B (93) and C (94) produced mortality in all cases, with salpichrolide B producing the highest effect. The fact that the reduction of the 2-en-l-one system increased toxicity is in agreement with the inhibition observed by Waiss and co-workers on Helicoverpa zea larvae exposed to withanolides and related esters isolated from Physalis peruviana [63,64]. In that case compounds with a reduced 2-en-l-one system exhibited higher activity in comparison with 4(3hydroxywithanolide E (119). The content of the salpichrolides in S. origanifolia was monitored by HPLC during plant development, reaching a maximum during summer (Dec 21 st to march 21 st in the southern hemisphere) when insect populations are higher [66]. These results in conjuction with the observed toxic and feeding deterrent activities suggest that these compounds may provide protection against predation by certain phytophagous insects acting as chemical defense. Feeding deterrant activity of the major components of J. odonelliana, jaborosalactone P (77) and jaborosalactone 10 (78), was studied against the stored grain pest Tribolium castaneum [44]. In this case, only jaborosalactone P (77) produced a significant delay in the development of neonatae larvae. Dinan and coworkers studied withanolides as potent ecdysteroid agonists and antagonists to assist in the further elucidation of the mode of action of ecdysteroids and, possibly, as novel invertebrate pest control agents [69]. Sixteen withanolides which had been isolated from Iochroma gesneriodes (Kunth) Miers (Solanaceae) were assessed for agonistic/antagonistic activity using the Drosophila melanogaster B l l cell line bioassay. Those possessing an oxygen-containing function at C-3 (hydroxy or methoxy) and an a,(3-unsaturated ketone in the side chain
1046
ring showed antagonistic activity, with 2,3-dihydro-3Phydroxywithacnistine (135) being the most active (ED50 2.5 x 10~6M versus 5 x 10~8 M for 20-hydroxyecdysone (20E)). Oxygen-containing functions at C-3 are rare among natural withanolides and in many cases they are artifacts of the isolation procedure (especially, methoxy groups), thus it is not clear if the antagonistic activity of the above mentioned withanolides is serendipitous or whether withanolides could be activated upon ingestion by insects [70]. Recently Dinan and coworkers surveyed 128 species of solanaceous plants for the presence of ecdysteroid agonist and antagonist activities. Only weak antagonist activity was associated with a few of the methanolic extracts, including those from species known to contain high levels of withanolides [71].
AcO
o
135
Cancer chemopreventive activity of withanolides The induction of the phase II drug-metabolizing enzyme quinone reductase (QR), using Hepa Iclc7 hepatoma cells, has been currently used to determine the potential cancer chemopreventive activity of withanolides [72]. Induction of QR activity was calculated from the ratio of specific enzyme activities of compound-treated cells in comparison with a solvent control. The concentrations required to double and quadruple QR activities in the cells, CD and CQ, respectively, were generated. To observe only the induction on QR and to avoid cytotoxic effects, the half-maximal inhibitory concentration of cell viability, IC50, was also determined. From the ratio between the IC50 and CD or CQ values, chemopreventive indices (CI) were calculated. Such measurements not only predicted anticarcinogenic activity but also provided a reasonable index of potency and toxicity.
1047
The first studies on cancer chemopreventive activity were performed by Kennelly and co-workers on withanolides isolated from Physalis philadelphica. The most potent compounds were found to be ixocarpalactone A (34), philadelphicalactone A (36), 4p\7p,20Rtrihydroxy-l-oxowitha-2,5-dien-22,26-olide (42), and ixocarpalactone B (38), all of which contained a 4p-hydroxy-2-en-l-one structural unit [73]. Lately, thirty-seven naturally occurring withanolides isolated from southamerican Solanaceae plants were evaluated for their potential to induce quinone reductase [74]. Jaborosalactone 1 (84), jaborosalactone O (71), jaborosalactone P (77), trechonolide A (64) and withaphysalin J (17), were demonstrated to be significant inducers with CD values in the range of 0.27-1.52 mM. In each subgroup of withanolides analyzed, it was found that some substituents lead to changes in quinone reductase activity. These results indicated that a functionalized methyl-18 plays an important role in improving QR activity. On the other hand, the presence of 5a-substituents resulted in lower activities. In general, spiranoid and trechonolide type withanolides exhibited good QR induction. In terms of CI values, some of the compounds described compared favorably with sulphoraphane, a known chemopreventive agent. Among these compounds, the spiranoid jaborosalactone P (77) was one of the most promising in terms of inducing potency and low toxicity. To further evaluate the potential of jaborosalactone P (77) a preliminary study was performed to test the capacity of this agent to induce steady-state levels of quinone reductase in multiple organ sites of BALB/c mice. Sulforaphane was used for comparison in this in vivo study. With Jaborosalactone Ptreated mice, a significant induction was observed in liver and colon, but not in lung, stomach, or mammary gland. The in vivo study confirmed the in vitro results, indicating that withanolides may function as potent phase II enzyme inducers. Activity-monitored fractionation of a chloroform-soluble extract of Deprea subtriflora using a quinone reductase induction assay led to the C-18 norwithanolides mentioned previously. Six of the active compounds obtained from this plant (44, 46, 47, 49, 52 and 53), presentes an a,Punsaturated ketone unit in ring A. Compound 55 -with a doubly unsaturated ring A ketone- was found to be inactive in the QR assay, while compound 54 was active [72]. It has been suggested that the presence of an a,P-unsaturated ketone unit in ring A of withanolides is important for inducing activity in the cell-based QR induction assay,
1048
however other structural features may compensate the lack of this functionality or block its beneficial effects. Withanolides 26 and 27 isolated by Minguzzi and co-workers from A. arborescens were very potent as monofunctional inducers of quinone reductase (CD value), but their selectivity (CI value) was marginal [22]. Phytotoxic activity Recently withanolides isolated from Iochroma australe and the norbornane-type withanolide jaborosalactol 18 (3) isolated from Jaborosa bergii, showed phytotoxic activity on monocotyledoneous and dicotyledoneous species. Iochroma australe extract and the major constituent (17S,20R,22R)-4p,7P,20-trihydroxy-l-oxowitha-2,5,24trienolide (24,25-dehydro-42) reduced growth of the radicle of the weeds Sorghum halepense (Monoct.) and Chenopodium album (Dicot.) [75]. Jaborosalactol 18 (3) showed significant inhibition of radicle growth at 2 x 1(T3 M on the dicotyledoneous species Chenopodium album, Ipomea purpurea and Lactuca sativa (phytogrowth inhibitory activity > 49%) [13]. On the other hand, in the monocotiledoneous species tested {Zea mays and Sorghum halepense) the phytogrowth effect of compound 3 was stimulatory. Thus, the authors suggest that compound 3 could act as a selective phytogrowth controller, stimulating radicle growth of monocotyledoneous species. Trypanocidal leishmanicidal and bactericidal activities. In the course of screening extracts from Bolivian plants against Trypanosoma cruzi, Leishmania spp., Bacillus subtilis and Staphylococcus aureus, Dunalia brachyacantha (Griseb.) Sleumer was found to be active. The bioassay-guided purification of the leaf extract led to the isolation of two known acetoxywithanolides (136 and 137), which displayed antiparasitic and antimicrobial activity (Table 4) [25]. This constitutes the first report of antileishmanial and antitrypanosomial (Chagas'disease) activities for steroidal lactones.
1049
AcO.
OH 136
137
Table 4. Antiparasitic activities of 18-acetoxywithanolide D (136) and 18-acetoxy-5,6-deoxy-5-withenolide D (137) [25].a Cone, (ng/tnl) 1JO
m
JU
Tc TT
La
TTT
TTT
+++
+++
J_L_L.
4-4—U
4-4-4-
TTT
TTT
TTT
+++
+++
25
0
^C
ZJ
10
Lb
+
Ld TTT
+++ 4-4-4-
TTT
+++
1
0 ++ + + a Tc= Trypanosoma cruzi, ii=Leishmania braziliensis, La= Leishmania amazonensis, Ld= Leishmania donovani.. 0; number of epimastigotes or promastigotes identical to control; +: 75% epimastigotes or promastigotes, with few degenerative forms; ++: 50% epimastigotes or promastigotes, with few degenerative forms; +++: total lysis of parasites.
ACKNOWLEDGEMENTS Financial support by CONICET (Argentina), Universidad de Buenos Aires, SeCyT-UNC, Agencia Cordoba Ciencia and FONCYT is gratefully acknowledged
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
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BIO ACTIVE SECONDARY METABOLITES RELATED TO LIFE-CYCLE DEVELOPMENT OF OOMYCETE PHYTOPATHOGENS MD. TOFAZZAL ISLAM AND SATOSHITAHARA Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 060-8589, Japan
ABSTRACT: Members of the oomycete genera, e.g., Phytophthora, Pythium and Aphanomyces, which are phylogenetically distinct from fungi, are the most devastating pathogens of plants, animals, fishes and humans. The zoospores of phytopathogenic oomycetes are believed to locate their host plants by chemotaxis, after which they undergo a series of morphological changes before penetrating the host tissues to establish the diseases. Bioassay-guided chromatographic separation visualized some host-specific plant signals which are not only responsible for chemotaxis but also trigger developmental transitions (encystment and germination) of zoospores on host surface to initiate infection. In contrast, nonhost plants possess diverse secondary metabolites that can directly affect motility and viability of the phytopathogenic zoospores, indicating their involvement in plant resistance against oomycetes. This review summarizes isolation, identification and bioactivities of diverse secondary metabolites identified in host and nonhost plants toward the most infamous phytopathogenic oomycete zoospores namely, Phytophthora, Pythium, and Aphanomyces. The possible ecochemical role and the mode of action of these active compounds are discussed in relation to the biorational control of oomycete phytopathogens. The importance of bioassay methods in isolating novel zoospore regulating molecules from the host and nonhost plants is discussed. In addition, a brief discussion on the chemical basis of host-specificity in animal, fish and human pathogenic oomycetes are also included.
INTRODUCTION Oomycetes (Peronosporomycetes in new classification [1]) are phylogenetic relatives of brown algae that cause many destructive diseases of plants, as well as several animal and human diseases [2,3]. They are mostly water or soil inhabiting organisms [4]. The members of oomycetes genera Phytophthora (Ph.), Pythium (Py.) and Aphanomyces are known as the most devastating pathogens of dicot plants [5]. For example, Ph. infestans, which causes the late-blight disease of potato, destroyed the Irish potato crop in 1845 and 1846, resulting in the historic Irish potato famine [6]. This is still a damaging disease, annually costing over $ 5 billion worldwide in crop losses and control measures [7]. Some members of
1054
oomycetes are pathogenic on species other than plants include: Py. insidiosum, which infects animals (swamp cancer); Lagenidium giganteum, which parasitizes the larval stage of mosquitoes is being used as potential control agent; Aphanomyces invadans, A. astaci and Saprolegnia spp. are the devastating pathogens of several fish species [1,2,8,9]. Phytopathogenic oomycetes give a special stage of motile spore with two dissimilar flagella called zoospore, in their life cycle (Fig. 1). Both flagella are ornamented with complex hairy structures probably for swimming, sensing and precisely docking on the host surface [1,5,10,11]. Zoospores of those plant pathogens are liberated from a mycelium or a sporangium within an hour; disease caused by these oomycetes can be multi-cyclic, resulting in severe epidemics that can destroy whole crops within a single season [12]. Zoospores show interesting responses to their host and nonhost roots as well as their secondary metabolites in addition to environmental physicochemical factors. However, very restricted chemical factors regulating the life cycle of such organisms have been known so far, even though understanding well about such chemical factors seems to be very significant to establish new techniques for control of those soil-borne oomycete phytopathogens still problematic in agriculture. Our knowledge of their biology is limited, but their physiology differs from that of fungi, and many fungicides are ineffective against oomycetes [7,14]. New approaches are needed to find novel targets and to develop the 'oomicides' for a sustainable and biorational management of those notorious phytopathogens [14]. •
a
PF
b
\' ^x^v\/)///
C
Fig. (1). Scanning (a) and transmission (b & c) electron micrographs showing the morphological features of an Aphanomyces cochlioides zoospore and its flagella. a. A reniform-ovate secondary zoospore with a shorter anterior (AF) and a longer posterior (PF) flagella. b. Terminal part of an anterior flagellum possessing tripartite tubular hairs, c. Fine tubular hairs on the tapered tip and surface of a posterior flagellum shaft. Circular objects in the background of micrograph (a) are pores (size: 0.6 |am) of SEMpore membrane.
Zoospores of the phytopathogenic oomycetes accumulate at the potential infection sites of host roots by chemotaxis [11,15-17]. Root released electrical currents (electrotaxis) are also found to participate with
1055
chemotaxis in homing responses of zoospores toward roots [18]. A few host-derived chemical signals have been identified as the chemotactic factors of phytopathogenic zoospores (Fig. 2) [19-23]. For example, indole3-carbaldehyde (1) from cabbage seedlings [19], and prunetin (2) from pea seedlings [20], is specific attractants for A. raphani and A. euteiches, respectively. Similarly, cochliophlin A (3) in roots and root exudates of spinach, and daidzein (4) and genistein (5) in soybean root exudates, are the specific attractants for A. cochlioides [21] and Ph. sojae zoospores [23], respectively. The other host-derived attractant so far reported is N-transferuloyM-O-methyldopamine (6) from the roots of Chenopodiwn album as a potent attractant of A. cochlioides zoospores [22]. All these compounds showed their attractant activity at concentrations as low as micromolar to nanomolar levels. Host-specificity in chemotaxis in oomycete zoospores has been described in some earlier reviews [13,17,25, 26]. Recently, some of the host-specific plant attractants were reported to trigger differentiation of zoospores as shown by the host roots suggesting the chemical basis of the host-specificity in phytopathogenic oomycetes [27]. Little is known about the signal transduction mechanism of zoospore chemotaxis and differentiation by host signals, whilst some stimuli related to general signal transduction have been reported. In recent reports, it was claimed that zoospore perceive host signal by a G-protein-coupled receptor and translate into responses via phosphatidic acid and/ or phosphoinositide signaling cascades [13,27,28]. Among the oomycetes, Aphanomyces species cause some of the destructive plant and fish diseases in the world [8, 29-32]. Species of the phytopathogenic Aphanomyces exhibit high degree of specialization and can infect a limited number of plant species [32, 33]. For example, most of the plants are resistant to the strains of A. cochlioides that infect sugar beet, spinach and a few other members of Chenopodiaceae and Amaranthaceae [32-34]. This phenomenon of nonhost resistance, the ability of a pathogen to cause a disease in particular species but not in others, has always intrigued plant pathologists but remains poorly understood especially in oomycetes [3, 35]. Survey of non-susceptible plants using A. cochlioides zoospore bioassay revealed that they possess diverse chemical weapons to defend themselves from the attack of oomycetes [36]. Here we review our research results concerning zoospore attractants, repellents, cytotoxins and inhibitors of zoospore motility together with bioassay systems to survey chemical regulators toward zoospores. The first part of this review described some new findings on the effects of hostspecific plant signals on chemotaxis and differentiation of oomycete zoospores. The potential role and mode of action of nonhost secondary
1056
metabolites in plant-pathogen compatibility is also discussed in the second part of this review in relation to the biorational control of oomycete phytopathogens on the basis of natural product chemistry. This updates our earlier reviews on chemotaxis of oomycete zoospores [13, 37] and homing responses of Phytophthora and Pythium by Deacon and Donaldson (1993) [17], and complements a recent review on molecular basis of recognition between oomycete plant pathogens and their hosts [26].
1: indole-3-carbaldehyde Host plant: Brassica campestris var. capitata Oomycete: Aphanomyces raphani
3: cochliophilin A Host plant: Spinacia oleracea Oomycete: Aphanomyces cochlioides
2: prunetin Host plant: Pisum sativum Oomycete: Aphanomyces euteiches
4: R =• H : daidzein 5: R * OH : genistein Host plant: Glycine max Oomycete: Phytophthora sojae
6: N-frans-feruloyl-4-O-methyldopamine Host plant: Chenopodium album Oomycete: Aphanomyces cochlioides Figure (2). Structure of some host-specific attractants for oomycete zoospores
HOST-SPECIFIC PLANT SIGNALS REGULATING THE LIFECYCLE DEVELOPMENT OF OOMYCETES Motile zoospores are an important means of initiating infection by oomycetes. Zoospores do not divide, however, but have high-affinity
1057
receptor-based recognition systems for locating hosts by chemotaxis [13,25,26]. The accumulated zoospores differentiate into adhesive cysts (a process called encystment), which in turn germinate to produce hyphae that are actually responsible for invading the host tissue [11]. Recent reports suggest that all these key pre-infection events of soil-borne zoosporic phytopathogens are triggered by chemical signals released from the host roots [24,26]. Zoospores Use Host^specific Chemical Signals to Target Roots Preferential chemotaxis toward hosts
Preferential chemotaxis followed by encystment of zoospores toward compatible hosts has been reported in many cases for plant pathogenic oomycetes. Encystment in vivo involves recognition of a host or substrate surface. Held (1973) was among the first who established this because zoospores of Rozella allomyces were attracted to both host (Allomyces) and nonhost (Blastocladiella) thalli; but encyst only on the host [38]. A scanning electron microscopic observation showed that significantly higher numbers of zoospores of Ph. cinnamomi encysted on the surface of a susceptible cultivar of avocado compared to that of the tolerant cultivar [39]. Fewer cysts were germinated on the roots of the tolerant cultivar than on the susceptible cultivar. Mitchell and Deacon (1986) observed that zoospores from Pythium graminicola and Py. arrhenomanes, which characteristically infect graminaceous hosts, preferentially accumulated behind root tips of grasses compared to dicots, whereas zoospores of the broad host range species Py. aphanidermatum and Py. ultimum did not show preference for grass roots [40]. Similarly, zoospores of a cotton pathogen, Py. dissotocum were attracted to cotton roots but zoospores of Py. catenulatum which is not compatible with cotton were not. The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola [41], and those of the nematode parasite Catenaria anguillulae accumulate at the mouth of the host [42]. All these interesting phenomena suggest that some host-specific chemical signals might play a vital role in locating host and differentiation of zoospores on host surface before penetration. On the other hand, saprophytic species selectively colonize of the host depending on their food sources [43,44,45].
1058
100 wm
Fig. (3). Response of Aphanomyces cochlioides zoospores toward spinach roots. A. Photomicrograph (dark field) of aggregated zoospores (dots close to root surface) just behind root cap of spinach root tip. B. A mass of cystospores (arrow) on spinach root [11].
Evidence of host-specific signals for zoospore chemotaxis
The question of specificity in chemotaxis is an important one as it relates to the contribution of chemotaxis, and subsequent steps of infection by zoospores, to host selection and host specificity [26]. Several host-specific attractant signals have already been identified for phytopathogenic oomycete zoospores. For example, indole-3-carbaldehyde (1) isolated from cabbage seedlings is a chemoattractant down to concentration of 1 nM for A. raphani zoospes [19]. Prunetin (2) isolated from the roots and root exudates of pea seedlings [20] is a potent attractant (down to 10 nM) for zoospores of the A. euteiches, while the zoospores of A. cochlioides are strongly attracted to host metabolites, cochliophilin A (3) and N-trans-femloyl-4-Omethyldopamine (6) at concentrations down to 0.1 nM and 10 nM, respectively [21,22]. Zoospores of Ph. sojae are attracted to daidzein (4) and genistein (5) from the roots and root exudates of its host, soybean, at a concentration down to 0.1 nM [23,24]. In contrast, some nonhost secondary metabolites including flavonoids displayed potent repellent activity toward pathogenic zoospores [13,36,46]. All these reports are interesting because they parallel the behavior of host-specific Rhizobium species, which also are attracted to the flavonoids of their hosts and can be repelled by nonhost flavonoids, the ability to recognize flavonoids released by their hosts appear to be also critical for first recognition step in the Rhizobium4eg\ime interaction [47]. So far, there are two best documented examples of specificity in chemotaxis and subsequent differentiation of oomycete zoospores by host plant signals. The first one is the attraction, encystment and germination of A. cochlioides zoospores to a rare flavone, cochliophilin A (3), and the other one is Ph. sojae zoospores to isoflavones daidzein (4) and genistein (5)
1059
[21,23,24,27]. In both cases, these compounds are present in the host seeds, roots and exuded by the roots. The details of each interaction between host signal and the respective pathogenic zoospores are described in the following section. Host-specific attractants for Aphanomyces spp. zoospores
The genus Aphanomyces consists of both saprophytic species as well as specialized parasites on plants and fishes [8,36]. In crops, they generally cause damping-off and root rot diseases, for example, A. euteiches on peas (Leguminosae), A. cochlioides on spinach, sugar beet and few other members of Chenopodiaceae and Amaranthaceae, and A. raphani on radishes (Cruciferae). Studies on the ecological chemistry of phytopathogenic Aphanomyces spp. revealed that the zoospores of those specialized pathogens locate their host plants guided by specific host signals released from the roots. The zoospores of phytopathogenic Aphanomyces attracted only to the potential infection sites of host roots but not to those of nonhost [36]. In a detailed study, Ui and Nakamura (1963) observed that the A. cochlioides was highly compatible with most of the members of Chenopodiaceae and a few members of Amaranthaceae (Table 1) [32]. Members of other plant families were less compatible or incompatible with A. cochlioides. When root extracts of 15 plant species belonging 10 families were subjected to A. cochlioides zoospore bioassay, interestingly, the crude extracts from the plants belonging to Chenopodiaceae and Amaranthaceae showed strong attractant activity indicating a direct correlation between host-pathogen compatibility and zoospore attractant activity toward plant root extracts (Table 1) [36]. At least three host-specific attractant signals (1-3) have been isolated for Aphanomyces zoospores by detailed bioassay-guided fractionation procedures [19-21]. Another potent attractant, JV-frans-feruloyM-0-mehyldopamine (6) for A. cochlioides zoospores was isolated from the roots of a host plant, Chenopodium album. These compounds show attractant activity only toward the respective pathogenic zoospores down to the nanomolar level. Other non-pathogenic oomycetes do not respond to these compounds. The third attractant for A. cochlioides zoospores was identified from the leaves of spinach as 5,4'-dihydoxy-6,7-methylenedioxyflavone (7) which is less active than the root attractant cochliophilin A (3). Although this compound has already been found as a glucuronide derivative in spinach, the ecological significance of 7 in host-pathogen relationship is yet to be clarified [49].
1060 Table 1: Responses of zoospores toward the extracts of plants belonging to different families and varying levels of compatibility with Aphanomyces cochlioides*
Plant
Degree of compatibility
Minimum active cone. (ppm)b
Chenopodiaceae
Spinacia oleracea L.*
attractant (30)
Chenopodium album L.*
attractant (30)
Beta vulgaris L. var. saccharifera*
attractant (100)
B. vulgaris L. var. cicla*
attractant (30)
Papaveraceae Papaver rheos L.
attractant (30)
Amaranthaceae Celosia cristata L.*
attractant (30)
Amaranthus retroflexus L.*
attractant and weak halting (100)
A. gangeticus L. Potulacaceae
attractant and halting (100)
Portulaca olercea L. Cruciferae
halting (100)
Raphanus sativa L. Graminae
repellent (500)
Zea mays L. Leguminosae
NA (1000)
Trifolium repens L. Solanaceae
NA(1000)
Lycopersicon esculentum L. Compositae
NA (1000)
Taraxacum officinalae L. Anacardiaceae
NA (1000)
Lannea coromandelica L. halting and lytic (200) a
Adapted from the reference of [8,32,36,37,48].
* Reported hosts.'+' and '-' signs indicate the degree of compatibility. and incompatibility of pathogen to the respective plant species, respectively. Acetone extractives.
b
1061 Host-specific plant signal, cochliophilin A
The specific signaling compound, cochliophilin A (3) for A. cochlioides zoospores has been found in the seeds, roots and root exudates of spinach [37]. Cochliophilin A (3) is also contained in the roots of two other hosts of A. cochlioides, C. album and sugar beet [50]. The detailed isolation and quantification procedures of compound 3 from the fresh spinach roots and root exudates were elaborately discussed in an earlier review [37]. It was estimated that fresh spinach roots contained approximately 1.9 x 10~5 mol/kg cochliophilin A (3) and exude sufficient amount (34 ng/plant/day) of this compound for attracting the zoospores of A. cochlioides [37]. So far, the distribution of 3 is completely restricted in the hosts (Chenopodiaceae) of A. cochlioides. Therefore, Tahara and Ingham (2000) suggested that the sensitive attraction of A. cochlioides zoospores to this rare flavone may account for a part of the mechanism that determines the host range of this phytopathogen [37]. This hypothesis is now strengthening when cochliophilin A (3) was found to trigger developmental transitions of zoospores at a physiologically relevant concentration [27]. Host-specific plant signals involved in host recognition as well as germination of pest propagules have been reported in many cases including soil-borne fungi [51], parasitic plants [52] and nematodes [53]. Structure-activity relationships
The specificity of A. cochlioides for a flavone, cochliophilin A (3) has been defined using a number of synthetic compounds with various levels of structural similarity to the flavone [37,54]. It revealed that A^ing oxygenation at C-5 and C-7 positions in the flavones with unsubstituted firing were the most important determinants of attractiveness. The 5-hydroxy group in flavones is known to form a strong hydrogen bond with certain metal ions but it is not known at present if this property is linked with the ability of 5 to attract zoospores. Although a limited number of compounds were investigated, the 6,7-methylenedioxy-substituted flavone, cochliophilin A (3) and its 6,7-dimethoxy derivative (8) showed almost equivalent stronger activity than those of other compounds tested. The presence of small alkoxy groups at C-6 and C-7 would seem to be effective in enhancing the zoospore attractant properties. Flavones with a 5-hydroxy7^nethoxylated A^ing, and an unsubstituted B^ing, exhibited stronger activity than chromone which lacked the B^ing, or apigenin-7,4'-di-Omethyl ether with a /?-methoxylated B^ing [37,54]. These results suggest that amongst flavones, strong activity may be related to the presence of an
1062
unsubstituted B^ing. In contrast, many other flavones and isoflavones showed little or no attraction [36,37]. For example, genistein (4) and prunetin (5) have respectively been reported as zoospore attractants of Ph. sojae and A. euteiches, respectively; these isoflavones were essentially inactive toward A. cochlioides at a concentration of 10"6 M [37]. An important finding of these studies was that A. cochlioides zoospores could respond to a wide range of phenolic compounds, albeit at significantly higher concentration than that for the flavones. Furthermore, in some cases the response observed was repulsion rather than attraction, for example, 8prenylated naringenin (9) and medicarpin (10) [36].
OCH, OCH, OH
OH
O
O
OH
OCH3
OH O 10
Recently, we tested some synthetic flavonoids those of which showed potent repellent activity toward Ph. sojae zoospores. Surprisingly, most of the compounds tested, showed strong attractant activity toward A. cochlioides zoospores, where some of them were more powerful attractants than the host-specific cochliophilin A (3) [36]. Although, the attractant property of those synthetic flavonoids were similar to cochliophilin A (3), however, very less number of attracted zoospores were encysted and germinated by those synthetic attractants than that of the natural plant signal (3). Structural requirements for attractant activity of synthetic flavonoids revealed that hydrophobic B-ring plus alkylated (methylated) A^ing in the flavonoid skeleton is responsible for strong activity. For example, 7hydroxy-5^nethylflavone (11), displayed 100 folds higher attractant activity than that of cochliophilin A (3). Similarly, a methoxylated B^ing at 4'position with unsubstituted A^ing (e.g. 4'-methoxyflavonol, 12, active at 1011 M) also showed potent attractant activity. Therefore, hydrophobicity in
1063
A^ing also increases attractant property. These observations raise the possibility that A. cochlioides zoospores can integrate a large amount of information about their chemical environment, over and above their attraction to host flavone (3). Although the detailed structure-activity studies of compounds related to the ferulamide attractant (6) have not been carried out. However, among the four regio-isomers tested, the natural ferulamide (6) displayed the strongest attractant activity. The substitution patterns of 4-OH or 3-OCH3 in amides are popular in nature, but they are less active than 4-O-methyldopamine. Compound 6 is the first naturally occurring amide possessing a rare substitution pattern (4-O-methyldopamine moiety) in the dopamine part may be indicative of the presence of a specific relationship between the host material and the mobile pathogen [54]. However, this compound (6) seemed to operate through a different receptor from cochliophilin A (3) because a background of one of them did not affect the response to the other. Similarly, Sekizaki et al. (1993) studied the specificity of A. euteiches zoospores for prunetin (2) using wide variety of isoflavones (non-, mono-, di-, and tri-substituted) [55]. They observed that a hydroxy group (but not a methoxy) at the C-5 position of an isoflavone is necessary to strongly attract A. euteiches zoospores. In addition, the presence of an additional hydroxy group at C-7 or C-4' enhanced the attractant activity, which was further increased by 7-O^nethylation, but slightly decreased by 4'-O^ethylation. The strongest activity amongst 26 isoflavones was associated with the natural attractant prunetin (2). The structural requirement for another host-specific attractant, indole-3 carbaldehyde (1) has been studied by using 16 compounds (namely, indole, indole-3-ethanol, indole-3-propionic acid, indole-3-butyric acid, indole-3acrylic acid, indole-3-acetic acid, indole-2-carboxylic acid, indole-3carboxylic acid, indole-5-carboxylic acid, acetoaldehyde, vanilline, nbutyraldehyde, cinnamaldehyde, citrol, citronellal and tryptamine hydrochloride) structurally related to compound 1 [19]. Only few compounds, namely, indole-3-carboxylic acid, indole-3-acrylic acid, indole3-acetic acid, and indole-2-carboxylic acid showed weak attractant activity toward A. raphani zoospores only at very high concentrations (>10"6 M). Tryptamine hydrochloride exhibited attractant activity at 10 |ag/ml however; at 100 |ug/ml it immobilized zoospores. Obviously, none of the compounds displayed any attracting activity at concentrations lower than 10"6 M suggesting that compound 1 is a host-specific plant signal which is an attractant at 10"9 M for A raphani zoospores. Indole-3-carbaldehyde (1) did not attract zoospores of A. cochlioides and A. euteiches which further
1064 strengthen the assumption that compound 1 is a host-specific one for A. raphani. Attractant for Phytophthora and other oomycete zoospores
Among the oomycete genera, Phytophthora and Pythium are known as the most devastating pathogen of dicot plants. Most of them have wide host range. Zoospores of those Phytophthora and Pythium species show a relatively nonspecific attraction to amino acids (particularly aspartate, glutamate, asparagine, glutamine, arginine and methionine), sugars (e.g. glucose) or volatile compounds (e.g. alcohols, isovaleraldehyde, valeraldehyde), all of which are common components of root exudates [17,26]. Attraction to these compounds may account for the nonspecific attraction of many Phytophthora and Pythium zoospores because, the threshold level for chemotaxis is usually higher than the micromolar order which may not practically exist in the rhizosphere. Many researchers have been studied on the preferential chemotaxis of Phytophthora and Pythium zoospores; however, convincing information available on the molecular mechanism of host recognition in those phytopathogens is limited. Species of Phytophthora and Pythium, especially those with restricted host ranges, appear to exhibit more specificity in their attraction toward root exudates. The most characterized example of specificity is the attraction of Ph. sojae zoospores to the isoflavones, daidzein and genistein [23]. The zoospores of Ph. sojae, which are chemotactically attracted to the isoflavones, daidzein (4) and genistein (4), released from the soybean roots [23,46] were used as the test organisms to check the response to a wide range of compounds possessing some structural similarity to genistein, including isoflavones, flavones, chalcones, stilbenes, benzoins, benzoates, benzophenones, acetophenones, and coumarins [46]. Of 59 compounds examined, 43 elicited some responses. A comparison of the chemotactic responses elicited by the various compounds revealed a primary role for the phenolic 4'- and 7-hydroxy groups on the isoflavone structure. An important finding of this study was that Ph. sojae zoospores could respond to a very wide variety of phenolic compounds, albeit at significantly higher concentration (1 uM) than for the isoflavones [26,46]. Tyler and coworkers (1996) observed substantial levels of genetic variation in the attraction of zoospores of different Ph. sojae genotypes to isoflavones [46]. Genetic crosses between the isolates showed that a single gene was responsible for the difference in attraction to genistein and other isoflavones [26]. The genetic differences of zoospores in response to nonisoflavone phenolics were determined by at least six additional
1065
independently segregating genes, supporting the notion that Ph. sojae has an extensive array of receptors capable of sensing the phenolic environment of the zoospores. Based on these findings, Tyler (2002) hoped that detailed mapping of these genes may provide a route to cloning the Ph. sojae receptors responsible for detecting isoflavones and other phenolic compounds [26]. Similar extensive array of receptors may exist in other oomycetes for sensing chemical environment in the rhizosphere. During the study on structure-activity relationships, Tyler et al. (1996) also surprisingly observed that a few flavonoidal compounds were acted as good repellents, notably methylated flavones (7-hydroxy-5-methylflavone 11, 7-hydroxy-3-methylflavone 13, 5,7-dimethoxyflavone 14, and 4',5,7trimethoxyflavone, 15) with mainly a hydrophobic B^ing [46]. Interestingly, one of the most potent repellents for Ph. sojae zoospores, 7hydroxy-5-methylflavone (15), was also reported to be the most potent inhibitor of the nodulation response of several genotypes of Bradyrhizobium japonicum [56]. However, all those alkylated synthetic flavonoids showed potent attractant activity toward A. cochlioides zoospores (particle method). In organisms that use klinokinesis as a chemotaxis strategy, such as Phytophthora species and many bacteria, attraction and repulsion are parts of a continuum of responses: attraction results from a lower-than-average frequency of turning, while repulsion results from a higher-than-average frequency of turning [16,46,57]. In Escherichia coli, the same receptors and the same signal transduction pathways mediate both attraction and repulsion [57]. OCH 3
OH
12
OCH3 H,CO H3CO H,CO H,CO
O
O 14
15
1066
Specific sensitivity of zoospores to the host-derived compounds has also been found in other pathogenic oomycetes. Kerwin et al. (1992) observed that Py. marinum exhibited encystment on the surfaces of red algae (its hosts) but not on green or brown algae (non-host) [58]. Galactose or anhydrogalactose contents in the surface of red algae were found to be responsible for such a specific response. Similarly, fish and mosquito larval parasites showed positive chemotaxis toward their host surface chemical constituents [59-62]. Developmental Transitions of Zoospores Triggered by Host-Specific Plant Signals A common characteristic among most of the oomycete and fungal plant pathogens is that each specialized on a narrow range of specific plants as hosts [51]. One adaptation to a specific host plant is the recognition of the host's chemicals which can be used to trigger genes or developmental pathways needed for pathogenesis. The production of characteristic secondary metabolites (e.g. flavonoids) by plants, particularly those exuded from roots (e.g. legumes), appears to be used as signals for various microbes, including symbionts as well as pathogens. Although zoospores of oomycetes rapidly differentiate after docking on the potential infection sites of host, but it has long been unknown whether some host-specific plant signal are involved in this developmental pathway. Differentiation of zoospore involves encystment of a zoospore and germination of a cystospore to a hypha. Recently, a few host-specific attractants were found to trigger differentiation of zoospores, essential for penetration into host roots (Table 2). Developmental transitions by cochliophilin A
Despite of the discovery of some host-specific chemoattractants of zoospores, it was a big question whether the same signaling molecule induces subsequent encystment and germination on host surface or these following events are regulated by different host signals. Moreover, it was unknown whether the stages of pre-infection are necessarily under separate control or a part of the signaling cascade. This is important because a success of infection depends on the completion of sequential events. Deacon (1996) suggested that zoospores might be induced to encyst by the effect of specific root surface components [25]. Evidence supporting the involvement of any host-specific plant signal in differentiation of
1067
pathogenic zoospores has been lacking for long. However, in vitro studies revealed that zoospores are encysted by root surface mucilage, fucosyl residues, pectin, alginate or specific polysaccharides, lectin or monoclonal antibodies specific for flagella [17]. However, in all those cases the threshold concentrations were high. Recently, investigation of the host factors triggering encystment and germination of A. cochlioides zoospores on spinach roots revealed that a gradient of the host-specific attractant, cochliophilin A (3) triggers encystment and germination of zoospores at a concentration approximately ten times higher than that observed to elicit chemotaxis (Fig. 4) [27]. The effects of three host-derived attractants identified for A. cochlioides zoospores, cochliophilin A (3), N-trans-feruloyM-O- methyldopamine (6) and 5,4'-dihydroxy-3,3'-dimethoxy-6,7-methylenedioxy-flavone (7) have been assayed by particle bioassay method. Only, the cochliophilin A (3) induced encystment of the attracted zoospores at a range of 10"8 to 10"6 M concentration in a dose dependent manner and formed a mass of cystospores on and around the Chromosorb W AW particles treated with 3. Initially the attracted zoospores became sluggish, moved in a circular fashion, halted and rapidly changed into round-shaped cystospores. Interestingly, the attracted zoospores landed and encysted on the surface of Chromosorb particle coated with 10~7 to 10"6 M solution of cochliophilin A (3). All encysted zoospores germinated (100%) on and around the particles within 30^4-0 min. The cystospores germinated adjacent to the particles coated with host-specific attractants showed germ-4ubes tropism toward the particles. The particles coated with lower than 10"8 M concentration of cochliophilin A exhibited only attractant activity but not induced encystment of zoospores. On the other hand, the control particles treated with solvent alone neither affected the normal motility of zoospores in the aquatic medium nor resulted in encystment of any zoospore. The other two host-specific attractants (6 and 7) did not induce encystment and germination up to 10"6 M concentrations.
Zoospore
Immature cystospore
Mature cystospore
Germinating cystospore
Fig. (4). Morphological changes (developmental transitions) of Aphanomyces cochlioides zoospores triggered by a host-specific plant signal, cochliophilin A (3). White bars = 1 |JM.
1068
The effect of cochliophilin A (3) on the encystment and germination of zoospore was evaluated by the direct application of 3 suspended in water at a range of 10"12 to 1CT6 M concentration [27]. The direct application of 3 into the zoospore suspension as a homogeneous solution at a range of 1CT12 to 10"8 M just stimulated the motility of zoospores for 10-15 minutes without resulting any encystment and germination. However, at higher concentrations (10~7 to 10"6 M) of cochliophilin A (3) in the above conditions, it showed no effect on the motility of zoospores. Interestingly, very slow release of 5xl0~12 to 5xl0~10 M cochliophilin A (3) solution to the zoospore suspension by a microsyringe showed strong stimulation of the motility of zoospores followed by encystment and germination. In most cases, the stimulated zoospores formed the clumps of aggregated cells scattered at the bottom of glass petri dish, and then encysted and germinated. It clearly indicates that a gradient of host signal is necessary for taxis and differentiation of zoospores which seems to reflect exactly the natural event at rhizosphere. Thus, our particle bioassay appeared to be a suitable method for studying chemotaxis and subsequent differentiation of zoospores where a gradient of chemical is essential for the response of cells [63]. The germ tubes of the cystospores germinated adjacent to the aggregate exhibited tropism toward the aggregate center. Tropic responses of hyphal germlings to host-specific signals have also been observed in Ph. sojae [24], and autoaggregation of zoospores in the absence of a host seems to be characteristic of many other oomycetes [64]. It is feasible that the hostspecific compounds might also induce the similar aggregation phenomenon. Aggregation of inoculated zoospores on a certain point of the host root might increase the vigor of the inoculums for successful infection. To understand the mechanism of halting response of the zoospores to cochliophilin A (3) followed by encystment and germination, we undertook a time-course observation of changes by scanning electron microscopy (SEM). SEM observation revealed that zoospores stimulated by 3 or spinach root tip underwent a similar sequence of morphological changes up to germination of cystospores. In both cases, the stimulated zoospores became almost round shape by shedding their flagella within 20 min of stimulation and soon became the enlarged cystospores (8.5-10.5 jam i.d.) bounded by a smooth cyst-coat [11]. The flagella of the zoospore were found to lose their fine structures immediately after detachment. The initial cystospore coated with a smooth cyst-coat rapidly changed into a mature cystospore (5.7-7.1 |im i.d.) coated with rough cell wall within 20-30 min and finally germinated within 40-60 min. Interestingly, the sequence of morphological changes of zoospores by cochliophilin A (3) was identical to those occurred during interaction with spinach roots.
1069
Therefore, the behavior of zoospores on and around Chromosorb particles coated with cochliophilin A (3) was identical to that of zoospores toward spinach roots. The amount of 3 in the spinach root (ca, 1.9 x 10"5 mol/kg fresh root) seems to be enough to initiate encystment of zoospores followed by germination which are regenerated by Chromosorb particles coated with a 10"8 M solution of 3 as a dummy of the spinach root [27]. These observations suggest that cochliophilin A (3) is indeed a host-specific plant signal which may play essential roles in both locating host roots and initiating encystment and germination. Interestingly, an almost similar phenomenon was observed in bacteria. As a signal for chemotaxis of rhizobia a concentration as low as 10"9 M luteolin is sufficient, and at 10"6 M concentration luteolin stimulates nod gene expression [65,66]. Furthermore, the growth and sporulation of A. cochlioides on a corn meal agar medium were unaffected up to 10 M concentration of cochliophilin A (3). This information supports that this oomycete can grow well and produces zoospores for further dissemination of pathogens to spread the disease through surrounding healthy plants. All these interesting features of host-pathogen interactions might have ecological significance, and may find useful application in the investigation of biochemical and molecular mechanism in pathogenicity where it is definitely desired to synchronize the development of pathogen with that of the host. Differentiation of zoospores by soybean isoflavonoids
The specific attractants of soybean pathogen, Ph. sojae also trigger encystment followed by germination of zoospores at the higher concentration than that needed for attracting activity. This phenomenon was confirmed by capillary bioassay methods. When a capillary tube containing 20 |jM of daidzein (4) or genistein (5) was introduced to the chemotaxis chamber and left undisturbed, zoospores rapidly plugged the capillary tube and other encysted around the mouth of the tube and germinated [23,24]. This phenomenon was identical to the responses of zoospores toward a soybean root tip. Addition of the solution of soybean isoflavones at a low concentration directly to the zoospore suspension before vortexing also significantly increased the germination ratio of cystospores than that of control indicating that host signals are effective in germination of pathogenic spores [67]. Flavonoids have also been reported to germinate many pest propagules of soil-borne fungi, parasitic plants and mycorrhizal spores [51].
1070 Table (2). Triggering Che mot axis and differentiation (encystment and germination) of Some Oomycete Zoospore by Host-specific Plant Signals Host-specific
Host plant
plant signal
Threshold concentration (n M) Oomycete
Taxis
Encystment
Germination
3-indolecarbaldehyde (1)' Cabbage
A. raphani
1
nt
nt
Prunetin (2)a
Pea
A. euteiched
10
nt*
nt
[20]
Cochliophilin A (3)b
Spinach
A. cochlioides
0.1
10
10
[21,27]
Daidzein (4)*
Soybean
Ph. sojae
1
100
100
[23, 24]
Genistein (5)a
Soybean
Ph. sojae
1
100
100
[23, 24]
Feruloyldopamine (6)b
C. albumm
A. cochlioides
10
na
na
[22, 27]
[19]
* nt = not tested; na = non-active; Jcapillary method; particle method.
4: R = H : daidzein 5: R = OH : genistein
Chemotropism of hyphal germlings
Factors that influence the direction of hyphal growth after germination of a cystospore are less explored than chemotaxis. Autoaggregation of zoospores in the absence of an available host is characteristic of some if not all oomycetes [64]. Zentmyer (1961) demonstrated that Ph. cinnamomi cysts that were adjacent to the root of their host germinated rapidly and grew in the direction of the root, but host-specific chemical signals regulating this behavior has been discovered very recently [24,27]. However, tropic responses of growing hyphae to nutrient sources have also been demonstrated in several saprophytic and parasitic oomyetes [68,69]. Recently, Morris et al. have investigated the role of the host-specific isoflavones, daidzein (4) and genistein (5) on chemotropic behavior of germinating cysts of Ph. sojae [24]. They demonstrated that hyphal tips respond chemotropically to 4 and 5, suggesting that hyphal tips from the zoospore cysts that have encysted adjacent to the root may use specific host isoflavones to locate their host. Thus chemotropic responses of oomycetes hyphae might also contribute to their effectiveness as plant pathogen.
1071 Receptors in zoospores
It is assumed that the responses of zoospores to different host-specific plant signals or environmental chemical stimuli are mediated by chemoreceptors in the zoospore of flagellar membrane. But no receptor has yet been purified and characterized for a zoosporogenic oomycetes. In a biochemical study, Sakasai analyzed a putative cochliophilin A (3) receptor protein in the membrane of A. cochlioides zoospores [70]. He designed a cochliophilin A analog, AF-bio (16) according to the results of structure-activity relationships analyzed by Kikuchi et al. [54] and Takayama [71]. The analog (16) consists of the required part structures as an attractant, a biotin part to be trapped by a horseradish peroxidase-avidin conjugate, and an azido group, which is for photoaffinity labelling of the zoospore protein(s). AF-bio (16) showed attractant activity toward A. cochlioides zoospores and competition against cochliophilin A (3) itself in the zoospore chemotaxis.
H
OH O OH O biotin part
attractant part 16
photoaffinity ligand
17
According to his preliminary experiments, a fresh zoospore suspension containing 16 was treated by UV-light, and then the membrane proteins were fractionated and subjected to SDS PAGE. The proteins in the gel were transferred to a polyvinylidine difluoride (PVDF) membrane and treated with a horseradish peroxidase-avidin conjugate. Peroxidase active region on the PVDF membrane was detected by ECL™ (enhanced chemi luminescence) method. Finally, he found AF-bio (16) binding protein at ca 70 kDa, presumably a reputed receptor protein for cochliophilin A (3), because the band disappeared completely when the zoospores treated with 3 for photoaffinity labelling in the presence of excess amounts of AF (17) lacking a biotin part structure. Further progress of characterization of this AF-bio binding protein is eagerly waited.
1072 Signal transduction pathways in zoospores
G-proteins are believed to be key components of signal transduction pathways in chemotaxis of many other motile cells [72]. Mastoparan is commonly used as a diagnostic agent for the participation of G-proteins in both animal and plant signal transduction pathways [73,74]. Interestingly, the heterotrimeric G-protein activator, mastoparan showed encystment of both A. cochlioides and Ph. infestans zoospores at a micromolar concentration [13,27,28]. The synthetic peptide analog Mas 17, predicted not to form an amphipathic helix at lipid interface because of the replacement of Leu-6 by Lys, is totally devoid of agonist activity. The concomitant application of mastoparan and the host-specific attractant cochliophilin A (3) appeared to further enhance encystment of zoospores and rapid germination of A. cochlioides cystospores. In addition, chemicals interfering with phospholipase C activity (neomycin) and Ca2+ influx/release (EGTA and loperamide) suppressed cochliophilin A and mastoparan induced encystment and germination. Changes of Ca2+ fluxes during differentiation of zoospores have been observed by early investigators [67,75]. By an X^ay microanalysis of individual encysted zoospores, Connolly and co-worker (1999) also demonstrated that the hostspecific plant signals, daidzein (4) and genistein (5) trigger a calcium influx in Ph. sojae [67]. These results suggest that the zoospore differentiation by host-specific cochliophilin A (3) might be mediated by G^irotein-coupled receptors to activate both phosphoinositide and Ca2+ second messengers pathways. Genes encoding a and |3 subunits of heterotrimeric G-proteins have already been characterized in Ph. infestans [76]. However, in a recent study, Latijnhouwers et al. demonstrated that differentiation of zoospores of late blight pathogen Ph. infestans is triggered not only by mastoparan, but also by di-octanoyl phosphatidic acid (DOPA), n-and sec-butanol but not terr-butanol [28]. Likewise, mechanical agitation of zoospores, which also induced encystment of zoospores, resulted in increased levels of phosphatidic acid (PA) as well as its phosphorylation product diacylglycerolphosphate (DGPP). They also found that the accumulation of PA during encystment by mastoparan and rc-butanol is caused by the stimulation of PLD but not PLC activity. They concluded that PLD is involved in zoospore encystment by mastoparan. It is known that encystment of phytopathogenic oomycete zoospores by mechanical agitation regenerate into next generation of zoospores instead of germination. We also observed that both n- and 5ec-butanol also induced encystment of A. cochlioides zoospores where the cystospores did not advanced to germination rather regenerated into zoospores. Whereas,
1073
induction of encystment by a host-specific plant signal synchronously germinate into hyphae within a few minutes. These raises a question whether encystment of zoospores by mechanical agitation or PA or nbutanol follow a different signal transduction pathway than encystment induced by a host-specific plant signal. Additionally, PA has been linked to a variety of plant treatments and responses, most of these involve biotic or abiotic stresses, suggesting a role for PA as a general stress-signalling molecule [77]. A further comparative study on lipid metabolism in zoospore after induction of encystment by host-specific plant signals and other chemicals (e.g., n-butanol) would give suggestive information of the signal transduction pathways in zoospores. Since the components of the pathway represent attractive targets for developing alternative disease control methods, agricultural practice may benefit from such kind of research results in the long term. Concluding Notes Much progress has been made in the past decade in understanding the signaling and interactions between root-infecting oomycetes and their hosts, and the findings indicate several points of potential general significance. A common characteristic among oomycetes and fungal pathogens of plants is that each specializes on a narrow range of specific plants as hosts. One adaptation to a specific host plant is the recognition of the specific host's chemicals which can be triggers for specific gene expression or developmental pathways needed for pathogenesis [51]. The production of characteristic plant secondary metabolites by plants, for example, flavonoids particularly those exuded from the roots, appear to be used as signals for various microbes, including symbionts as well as pathogens. The phenomenon of host recognition through host-specific plant secondary metabolites which function as chemical signals directing several key steps in the early stages of the infection response of oomycete phytopathogens is summarized as follows: a)
They mediate chemotaxis of swimming zoospores toward the root tips [11,23], where most of the signaling compounds are exuded by the root [37,78].
b)
Exposure of zoospores to elevated levels of host-specific attractant signals cause encystment followed by 100% germination of cystospores on host roots as well as on
1074
artificial surfaces such as Chromosorb particle, capillary tube or plastic membrane [24,27]. c)
Host-specific attractants also induce chemotropic growth of germlings toward the roots [11,24,27].
d)
Zoospore perceives host-signal by a G-protein-coupled receptor and then translate into responses (chemotaxis and differentiation) via phosphoinositide/ Ca2+ or phosphatidic acid second messengers pathways [27,28].
^ Fig. (5). Chemotaxis and subsequent differentiation of Aphanomyces cochlioides zoospores by host-specific plant signal cochliophilin A and host (spinach) roots, a. Zoospores aggregated, encysted, and germinated on and around a Chromosorb W AW particle coated with lxlO"6 M cochliophilin A (3). Cystospores germinated adjacent to the particle showed germ tube tropism toward the cochliophilin source; b. A SEM micrograph showing cystospored adhered and germinated on the surface of a cochliophilin A coated particle; c. Cystospores germinated on a host (spinach) root. White bars = 100 |im.
DIVERSE NONHOST SECONDARY METABOLITES AFFECTING MOTILITY AND VIABILITY OF ZOOSPORES In contrast to susceptible plants, non-susceptible plants may possess some chemical weapons to defend themselves from the attack of zoosporogenic oomycetes [79]. This hypothesis has been tested by surveying physiologically active secondary metabolites in more than 200 nonhost plant extracts using A. cochlioides zoospores by particle bioassay method [80]. The crude extracts showed a wide range of biological activities toward zoospores, and the active principles of several plant extracts have been identified by bioassay-guided chromatographic techniques. This section summarizes our research findings along with current knowledge on nonhost plant secondary metabolites in relation to their resistance against oomycete phytopathogens.
1075
Survey of Physiologically Active Secondary Metabolites in Nonhost Plant Extracts In recent years, there has been renewed interest in examining interactions between nonhost plants and oomycetes [3]. The molecular basis of nonhost resistance remains one of the major unknowns in the study of plant-microbe interactions. Plant disease resistance can be conferred by constitutive features such as structural barriers or performed antimicrobial secondary metabolites. Performed barriers and compounds such as saponins are ubiquitous in plants and play important roles in nonhost resistance against filamentous fungi [35,81]. Studies concerning nonhost resistance against oomycetes by plant secondary metabolites are very few. Screening extracts of nonhost plants revealed that nearly half of the extracts had direct effects on motility and viability of A. cochlioides zoospores (Table 3). Although, some of the nonhost plant extracts exhibited attractant activity, however, none of them showed attractant and subsequent differentiation of zoospores as shown earlier by cochliophilin A (5) [27,36]. In addition nonhost extracts exhibited some deleterious activities for example, repellent, stimulant, halting, lysis etc. against the zoospores. Some unusual activities like sudden inhibition of motility by Portulaca oleracea (Portulacaceae), attraction and halting by Amaranthus gangeticus (Amaranthaceae), motility inhibition and subsequent lysis of zoospores by Lannea coromandelica (Anacardiaceae) and Ginkgo biloba (Ginkgoaceae), and negative chemotaxis by Dalbergia odorifera (Leguminosae) and Magnolia kobus (Magnoliaceae) extracts were noticeable (Table 3) [36,82]. These interesting effects of nonhost plant extracts toward phytopathogenic oomycete zoospores prompted us to investigate the active principles by detailed chemical studies. Screening results thus indicated that many nonhost plants might use secondary metabolites to directly defend themselves from the attack of oomycete phytopathogens. Isolation of various nonhost defense factors (chemical weapons) against oomycetes may give some new interesting targets for controlling oomycete phytopathogens. Based on the screening results, we identified the active principles in nonhost plant extracts by detail bioassay-guided chemical fractionations, and the results are reviewed in the following sections. The modes of actions of the isolated compounds on zoospores are also illustrated.
1076 Table 3. Activities of Some Nonhost Plant Extracts3 toward Aphanomyces cochlioides Zoospores Plant name
Family
Achyranthes sp. Amaranthus gangeticus A. caudatus A. tricolor Lannea coromandelica Mangifera indica Catharanthus roseus Basella alba Terminalia arjuna T. chebula Attractylodes lancea Aucklandia lappa Cyperus rotundus Phyllanthus emblica Ricinus communis Ginkgo biloba Leucas zeylanica Leonurus heterophyllus Akebia quinata Pueraria lobata var. chinensis Dalbergia odorifera Allium chinensis A. cepa A. sativum Magnolia kobus Hibiscus rosa sinensis Azadirachta indica Sinomenium acutum Papaver somniferum Ampelygonum chinense Portulaca oleracea Nigella sativa Paeonia suffruticosa Aegle marmelos Capsicum annuum Abroma augusta Cuminum cyminum Foeniculum vulgare Vitex negundo Curcuma longa Zingiber officinale Elettaria cardamomum
Amaranthaceae
Anacardi aceae Apocynaceae Basell aceae Combretaceae Compositae Cyperaceae Euphorbiaceae Ginkgoaceae Labiatae Lardi zabal aceae Leguminosae Li li aceae
Magnoliaceae Malvaceae Meliaceae Menispermaceae Papaveraceae Polygonaceae Portulacaceae Ranunculaceae Rutaceae Solanaceae Sterculiaceae Umbelliferae Verbenaceaee Zingiberaceae
Plant organ
Types of activity* (MAC Mg/ml)
stem bark leaves whole plant aerial part rhizome root rhizome aerial part unripe fruit whole plant aerial part root heartwood bulb
fruits aerial part stem whole plant root seed root bark aerial part whole plant aerial part seed ripe fruit aerial part rhizome
+ = attractant; -= repellent; s = stimulant; h = halting motiliity; b = bursting zoospores (cell lysis). MAC - minimum active concentration. *Particle bioassay method was used to test the activity of extracts. a Plant materials were ground or cut into small pieces and extracted with 70% acetone.
+ & h (200) + & h (30) + & h (50) + & h (30) h & b (200) -(200) s(100) + (30) -(500) -(500) -(1000) (1000) -(1000) -(200) - & h (200) h & b (200) + & s (500) + (500) + (200) + (500) -(200) + (500) (1000) + (1000) -(200) + (500) -(100) + (200) + (200) -(200) + & h (200) s (500) -(200) + (200) + (30) s (500) + (500) -(500) s (500) -(100) s (500) -(500)
1077
Zoospore Motility Inhibitors in Portulaca oleracea Isolation of active principles
To identify the zoospores motility inhibitory principles from the common purslane, Portulaca oleracea, 1.15 kg of fresh roots were first extracted with MeOH and then subjected to chemical fractionation using n-hexane, diethyl ether and EtOAc (Scheme 1) [83]. The diethyl ether extract (1.72 g) Fresh roots of Portulaca oleracea (1.15 kg) extraction with MeOH Coned. MeOH extracts (50% MeOH)
Hexane layer
MeOH-H2O layer MeOH evaporation Ether extraction
Aqueous layer (780 mg)
Ether extract (1.72 g)
EtOAc extraction
SiO2 C.C. in EtOAc^vleOH-H 2 Oconc.NH4OH = 60:15:5:1
Aqueous layer Repellent fraction (small amount)
EtOAc extract SiO 2 C.C. (CHCl3-MeOH = 2:l)
Stimulant fraction (321 mg)
Repellent fraction (42.2 mg)
SiO2 C.C. (CHCl 3 -MeOH=20:l) Stimulating fraction (30.2 mg)
CHC13 layer
HPLC (CHCl 3 -MeOH=10:l) Stimulant (19, 12.5 mg)
Partition between CHC13 and aq. 2N HC1 Aqueous layer
SiO 2 C.C. (CHCl3-MeOH-H2O = 65:25:4)
Repellent (20, 8.5 mg)
Scheme 1. Isolation procedure for compounds in Portulaca oleracea roots exhibiting stimulant and repellent activity on zoospore of A. cochlioides. SiO2 C.C. : silica gel column chromatography.
1078
was applied to silica gel (200 g) column chromatography using EtOAcMeOH-H 2 O-conc. NH4OH 60:15:5:1 to give 12 fractions (100 ml each) in which two active components, a stimulant (fractions 2-4) and a small amount of repellent (fraction 6) were detected. Fractions 2-4 were rechromatographed and the stimulant was finally purified by HPLC using an Inertsil column (6.0x250 mm) in CHCl3-MeOH 20:1, flow rate 1 ml/min, to yield 12.5 mg of the stimulant (?R ca 16.3 min). As the amount of repellent in the ether extract was insufficient for further purification, the EtOAc extract (Scheme 1) was used as an alternative source. This extract was initially applied to a silica gel (60 g) column and the repellent was eluted with CHCl3-MeOH 2:1. Three repellent fractions (6-8, 40 ml each) were combined, the volume reduced to near dryness in vacuo, and the residue redissolved in EtOAc and washed with 2N HC1. The EtOAc-soluble constituents were further purified by passing through a silica gel Sep-Pak column (3 cc) with CHCl3-MeOH-H2O 65:25:4 as eluting solvent to give the repellent (8.5 mg). Structure elucidation ofmotility inhibitors
HR-EI-MS indicated the empirical formula C18H19O4N for the stimulant, whilst the 'H-NMR spectrum revealed two hydroxy groups [5 7.88 (1H, s) and 5 8.09 (1H, s)], seven aromatic protons [5 6.7-7.2 (7H)], one methoxy group [5 3.88 (3H, s)], two methylene groups [5 2.74 (2H, t, 7=7.3 Hz) and 5 3.48 (2H, q, .7=7.3 Hz)], and two olefmic protons [5 6.47 (1H, d, 7=15.5 Hz) and 5 7.42 (1H, d, 7=15.5 Hz)]. The coupling constant of 15.5 Hz indicated the presence of a frans-distributed olefinic bond. The detection of protons assignable to a methoxy, and a hydroxy group, an olefinic group and a l,2,4^risubstituted benzene, as well as an EI-MS fragment at m/z 177, strongly indicated the presence of a feruloyl part structure. The remaining four aromatic protons [5 6.75 and 7.06 (both 2H, d, J=8.4 Hz)] and four aliphatic protons attributable to two methylene groups were assigned to those of tyramine. Most of these signals were very similar to those of Nfran.s-feruloyl-3-O-methyldopamine (18) and iV-frans-feruloyM-O-methyldopamine (6) previously isolated from Spinacia oleracea [84] and Chenopodium album [22]. The stimulant was thus considered to be N-transferuloyltyramine (19). The identification was confirmed by acylation of commercially available tyramine with ferulic acid in the presence of N,N'dicyclohexylcarbodimide to yield Af-frans-feruloyltyramine (19) which possessed physicochemical properties indistinguishable from those of the natural stimulant. The stimulant activity of natural iV-?rans-feruloyltyramine
1079
(19) towards the zoospores of A. cochlioides was also identical to that observed for the synthetic compound (19). The purified repellent, containing minute amounts of a homologue (or homologues), gave a positive response to the Dittmer test which indicated the presence of a phosphate group in the molecule [85]. In the 31P-NMR spectrum, a phosphorus atom resonated at 4.54 ppm (internal standard: triphenylphosphine). Alkaline methanolysis of the repellent was yielded methyl linoleate indistinguishable from an authentic sample. In the ! HNMR spectrum, signals assignable to two double bonds via one methylene (-CH=CH-CH2-CH=CH-), and a methyl doublet coupled with 31P via an oxygen atom [5 3.66 (3H, d, 7=10.9 Hz)] were detected. The molecular formula of the repellent was found to be C22H41O7P by HR-FAB-MS (negative ion mode: [M-H+]~, m/z 447). From an analysis of all the available physicochemical data, the repellent was considered to have structure 20 [83]. This structure was confirmed as follows. Commercially available 1oleoyl-24ysophosphatidic acid (21) was methylated with diazomethane in ether to yield the monomethyl and dimethyl esters, which were respectively positive and negative in the Dittmer test. Chromatographic and spectroscopic properties of synthesized monomethyl ester (22) were in good agreement with those of the natural repellent except for minor features reflecting differences in the fatty acid part of each molecule. Biological activity and the possible function of the motility inhibitors
Compounds 19 and 20 along with some acylated phosphatidic derivatives in addition to 1,2-dioleoylphosphatidic acid, were tested using the particle bioassay [83]. Commercially available l-oleoyl-2lysophosphatidic acid (21) possessed repellent activity which was enhanced by monomethylation. When A. cochlioides zoospores were pre-treated with an excess of the natural stimulant N-trans-feruloyltyramine (19), and then exposed to Chromosorb W AW particles coated with various test compounds, it was found that l-oleoyl-24ysophosphatidic (21, 100 ppm) and its monomethyl ester (22) (10 ppm), as well as the natural repellent 1linoleoyl-2-lysophosphatidic acid monomethyl ester (20, 30 ppm), effectively inhibited zoospore motility [83]. However, l-oleoyl-2lysophosphatidic acid dimethyl ester (23) and 1,2-dioleoylphosphatidic acid tested with and without the stimulant (19) showed neither repellent nor motility inhibitory activity. The bioassay revealed that compounds possessing repellent activity are monoacylated phosphatidic acid derivatives containing at least one hydroxy group on the phosphoryl unit [83].
1080
HO OCHi 19 (natural stimulant)
H
°
H-C-O' H-C-OH o H-C-O-P-OH H 20 (natural repellent) O H H-C-O' H-C-OH H-C-O-R H O 21: R=—P-OH OH
O 22: R = —P-OH OCH3
23: R
O P-OCH3 OCH3
The characteristic behavior of Aphanomyces zoospore movement is shown in Fig. 6, where zoospore movement is inhibited in the area close to a Chromosorb W AW particle treated with a mixture of the stimulant and repellent factors from roots of P. oleracea, whilst in area remote from the
1081
particle, they are still swimming quite actively. Based on the bioassay results, we conclude that a mixture of the stimulant, N-transferuloyltyramine (19), and the repellent, l-linoleoyl-24ysophosphatidic acid monomethyl ester (20), in Portulaca root is responsible for inhibiting the motility of zoospores of A cochlioides.
Fig. (6). Photomicrograph of zoospores of A. cochlioides after exposure to a mixture of stimulant, N-transferuloyltyramine (19) and the repellent, Hinoloyel-24ysophosphatidic acid monommethhyl ester (20) released from Chromosorb particle treated with a 1000 ppm and a 100 ppm solution of 19 and 20. Photograph was taken through a microscope with an exposure time of 0.5 s. Dots close to the particle: inhibited zoospores. Lines in the area remote from the particle: traces of swimming zoospores.
The behavior of zoospores treated with a mixture of these pure compounds was very similar to the effect observed when zoospores were exposed to segments of fresh roots of P. oleracea suggesting that both compounds exude from the roots. Nevertheless, natural compounds possessing important in vivo functions, which remain to be fully elucidated. When bioassayed using the particle method, it was found that cochliophilin A (3), like the stimulant iV-frans-feruloyltyramine (19), could act together with the natural repellent 14inoleoyl-24ysophosphatidic acid monomethyl ester (20) to completely inhibit zoospore motility. Under the microscope, the treated zoospores were first seen to become stationary, and then settle at the bottom of the petri dish where they encysted to give cystospores. These cystospores germinated within 1-2 h, although germination would not normally be expected in the absence of a host plant. This is the first report on the inhibition of zoospore motility as a result of
1082
the interaction of a zoospore stimulant (iV-frans-feruloyltyramine, 19) and a repellent (14inoleoyl-24ysophosphatidic acid monomethyl ester, 20). The data also indicate a new biological action for lysophosphatidic acid, derivatives of which are already known to exhibit chemoattractant effects on the amoeba, Dictyostelium discoideum [86]. The biochemical properties of lysophosphatidic acids are described in detail in the latest review [87]. Recent identification and cloning of lysophosphatidic acid-specific receptor has led to the elucidation of G-protein and signaling pathways through which lysophosphatidic acid functions [88]. Recently, di-octanoyl phosphatidic acid (DOPA) was found to cause encystment of 100% of Ph. infestans zoospores at 15 (ig/ml within 10 min [28]. Hydroxylated iVcinnamoyl-/?-phenylethylamine derivatives including JV-rrans-feruloyltyramine (19) are relatively widespread in higher plants [89]. Their physiological functions are of general interest because their biosynthesis from the corresponding acyl-CoA and amine derivatives, under the influence of enzymes such as tyramine feruloyl transferase, is stimulated in response to pathological infection [90,91]. Isoflavonoidal Repellents from Dalbergia odorifera The extracts of a famous Chinese herbal medicine, the heartwood of Dalbergia odorifera displayed potent repellent activity toward A. cochlioides zoospores. Three isoflavonoids were isolated as the active factors from the extract by a series of column chromatography followed by preparative TLC [36,82]. Their chemical structures were assigned on the basis of physicochemical data including 2D NMR. Structure elucidation
The first isolate gave an intense molecular ion peak at m/z 270 ([M]+, 100%) in the FD-MS spectrum and analysis of HR-EI-MS established the molecular formula of 10 as C16Hi404. The UV, EI-MS, *H- and 13C-NMR data were found to be reasonably matched with those reported for medicarpin [92,93]. Thus the structure of 10 was confirmed as (±)medicarpin (3-hydoxy-9-methoxypterocarpan, 10) ([oc]29D 0° in MeOH, c = 0.045). The HR-EI-MS of the compound 24 exhibited the exact molecular mass (calcd., 286.0841, obsd., 286.0834) corresponding to the molecular formula C16Hi405. The UV, EI-MS, ! H and 13C-NMR data agreed with those of the reported (-)-claussequinone (24) [94]. The optical data recorded for 24 was ([a] 28D -31.5° in MeOH, c = 0.0069). The HR-EI-MS and the
1083 !
H-NMR spectra of compound 25 estimated its molecular formula as C16H12O4. The ] H- and 13C-NMR assignments were compared with those of the literature and all chemical shifts and coupling patterns were found to be identical with those of formononetin [92,95]. Thus the 25 was confirmed as formononetin (7-hydoxy^T^nethoxyisoflavone, 25). The compound 25 was acetylated and the bioactivity of acetylated formononetin (26) was also evaluated.
10. (±)-medicarpin
24. (-)-claussequinone
25. R = OH, formononetin 26. R = OAc
Biological activity The bioactivity of compounds 10 and 24-26 were evaluated by particle method. The possible combinations of these four compounds were also tested. All these four compounds (10 and 24-26) showed different activities to the motility of the zoospores. (i)-Medicarpin (10) showed repellent activity at 150 |ug/ml, while (-)-claussequinone (24) and formononetin (25) showed stimulating and attracting activities at 100 and 50 |Jg/ml, respectively. Mixture of these three isoflavonoids (1:1:1, w/w) exhibited repellent activity at 50 |ug/ml. The repellent activity of (i)-medicarpin (10) was enhanced in the presence of 24 and 25. Compounds 10, 24 and 25 are known to be antimicrobial as well as bioregulating in human physiology [96-98]. The negative chemotaxis of isoflavonoids may be interesting because a plant flavone, cochliophilin A (3), is a host-specific plant signal for A. cochlioides. Medicarpin has been found as phytoalexin in many legumes [99]. Antimicrobial activities of these three isolates (10, 24 and 25) have been reported but the repellent activity of those isoflavonoids toward zoospores has not been claimed. Negative chemotaxis of zoospores from isoflavonoids those were found in many plants, raises questions on the occurrence of this phenomenon particularly during plant/parasite interactions.
1084
Motility Inhibitory and Zoospore Lytic Factors from Unripe Ginkgo Fruits The maidenhair tree, or Ginkgo, is a gymnosperm that has been described as a 'living fossil' because it is known to have existed early in the Jurassic period 170 million years (Myr) ago [100]. It is one of the most important medicinal plants that received a considerable interest [101-103]. During the survey of physiologically active secondary metabolites in traditional medicinal plants toward zoospores of A. cochlioides, we observed that the EtOAc soluble extracts of unripe Ginkgo biloba fruits induced potent motility inhibition followed by lysis of the zoospores. Anacardic acids in the Ginkgo extracts were found to responsible for such a characteristic biological activity [104]. Isolation of the active principles along with their biological activities toward zoospores is discussed here. Isolation of anacardic acids, cardol and cardanol
The whole unripe Ginkgo fruits (15 kg) were extracted with MeOH (10 1) and the MeOH extract was concentrated in vacuo to remove the solvent. The resulting aqueous solution was diluted with deionized water to 4 1 and extracted successively with n-hexane (4 1) and then EtOAc (4 1). The EtOAc solubles (ca. 70 g) were chromatographed on a silica gel (900 g) column and the constituents were eluted with a mixture of n-hexane and EtOAc (15:1 v/v) to yield 40 g of an anacardic acid mixture. A part of the anacardic acid mixture (1.5 g) was refluxed in cone. H2SO4-MeOH (1:20 v/v) followed by silica gel column chromatography to yield a mixture of methyl esters (975 mg) and a slowly eluting non-derivatizable constituent (99 mg). The mixture of methyl anacardates was applied to medium-pressure column chromatography using an ODS column eluted with 3% H2O/MeOH to give two major peaks (fr. 1 and fr. 2, 478 and 360 mg, respectively). The major component in fr. 1 was further purified by HPLC (Prep-ODS column, 1% H2O/MeOH, flow rate 5 ml/min, l i?-66.6 min) to yield 281 mg of first methylated product. From the latter fraction, two major peaks were separable by HPLC (Prep-ODS column, 3% H2O/MeOH) to give 280 mg of crude second and 60 mg of crude third methylated products, respectively as oils. The second and third methylated products were purified from each HPLC fraction by repeated HPLC (100% MeOH, flow rate 5 ml/min) as colorless oils (180 and 24 mg, respectively). Methylated products (1st, 2nd and 3rd) thus purified were separately hydrolyzed in MeOH-aq. 2 M KOH (1:1) and the resulting products purified by preparative TLC (n-
1085
hexane-EtOAc-HCOOH, 14:2:1 v/v/v) to yield 27, 28 and 29, respectively in good yields. 21:l-io -Cardol (30) containing fraction (66 mg) eluted from the first silica gel column followed after the anacardic acid mixture was purified by preparative TLC in CHCl3-EtOAc-HCOOH (60:10:3, v/v/v, Rf 0.27, 35 mg). 21:l-w7-Cardanol (31) found as the unchanged constituent (99 mg) in the methylation reaction mixture from 1.5 g of the crude anacardic acid mixture was separated by preparative TLC (Rf 0.65 in CHCl3-EtOAc-HCOOH = 60:10:3), and finally purified by HPLC (Prep-ODS column, 3% H2O/MeOH, '/J-53.3 min) to give 40 mg of a colorless syrup. The structures of all derivatives were confirmed by spectroscopic methods [104].
27: R r
, R2=R3=H, (22:lco7-anacardic acid)
32: R r
, R2=H, R3=Me
28: Rj=
, R2=R3=H, (24: Ito9-anacardic acid)
29: Rj=
, R2=R3=H, (22:0-anacardic acid)
R
CF,
30:R 1 =OH(21:lco 7 -cardol) NO 2 7
31: R r H (21:lco -cardanol)
33: fluazinam
Biological activities of anacardic acids and related compounds
In homogeneous solution method [105], the active EtOAc solubles of unripe Ginkgo fruits exhibited lytic activity at a range of 0.1-1 |ag/ml
1086
toward A. cochlioides zoospores. After adding the sample solution into the zoospore suspension, cells quickly became immobile or moved in an unusual circular fashion for a few minutes and then halted. Initially the halted zoospores became round-shaped spores by losing their flagella and part of them was burst gradually. Relatively high motility inhibition and lysis-inducing activities against A. cochlioides zoospores were expectedly observed in 22:l-oo72-O-methylanacardic acid (32) and 22:l-w7-anacardic acid (27) as with a reputed fungicide fluazinam (33) [106]. 21:l-o:7-Cardol (30) and 24:l-co9-anacardic acid (28, a homologue of 27) were a little less active. Among the studied compounds, the halting and lytic activities were observed in decreasing order of 33 > 32 > 27 > 30 > 28 > 29. Compound 27 at 10~6 M caused 96% motility inhibition in 20 min and 67% lysis of the zoospores within 3 h. To achieve similar lytic activity, relatively longer time (6 h) was required for 30 and 28. In contrast, 22:0-anacardic acid (29 with a saturated 27 at the aliphatic side chain) exhibited relatively a weak activity and 21:1-w7-cardanol (31, a decarboxylative product of 27) was thoroughly inactive up to 10"4 M. Table 4. Motility Inhibitory and Zoospore Lytic Activities Induced by Ancardic Acids, Related Compounds and a Synthetic Pesticide Tested compounds*
Motility inhibition (%)
Lysis (%)
10 min
20 min
30 min
60 min
120 min
27
75
98
100
31
47
68
28
55
72
99
24
34
43
29
36
54
99
4
14
28
30
56
82
99
28
39
54
32
80
100
100
36
51
72
40
58
79
0
0
0
33
84
100
100
Control
2
4
7
ISO min
Final concentration of each compound in zoospore suspension was 10 M. Adapted from reference [104]. *The number of tested compounds is corresponding to that in the text.
To understand the mode of action, we examined the morphological changes of zoospores interacting with anacardic acid (27) (Fig. 7). Time-course scanning electron microscopic observation revealed that anacardic acid (homogeneous solution method, 5xlO"5 M) first damaged fine hairs of flagella and thus halted the zoospores within few min. The affected zoospores immediately turned into a nearly
1087
round-shaped spores (10 min after treatment) leaving or after complete destruction of one or both flagella. The immobile round spores appeared to be dehydrated and squeezed within 20 min after treatment (Fig. 7a). The membranes of dehydrated spores ruptured at a single point through which the cellular materials gradually released into water (Fig. 7b, c). Finally, all inner materials of the affected cells were came out and dispersed into water within 60 min (Fig. 7d).
a
b
r
Q
liun
Fig. (7). SEM micrographs showing lytic activities of 22:l-co7-anacardic acid (27) at 5xl0' 5 M [104]. Circular objects in the background are pores (size: 0.6 |jm) of SEMpore membrane.
Structural requirements for motility inhibition and lysis activities of anacardic acids and related compounds were briefly studied by testing several derivatives of anacardic acids and related compounds [104]. In respect of structure-activity correlation, active compound possessed common part structures, an aliphatic side chain with one olefinic bond and a carboxy group on the aromatic ring, which are likely to be necessary to show the activity. Structural modification of 27, for example 2-O-methylation (32) could improve their activities. Cardol (30) having no carboxyl group, but two hydroxyl groups on the aromatic ring also exhibited noticeable antizoosporic and antibacterial activities, whilst the content of 30 in the crude extract of Ginkgo fruits was very small (<0.2%). However, at present we have no structural information what contributes to such biological activities of 30. In particular, the quantitatively major compound 27 in the EtOAc solubles of Ginkgo fruits revealed to be a predominant factor in quality for the motility inhibitory and zoospore lytic properties of the Ginkgo metabolites. Two classes of Ginkgo constituents, anacardic acids (27 and 28) and cardanol 30, both possessing significant lytic activity toward A. cochlioides zoospores also exhibited growth inhibition of Bacillus subtilis in a paper disc bioassay method. These results indicated the presence of a certain link between the lysisinducing activity to oomycete zoospores and antibacterial activity against B. subtilis [104,107]. Further understanding of the mode of action of anacardic acids and related compounds against oomycete zoospores should provide important knowledge required for the biorational control of the notorious soil-borne zoosporogenic phytopathogens.
1088
Zoosporicidal Polyflavonoids from Lannea coromandelica Lannea coromandelica L. (Anacardiaceae) is a deciduous tree widely distributed in Bangladesh, India and some other tropical countries. Plants belonging to this genus are used in folk medicine for treatment of elephantiasis, impotence, ulcers, vaginal troubles, halitosis, heart disease, dysentery, gout and rheumatism [108]. Stem bark extracts of L. coromandelica exhibited potent zoospore motility inhibitory activity followed by characteristic lysis. Identification of active principles and their mode of action toward zoospores are briefly discussed here. Isolation of the factor responsible for motility inhibition and lysis of zoospores
The MeOH fraction of acetone extracts of Lannea stem bark showed bioactivity, and hence subjected to bioassay-guided fractionation by different column chromatography including SiCh gel, Sephadex LH-20, and RP-18 CC. However, none of the chromatographic techniques were found suitable for separating the active factor(s) due to its high polarity and complex behavior in chromatography. In course of chromatographic studies, five inactive dihydroflavonols were identified and their structures were elucidated by spectroscopic methods, where two of them were new natural products [109]. The active fraction (MeOH solubles from the 80% acetone extracts) was a tan colored amorphous powder, soluble in 80% aqueous acetone and gave a sharp UV absorption maximum at 280 nm (in MeOH). The FD-MS and FAB-MS were found ineffective to get information of the molecular weight of bioactive constituents in the column fractions. *H NMR of the bioactive SiO2 gel column fractions or MeOH solubles gave very broad peak at the aromatic region. The 13C NMR also gave some broad peaks at 5 25-85 ppm and 96-160 ppm indicating the presence of polyflavonoid tannins in the bioactive fractions [110]. Based on the physicochemical properties including *H and 13C NMR data of the active fractions, we assumed that the active principle in Lannea extracts might be a mixture of complex polyflavonoid tannins, which was highly stable in hydrolysis. To get definite evidence, we tested the activity of two popular commercial tannins (Quebracho and Mimosa) in our bioassay. Interestingly, 80% acetone extracts of both commercial tannins showed identical halting and lysis activities and supported our assumption that active principle in L. coromandelica extract is also polyflavonoid tannins.
1089 Characterization ofLannea coromandelica polyflavonoid tannins by MALDI-TOF-MS
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has recently been successfully used in determination of aspects of the structure and characteristics of the polyflavonoid tannins, which are too difficult to determine, by other technique [111]. We applied this method to determine the structural features of Lannea tannin along with known commercial Quebracho tannin [111].). The Quebracho tannin gave clear spectrum showing the degree of polymerization of the building units and oligomer series with masses of the repeat units of 272.3 and 288.3 Da [111]. The predominant repeating units in this tannin are 272 Da, indicating that this tannin is predominantly consisting of profisetinidin-type unit. The flavonoid repeating units present in the polyflavonoid tannins could be A, B, C and D having masses of 258.3, 274.3, 290.3 and 306.3 Da, respectively (Fig. 8). Combinations of these masses can be used to calculate the masses of the profisetinidin/prorobinetinidin-type of polyflavonoid tannin oligomer peaks in the spectra according to the expression, M + Na+ = 23.0 (Na) + 2.0 (endgroups, 2 x H) + k(256.3A) + 1(272.3B) + m(288.3C) + n(304.3D) (k, 1, m, n, are integral numbers) [111]. As can be seen in the spectra, there are more peak series, which are due to different endgroups. They have the same repeat units, for example, 586-314 and 1450-1178 Da in Fig. 9. MALDI-TOF-MS analysis of Lannea extracts gave clear spectra exhibiting the degree of polymerization of the building units and oligomer series with masses of the repeat units of 256.3, 272.3, 288.3 and 304.3 Da (Fig. 9). For each oligomer, substructures with mass increments of 16 Da appear, suggesting different combinations of various substructures [111]. The MALDI-TOF-MS analysis also indicates the presence in the tannin of oligomers to the maximum of nonamer (2506.3 Da). Interestingly, a monomer peak at 314.2 Da is composed of one C type unit plus 2H endgroups plus Na+. The peak at 458.2 Da is obtained from the 568.2 Da dimer by elimination of a catecholic B-ring (568.2-110 = 458.2). Similarly, a major peak at 552.2 Da is also explained by the presence of a dimer composed of an A-unit plus a B-unit plus 2H endgroups plus Na+. There are however, some cases in which unequivocal assignment of the structure can indeed be done. This is the case of angular tannins, namely oligomers in which a repeating unit of type D is bound through both its 6 and 8 A^ing
1090 (OH)
HO (OH)
OH
OH (OH) A: B: C: D:
Flavan-3-ols repeating unit
R, R, R, R,
= R2 = R3 = H and R 3 = H, R 2 = OH = R 2 = OH, R 3 = H = R 2 = R 3 = OH
OH
OH
OH A possible linear type trimer (M + Na = 809.9) (central building unit shows linear-type substitution)
HO
OH HO
OH
R = H or OH
A possible angular-type trimer (M + Na = 905.9) (central building unit shows angular-type substitution) Fig. (8). Possible structural units (A-D) of Lannea coromandelica (stem bark) polyflavonoid tannins, and structures of typical linear-and angular-type trimers.
35000-
30000-
25000^
20000-
15000-
10000-
500
1000
1500 Mass (m/z)
2000
2500
Fig. (9). MALDI-TOF mass spectrum of Lannea coromandelica extract. Insets showing expanded form of some important parts of the MALDI-TOF-MS spectrum [113]. For each oligomer, subsructures with mass increments of 16 Da appear, indicating different combinations of various substrutures.
1092
sites to B and C type units, with its C4 sites equally bound and unbound [111]. The MALDI-TOF-MS analysis shows the existence of fragments of angular tannins by the presence of definite peaks at 904.1, 1178.3, 1194.3 and 1211.5 Da in Lannea tannin extract (Fig. 9). Thus the Lannea tannin is angular-type one which is partly similar to Mimosa but rather different from Quebracho [111]. The predominant repeat units in Lannea tannin are 288 Da, indicating it to be predominant prorobinetinidin-type polyflavonoid tannin. Although the presence of a high proportion of phlobatannin in L. coromandelica stem bark has been reported earlier [112], but so far, report on the structural characterization of Lannea tannin has not been published. Therefore, it is the first report on the structural features of the polyflavonoid tannin present in L. coromandelica [113]. Motility inhibition and lytic activities of polyflavonoid tannins against zoospores
Light microscopic observation revealed that the Lannea extract and commercial ployflavonoid tannins show zoosporicidal activity against A. cochlioides almost in similar manner. In all cases, initially zoospores were halted and the cellular materials rapidly fragmented and formed globular structures. These globular structures achieved Brownian movement, and finally dispersed into the surrounding water medium by bursting the cell membranes within 60 min. The motility inhibition and lytic activities of Lannea and commercial polyflavonoid tannins are presented in Table 5. It appeared that both Lannea extracts and commercial tannins caused motility inhibition followed by lysis of zoospores in a dose dependent manner at a range of 0.1-50 |ig/ml concentration (Table 5). Among the three extracts, Lannea showed the higher halting and lytic activity (MIC 0.1 fig/ml) than the two commercial tannins extracts (both MIC ca 0.5 ug/ml). Both halting and lytic activity was increased with time and the highest activity was achieved within 60 min of treatment. When previously encysted spores (cystospores) were exposed to 5 (Jg/ml of Lannea or commercial tannin extracts, they were deformed and did not germinated or regenerated zoospores even after 12 h seemingly being killed. Methylation or acetylation of Lannea extracts yielded completely non-active products (inactive at 100 fig/ml) indicating that hydroxyl groups in the polyflavonoid tannin may be essential for motility inhibition and lytic activity.
1093 Table (5). Zoosporicidal Activity of Polyflavonoid tannins. Polyflavonoid
Dose
Zoosporicidal Activity (%)*
Tannin
(ug/ml)
M.I.a
C.L.b
Lannea
0.1
25
7
0.5
68
28
5
100
57
50
100
89
0.5
40
10
5
79
35
50
100
60
0.5
45
15
5
81
39
50
100
71
-
9
0
Quebracho
Mimosa
Control
Adapted from [113]. aM.I, motility inhibition; C.L., cell lysis; * Activity recorded 60 min after treatment. Quebracho and Mimosa tannins showed no activity at 0.1 |ug/ml.
Morphological changes of zoospores interact with polyflavonoid tannins
Time-course SEM observation revealed that both Lannea extract and commercial polyflavonoid tannins caused lysis of zoospores in a similar manner (Fig. lOa-h). It appeared that tannin extracts first attacked the tripartite tubular hairs (TTHs) (characteristic hairy structures responsible for swimming) of the anterior flagellum as well as the fine structures of the posterior flagellum (responsible for swimming) [10]. The tannins reacted with TTHs, and precipitated them within 5 min (Fig. 10a). Thus, zoospores became paralyzed and rapidly halted. The surface of the affected zoospores became relatively smooth, rounded and rapidly burst (Fig. lOb-d). The inner cellular materials of the spores fragmented and formed unique globular structures and soon dispersed into surrounding
1094
Lysis of zoospores by Lannea tannins
Lysis of zoospores by Quebracho (e-g) and Mimosa tannins (h)
Fig. (10). Scanning electronic micrographs showing zoosporocidal activity of Lannea coromandelica (a-d) (5 Hg/ml) and two commercial polyflavonoid tannins (10 ng/ml) toward zoospores of Aphanomyces cochlioides [113]. (a) fine structures of the flagella of zoospores are precipitated (5 min), (b, c) a zoospore burst after fragmentation of cellular materials (30 min), (d) characteristic fragments of lysed zoospore (60 min), (e-g) characteristic zoosporicidal effects of Quebracho tannin (30 min), (h) typical zoosporicidal effects of Mimosa tannin (30 min). Circular objects in the background are pores (size 0.6 pm) of SEMpore membrane.
Medium within 60 min (Fig. lOd). Zoosporicidal activities of other two commercial tannins, Quebracho and Mimosa were similar to that of Lannea tannin. In contrast, 22:lto7-anacardic acid (27) isolated from the Ginkgo biloba caused shrinkage of zoospores followed by bursting at a single point of each spore without forming any characteristic fragmentation of cellular materials [104]. The morphological changes (fragmentation of cellular materials and formation of unique structures) of zoospores by polyflavonoid tannins observed in this experiment are similar to the characteristic features of apoptosis [114]. Characteristics fragmentation of nuclear DNA was also observed in tannin induced spores by fluorescence microscopy [36]. However, no clear ladders of DNA fragments were found in agarose gel electrophoresis of DNA extracts of tannin affected zoospores. Recently, gallotannin was found to induce apoptosis in a human colon cancer line (T84) at 10 ng/ml [115]. The cystospores induced by mechanical agitation or host plant signal also affected by the extract where they completely deformed and cracked down but no clear fragmentation of cellular materials was observed. Tannins are secondary metabolites distributed widely in the plant kingdom, which have been closely associated with plant defense mechanisms towards phytopathogens, insects and mammalian herbivores
1095
mainly due to their properties of denature proteins. Recently, direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep have been demonstrated [116]. Kiuchi et al. (1988) also found that tannins, both condensed and hydrolyzable, caused bursting of the second-stage larvae of dog roundworm (Toxocara canis) [117]. Lannea stem bark contains high proportion of polyflavonoid tannins (ca. 13%) and, thus raises a possibility of using those naturally occurring compounds as a zoosporicidal agent. To the best of our knowledge, this is the first report of zoosporicidal activity of natural polyflavonoid tannins against an oomycete phytopathogen [113]. Further studies on the zoosporicidal mode-of-action of polyflavonoid tannins and their effects on other phytopathogenic oomycetes are needed for considering their practical use as a naturally occurring oomicidal agent. Regulation of Developmental Transitions of Zoospores by Amaranthus gangeticus Metabolites Amaranthus gangeticus (Amaranthaceae) is a popular leafy vegetable in Bangladesh and some other tropical countries which is rich in vitamins and minerals. Some members of Amaranthaceae, for example, A. retroflexus, A. blitoides, Celosia cristata and Gomphrena globosa are reported to be the hosts of A. cochlioides [119]. During the screening of plants extracts using A. cochlioides, we observed that extracts of a nonhost A. gangeticus caused attractant followed by sudden inhibition of motility (halting) of zoopores. Bioassay-guided chromatographic separation of A. gangeticus constituents revealed that the taxis and subsequent motility inhibition of zoospores were regulated by the cumulative effects of two chemically different factors. The attractant was identified as iV-rrans-feruloyM-O^nethyldopamine (6) (our unpublished data), and the motility-inhibiting factor as nicotinamide (34) [105]. OH O
OCH,
NH,
OCH3 6. iV-fra/w^eruloyM-O^ethyldopamine
34. nicotinamide
1096
Bioassay by particle method revealed that compound 1 showed attractant activity up to lxlO"8 M concentration in a dose dependent manner without halting motility of zoospores even at a very high concentration (lxlO 5 M). The direct application of compound 6 as a homogenous solution had also no effect on the motility of zoospores. On the other hand, nicotinamide (34) showed immediate halting activity followed by encystment in both particle (MIC lxlO 5 M) and homogenous solution methods (MIC 2X10"8 M). Interestingly, cysts produced by 34 regenerated zoospores (85-90%) instead of germination within 2-3 h in homogeneous solution method or only 20-30 min (ca 90%) in particle method. However, concomitant application of compounds 6 (10 M) and 34 (10~5 M) showed encystment of zoospores followed by germination (100%) of cystospores within 30-35 min (particle method). Scanning electron microscopic (SEM) observation revealed that nicotinamide induced cysts regenerate zoospores leaving their smooth cyst coat whereas cysts produced by the concomitant application of compounds 6 and 34 germinated as shown by host-specific plant signal, cochliophilin A (3) [36]. When an excised root of a 6 days old seedling of A. gangeticus was immersed into the zoospore suspension in a small Petri dish, all zoospores around the root tip were immediately halted. The halted spores encysted and then regenerated after 3 h indicating the possibility of exudation of nicotinamide (34) predominantly from the root of A. gangeticus. On the other hand, zoospores were specifically attracted to the root cap regions of Celosia cristata (a host), aggregated and encysted to form a mass of cystospores within 30 min. Almost 100% cysts were germinated within 40 min instead of regeneration. However, different levels of motility inhibition followed regeneration zoospores were commonly observed in case of all nonhost species in Amaratnaceae. Preliminary studies (TLC examination of root extracts and bioassay) revealed that all nonhost roots (Amaranthaceae) contain high proportions of nicotinamide (34). In contrast, C. cristata (host) roots contain high proportion of iV-frans-feruloyM-O^ethyldopamine (6) with low amount of nicotinamide (34). It appeared from the results that the ratio of 6 and 34 in the root exudates of Amaranthaceae might determine the compatibility of pathogen to host. Therefore, studies on the contents of N^A•a/^5-feruloyl-4-O^nethyldopamine (6) and nicotinamide (34) in host and nonhost members of Amaranthaceae, and their exudation from the roots may clarify the ecochemical roles of these two secondary metabolites in plant-pathogen compatibility. Motility-inhibiting activity of nicotinamide (34) has been first observed against the zoospores of A. cochlioides. However, a further test using some
1097
kinds of zoospores revealed that both A. cochlioides and A. euteiches showed high sensitivity to nicotinamide (34) (5 x 1CT7 M), while the zoospores of Py. aphanidermatum strain 71-81, Py. aphanidermatum strain 72-22, and Ph. infestans strain Pio-761-S exhibited a weak response to nicotinamide at 4.1 x 10"* M, 1.5 x 10~5 M, and 1 x 10"1 M, respectively [105]. Table 6. Concentration of Nicotinamide (34) to Inhibit Motlity of Peronosporomycetes (Oomycetes) Zoospores [105]. Species
Concentration
Ahanomyces cochlioides
0.1
A. euteiches
1
Pythium aphanidermatum strain 72-22
82
Py. aphanidermatum strain 71-81
410
Phytophthora infestans strain Pio-761-S
410000
*Concentration of nicotinamide in the zoospore suspension.
Yamashita and co-workers reported that nicotinamide exhibit attaching repellent activity against the blue mussel (Mytilus edulis) [119]. Nicotinamide (34) is a product of NAD+ cleavage by mono (ADP^ibosyl) transferase, and it serves as an effective inhibitor of the enzyme activity [120]. It was also observed that nicotinamide (34) directly inhibited vascular smooth muscle cell contraction, which was suggested to act via blockage of external Ca2+ entry or release of Ca2+ from intracellular stores [121]. Ca2+ ion is also critical for motility and differentiation of zoospores, therefore, zoospores motility halting activity of nicotinamide (34) may be related to the blockage of Ca2+ influx/efflux in zoospores. Nicotinamide (34) and structurally related 51 compounds were subjected to the halting activity bioassay to elucidate the structure-activity relationships [105]. The highest activity was recorded in thionicotinamide (35) followed by pyrazinamide (36) and nicotinamide (34). Nicotinamide adenine dinucleotide (NAD) (oxidized form), nicotinamide adenine dinucleotide phosphate (NADP) (oxidized form), |3 nicotinamide mononucleotide (oxidized form) showed halting activity at ca. 10"7 M
1098
concentration. Other compounds showed weak halting activity while some others (37-42) were inactive at 10~2 M concentration (Fig. 11). Active at shown concentration
NH2
8.2 x 10"8 M
7.2 x 10"9 M
M
Inactive at 1 x 10 M concentration
Fig. (11). Structures of nicotinamide related compounds and their threshold concentrations for showing halting activity against Aphanomyces zoospores.
None of the tested compounds exhibited any antagonistic effects against the motility halting activity of zoospores by natural nicotinamide. The structure-activity relationships among nicotinamide derivatives and structurally related compounds are summarized as follows: (a) the aromatic ring containing at least one nitrogen atom, (b) carbonyl-like group adjacent to the aromatic ring and (c) hydrogen atoms on the amide group are responsible for the strong activity. Thus the structural requirements for the zoospore halting activity are quite less versatile.
1099
Repellent Activity of Mammalian Sex Hormones, Phytoestrogens and Xenoestrogens One of the remarkable biological activities of isoflavonoidal compounds has been recognized as an estrogenic one. The fact that a pterocarpan, medicarpin (10) exhibited repellent activity toward Aphanomyces zoospores prompted us to investigate the effects of estrogenic compounds toward zoospores. We also found that a reputed xenoestrogen [122], Bisphenol A (BPA) (43) exhibited potent repellent activity against the zoospores of A. cochlioides. Based on these finding, we tested a number of xenoestrogens, mammalian estrogens and androgen, and some natural estrogenic mimics, for example, diethylstilbestrol (DES) (44), 17ot-estradiols (45), 17(3estradiol (46), estrone (47), estriol (48), dienestrol (49), pregnenolone (50), progesterone (51), testosterone (52), mono- and di-O-methyl ether of DES (53, 54)and some natural estrogenic mimics, for example, miroestrol (55), formononetin (25), naringenin (56), and three prenylated naringenins (9, 57, 58) on the motility behavior of A. cochlioides zoospores [123]. Most of the estrogenic and androgenic substances exhibited a repellent activity against the motility of the zoospores, whereas, progesterone (51) and the natural mimic miroestrol (55) were practically inactive at 1000 ug/ml. The highest activity was recorded in DES (44), 17p-estradiol (46) and estriol (48) (active at 0.5 ug/ml), followed by 17cc-estradiol (45), estrone (47), dienestrol (49) (active at 1.0 ug/ml), testosterone (52) (active at 50 ug/ml) and pregnenolone (50) (active at 100 ug/ml). It revealed that most of the active estrogenic compounds except pregnenolone (50) showed higher repellent activity than that of an androgen, testosterone (52) The xenoestrogen, bisphenol A (43) showed clear repellent activity at 5 ug/ml under the same bioassay condition (particle method). In the present study, the estrogenic and repellent activities of known estrogenic compounds revealed to be correlated. When we examined for information of receptor, the receptor for estrogenic repellents seemed not to be affected directly by attractants (e.g. cochliophilin A, 3), because the repellent activity of estrogens was observed in the zoospores suspended in the homogenous solution of host^specific cochliophilin A. Our structure-activity studies revealed that aromatization of the A-ring with a free hydroxy group at C-3 position of a steroidal structure is necessary for higher repellent activity [123]. Surprisingly, methylation of diethylstilbestrol (DES) yielded completely different activity i.e. both mono- and di-O-methyl ethers of DES (compounds 53 and 54) showed attractant activity. Moreover, the attracted zoospores were found to be
1100
Activities of xenoestrogens/environmental pollutants
HO
H3CH2C -
43 Bisphenol A (repellent, 0.5 ng/ml)
=
=
^4: Ri R2 OH, Diethylstilbestrol (repellent, 1 |ug/ml) i = 0 H > R2 = 0 C H 3 (attractant/stimulant, 5|ug/ml) 54: R[ = R2 = OCH3 (attractant/stimulant, 1 |ag/ml)
53: R
Activities of mammalian sex hormones (estrogens and androgen) OH
H
HO 45: 17oc-Estradiol (repellent, 1 ng/ml) 46: 17p-Estradiol (repellent, 0.5 pg/ml) 0
OH
HO 47: Estrone (repellent, 1 ng/ml)
48: Estriol (repellent, 0.5 |Jg/ml)
H3C HO
49: Dienestrol (repellent, 5 |ig/ml)
50: Pregnenolone (repellent, 100 )H
51: Progesterone (inactive, 1000 ng/ml) 52: Testosterone (repellent, 50 |ig/ml)
1101 Activities of natural estrogenic mimics (phytoestrogens) H2
,OH
o O
55: Miroestrol (inactive, 1000 ug/ml)
a
25: Formononetin (attractant/stimulant, 50 |ag/ml)
0H
56: naringenin (repellent at 10 ug/ml)
9: 8-prenylated naringenin (repellent at 1 ug/ml) OH
HO
57: 6-prenylated naringenin (attractant at 10 |ag/ml)
58: 3'-prenylated naringenin (repellent at 10 pg/ml)
encysted and then germinated in the presence of di-O^nethyl ether of DES (54). Similarly, 8-prenylated naringenin (9), which is known as a potent phytoestrogen [124], also exhibited equivalent repellent activity (active at 1 |ug/ml) as shown by 17|3-estradiol (46), whereas, 6-prenylated naringenin (57) displayed attractant activity at 10 |ag/ml. Both naringenin (56) and 3 ' prenylated naringenin (58) exhibited repellent activity at 10 |Jg/ml concentrations, whereas, another natural estrogenic mimic, formononetin (25) had attractant/ stimulant activity (50 |ug/ml). It may be interesting for further investigation how minor modifications in the structure of DES or prenylated^iaringenin completely change the property of biological activity.
1102
So far, the repellent activity of estrogenic compounds toward trivial oomycete zoospores has not been reported [123]. The major mammalian sex hormones (both androgens and estrogens), like 17p-estradiol (46), estrone (47), estriol (48), testosterone (52) have been isolated from several higher plants [99,125-127]. Basically, steroid hormones are a group of substances derived from cholesterol which exert a very wide range of effects on biological processes such as growth, metabolism and sexual differentiation [128]. However, our current knowledge of their effect on non-mammalian biological systems like microorganisms is limited. Both growth-inhibiting and growth promoting effects of steroidal hormones have been observed by Fitzgerald and Yotis [129], but more interestingly, testosterone (52) and 17|3-estradiol (46) have been found to have sex hormone activity on yeast [130]. An insect repellent steroid was isolated from the Peruvian weed Nicandra physalodes [131]. Antheridiol (59) has been identified as the chemotactic hormone of the water mold, Achlya bisexualis (Saprolegniaceae) [132]. A steroid-binding protein (steroid receptor) has already been isolated from the A. ambisexualis [133].
O
HO
59: antheridiol Heftmann et al. observed that a structurally related sterol of antheridiol, 5astigmast-22-en-3P-ol, which is produced by Dictyostelium discoideum, triggers the remarkable differentiation, which this slime mold undergoes [134]. 17p-Estradiol (46) was found to stimulate hyphal growth of endomycorrhizal fungus, Glumus intraradices [135]. Endocrine disrupters {e.g., bisphenol A) are supposed to be the pollutants in our environment and pose a serious concern in human health. The minute amount of these compounds in our environment is difficult to detect mainly due to lack of a simple and sensitive bioassay method. In the present study, the estrogenic and repellent activities of known estrogenic compounds revealed to be correlated. The particle bioassay method is very simple and
1103
convenient for testing the motility behavior of fungal zoospores. Thus, the particle test appears to be a useful method for detecting repellents as estrogens from the natural sources or for pre-screening the detection of estrogenic activity in the environmental samples. Therefore, it may be important to carry out further work to evaluate the usefulness of this repellent test for the bioassay-guided isolation of environmental pollutants or phytoestrogenic compounds. Furthermore, high repellent activity of mammalian sex hormones towards fungal zoospores may be biologically very interesting because such high negative chemotaxis was not yet reported for any zoosporic phytopathogen. It may be important for biorational control of oomycetes and/or in studying the molecular basis of chemoresponses of zoosporic plant pathogens. Most of the mammalian estrogens have also been reported in plants. However, none of them has been found in the known hosts of A. cochlioides. The mechanism of high repellent activity by mammalian sex hormonal substances on oomycete zoospores is difficult to explain by our current knowledge. This negative chemotaxis raises questions on the occurrence of this phenomenon particularly during early stage of plant-parasite interactions, and the speculation that minor constituents of phytoestrogens might contribute to defense of non-host plants against pathogens. Bioactivities of Some Other Natural Products Including Microbial Metabolites toward Zoosporic Phytopathogens Plant secondary metabolites
The crude extract of a Chinese herbal medicine, Akebia quinata displayed potent stimulant/attractant activity toward A. cochlioides zoospores. Bioassay-guided investigation revealed that the active principles are the mixture of N-trans and cw^"eruloyltyramine (unpublished). Several lignans were isolated as the repellent factors for A. cochlioides zoospores from the ripe fruits of Magnolia kobus (unpublished). Similarly, Jensen et ah, (1998) isolated a major flavone glycoside (60), from the marine angiosperm Thalassia testudinum which effectively inhibits fouling and growth of parasitic fungal zoospores (Schizochytrium aggregation) [136].
1104 OH ,OH
HO OH
O
60: luteolin 7-O-$-D-glucopyranosyl-2"-sulfate (attachment deterrant)
This marine plant is usually suffered from periodic infections caused by S. aggregatum and other zoosporic fungi. However, the whole leaf concentration of the flavone glycoside reaches 4 mg/g wet tissue, which is more than sufficient to reduce growth of the above fungus by 50%. The fact that the flavone is present in a water-soluble form as a sulfate suggests that it may also be excreted from the plant to ward off fouling fungal zoospores in the marine environment [137]. Saponins are known to act as constitutive plant toxins against microbial pathogens. Although oomycetes can not synthesize sterols but they contain sterols acquired from the growing media/victim hosts which is important for their sexual and asexual reproduction. Thus zoospores are also sensitive to saponins. For example, avenacin (61), from the roots of oat (Avena sativa) and the grass Arrhenatherum elatius caused lysis of several species of oomycete zoospores namely, Allomyces arbuscula, Aphanomyces sp., Ph. cinnamomi, Py. aphanidermatum, Py. arrhenomanes, P. graminicola, Py. intermedium, Py. ultimum var. sporangiferum and Saprolegnia litoralis [138].
O
HO
61: avenacin A-l (Zoospore lytic factor)
NHMe
1105
When oat roots were used for bioassay, zoospores were first attracted and then caused lysis which raises the possibility that oat crops and their residues might be used to reduce soil populations of zoosporic plant pathogens. The cystospores were insensitive to concentrations of saponins or oat root extract that lysed zoospores. The zoospores lytic principle in oat root extract/exudate was believed to be a saponin, avenacin (61). The effects of avenacin (61) or root extracts on zoospores were identical to those of /?-aescin and consistent with a role of membrane active agents. Toxic effects of saponins on zoospores were also observed in Pseudoperonospora humuli Miyabe & Takah. and Phytophthora spp. [139,140]. A saponin, 3-O-[P^D"glucoPyranosyl(l ~* 6)-p^-glucopyranosyl]-20-OP^)-glucopyranosyl-3p, 12p, 20 (S)-trihydroxydammar-24-ene (62) isolated from a non-host Chinese traditional medicinal plant, Panax notoginseng caused motility inhibition and subsequent death of A. cochlioides zoospores [141].
HO
HO
OH
62: notoginsenoside K (motility inhibitor)
The antimitotic natural product, taxol was found to show toxicity toward the germinating zoospore cysts of the oomycetes Ph. capsici and A. cochlioides [142]. The mechanism of action of taxol was shown to involve inhibition of mitosis, presumably resulting from an effect on disassembly of microtubules. The importance of the side chain was shown by much lower the activity as compared to taxol of analogues lacking all or part of the side chain. The effect of stereochemistry at the C-2' position on fungitoxicity towards oomycetes was similar to that reported in mammalian microtubule assembly. The other natural product shown growth inhibiting activity against oomycetes is pipernonaline (63) from Piper longum L. [143]. Mycelial growth inhibitory activity was shown by an alkaloid, sanguinarine (64) and a furan-containing diterpene (65) isolated from roots of Chelidonium majus L. var. asiaticum and Salidago gigantea (Aiton) var. leiophylla, respectively [79]. The threshold concentrations for compounds
1106
64 and 65 to inhibit mycelial growth of A. cochlioides were 0.25 and 2.5 jag per disc, respectively.
2
-
9
7
63: pipernonaline (mycelial growth inhibitor)
CHO
64. (mycelial growth inhibitor)
65. (mycelial growth inhibitor)
Oomycete phytopathogens can not synthesize sterols, but acquire them from the victims [144]. In a recent investigation, sitosterol (66), a predominant sterol in soybean shoots had direct positive effects on growth of mycelia and production of oospores in Ph. sojae. Interestingly, cycloartenol (67), a predominant sterol in seed showed harmful effects to growth and oospore production of the same organism [145]. These findings clearly demonstrate the importance of sterols in plant-oomycete interactions and offer a possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life-cycle [145]. A characteristic plant response to microbial attack is the production of endo-p-glucanases, which are thought to play an important role in plant defense, either directly, through the degradation of |3-l,3/l,6-glucans in the pathogen cell wall, or indirectly, by releasing oligosaccharide elicitors that induce additional plant defences. Recently, Rose and co-authors discovered that soybean pathogen Ph. sojae secreted a novel class of proteins during interaction with host plant, termed glucanase inhibitor proteins (GIPs) as the counterdefensive weapon [146]. GIPs specifically inhibit the endoglucanase activity of the plant host and potentially function as important pathogenicity
1107
determinants.
HO
66: sitosterol (growth regulator)
67: cycloartenol (growth regulator)
Antibiotics and conventional fungicides
Several rhizoplane microbes including bacteria showed antagonistic effects on the growth and life-cycle development of soil-borne oomycete phytopathogens. Their active mechanisms mostly involved in antibiosis and/ or competition in colonization in the host roots. For example, three antifungal antibiotics, designated xanthobaccins A (68), B, and C, were isolated from the culture fluid of Xanthomonas sp. strain SB-K88, a rhizobacterium of sugar beet that effectively suppresses damping-off disease [147]. About 3 jig of xanthobaccin A (68) was detected in the rhizosphere of each sugar beet plant when sugar beet seeds were inoculated with strain SB-K88.
ONa
68. xanthobaccin A (mycelia growth inhibitor)
1108
Direct application of purified 68 to seeds suppressed damping-off disease in soil naturally infested with Pythium spp. The macrocyclic lactam antibiotic, xanthobaccin A (68), which has a characteristic 5,5,6-tricyclic skeleton and tetramic acid chromophore [148], displayed mycelial growth inhibition of A. cochlioides, Py. ultimum, and Ph. vignae f. sp. adzukicola at a range of 0.110 (Jg/ml. Phomalactone (69), produced by the fungus Nigrospora sphaerica, was tested in vitro against nine plant pathogens, and specifically inhibited the mycelial growth of Ph. infestans, with an MIC value of 2.5 ng/ml [149]. Its inhibitory activities against sporangium and zoospore germination of Ph. infestans were similar to those against Ph. capsici. In vivo, at 100 and 500 |ug/ml, it reduced the development of tomato late blight caused by Ph. infestans. A glycolipid antibiotic, rhamnolipid B, 3-(3-[L-rhamnopyranosyl(1 -• 2)-a-L^-hamnopyranosyloxy]-decanoyloxy)-decanoic acid (70), isolated from Pseudomonas aeruginosa strain B5 showed lytic activity against zoospores of the late blight pathogen, Ph. capsici at 10 |ag/ml and displayed equivalent efficacy against phytophthora blight when pepper plants (grown in glasshouse) treated just before inoculation with Ph. capsici [150]. This microbial product also exhibited inhibitory activity against the germination of zoospores and hyphal growth of that oomycete at a higher concentration, 50 |Jg/ml. However, this compound had no effect on cystospores surrounded by cell wall. OH H,C,
OH OH
H,C
HOOC 69: phomalactone (mycelial growth inhibitor)
70: Rhamnolipid B (zoospore lytic factor)
1109
The rhamnolipid B (70) has the molecular structure of biosurfactants, comprising a hydrophilic part (rhamnose moiety) and a hydrophobic part (P-hydroxydecanoate moiety) [151]. The biosurfactant property of this glycolipid was supposed to confer the ability to intercalate into and disrupt the zoospore plasma membrane [152,153]. However, other glycolipids such as sophorolipids and trehalose lipids, which have chemical properties similar to the rhamnolipids, did not show zoosporicidal activity at concentrations up to 1000 |ug/ml. A unique chlorinated macrocyclic lactone, named oocydin A (71) was isolated from an epiphytic bacterium, Serratia marcescens on an aquatic plant Rhyncholacis pedicillata [154]. MIC of approximately 0.03 jag ml"1 was noted for oocydin A against vegetative growth of oomycetes like Py. ultimum, Ph. parasitica, Ph. cinnamomi, and Ph. citrophora. This interesting compound had either minimal or no activity against other fungi indicating it to be a selective oomicide. However, its effect on zoospores or oospores of oomycetes is unknown. Jesterone (73) and hydroxy^esterone (74) isolated from an endophytic fungus Pestalotiopsis jesteri exhibited growth inhibitory activities against oomycetes [155].
-O
CH, COOH O
71: oocydin A (mycelial growth inhibitor)
HO HO
72: jesterone (mycelial growth inhibitor)
7
3: hydroxy-jesterone (mycelial growth inhibitor)
1110
Some cationic lytic peptides, for example, MSI-99 significantly reduced germination of zoospores of Ph. infestans. In the leaf disk assay, pretreating spores of Ph. infestans with the peptide at concentration down to 10 |ug/ml prevented development of any late blight lesions on tomato leaf disk [156]. It was suggested that MSI-99 can be used as a trans gene to generate tomato lines with enhanced resistance to late blight disease of this crop. Forty-nine compounds including commercial pesticides and natural products were tested in vitro for oomicidal activity against hyphae and zoospores of the fish pathogenic oomycete A. invadans [157]. No compounds tested proved as malachite green, but some low-toxicity natural products like, coconut diethanolamide, propolis, neem seed extract, tea tree oil and D-limonene exhibited mycelial growth inhibition as well as halting activity against zoospores. A new class of fungicide (oxazolidinediones), famoxadone inhibited sporangial differentiation and zoospore release and caused lysis of zoospores within minutes. Doses in the order of 0.01 |ag/ml were sufficient to lyse zoospores of both Plasmopara viticola and Ph. infestans [106]. The nonprotein amino acid, /?-aminobutyric acid (BABA) protected Arabidopsis against the oomycete pathogen Peronospora parasitica through activation of natural defence mechanisms of the plant such as callose deposition, the hepersensitive response, and the formation of the trailing necroses [158]. Seed treatment with BABA also shown to protect Pennisetum glaucum (pear millet) systematically from the attack of Sclerospora graminicola [159]. Concluding Notes Results discussed above suggest that nonhost plants or rhizospheric microorganisms possess some 'chemical weapons' for affecting life-cycle development of soil-borne phytopathogenic oomycetes. Research on isolation of oomicidal constituents from the nonhost plants or nonpathogenic rhizoplane microorganisms would lead some interesting novel targets for designing biorational regulators against zoosporic phytopathogens.
1111 Table 7. Summary of Diverse Stimuli Triggering Characteristic Behavior or Morphological Changes toward Oomycete (Peronomycete) Zoospores (1) Host-specific plant signal triggering life-cycle development of Oomycete Phytopathogens a) indole-3-carbaldehyde (1) Aphanomyces raphani b) prunetin (2) A. euteiches c) cochliophilin A (3) A. cochlioides d) Feruloyldopamine (6) A. cochlioides e) daidzein (4) and genistein (5) Ph. sojae (2) Non-specific (general) attractants for oomycete zoospores a) amino acids b) carbohydrates c) alcohols d) aldehydes (valeraldehyde, isovaleraldehyde) e) fatty acids f) nonhost/synthetic flavonoids (e.g., synthetic methylated flavones are powerful attractants for A. cochlioides zoospores) (3) Inorganic and physical stimuli a) taxis toward gradient of H* or root released electric charges b) encystment by mechanical stimulation c) thigmotropism or contact sense (growing hyphal recognition to surface structure) (4) Repellents toward oomycete zoospores a) synthetic/nonhost flavonoid compounds b) environmental pollutants (e.g., bisphenol A, diethylstilbestrol) c) synthetic and natural mammalian estrogens d) phenylpropanoids (lignans) (5) Motility stimulants a) isoflavonoids b) N-trans or cis feruloyl-tyramine c) synthetic pesticides (6) Zoosporicidal substances a) antibiotics b) polyflavonoid tannins, anacardic acids from nonhost plants causing zoospore lysis (7) Motility inhibitors or encystment triggering substances a) nicotinamide and related compounds b) mastoparan c) l-linoleoyl-2-lysophosphatidic acid+N-trans -feruloyl-tyramine d) n -BuOH, sec -BuOH
CHEMICAL BASIS FOR THE HOST-SPECIFITY IN ANIMAL, FISH AND HUMAN PATHOGENIC OOMYCETES Several oomycete species, for example, Py. insidiosum, A. invadans, A. astaci, and Saprolegnia sp. are also cause of life-threatening infections in animals, fishes, and humans [160,161]. Although most of those organisms have limited host range, but chemical basis of their host-specificity is vastly unknown. Zoospores of those non-plant pathogens are also attracted to the
1112
potential infection sites of host, encyst, germinate and then initiate infection processes. Zoospores of the human and animal pathogenic oomycete, Py. insidiosum had a strong tropism for skin tissue of horse and human hairs. The accumulated zoospores favorably encyst and germinate on the host tissues [160]. Similarly, zoospores of the fish pathogenic fungus Saprolegnia diclina showed positive chemotaxis toward concentration gradients of chorionic membrane extracts from live eggs of the brook trout, Salvelinus fontinalis [59]. A mosquito larval parasite, Lagenidium giganteum, selectively colonizes and encysts on culicid hosts [60-62]. It was found that specific conformations of chitin and chitosan in mosquito larvae could effectively cause encystment of the zoospores of this mosquito parasite [62]. IMPORTANCE OF BIOASSAY METHODS FOR IDENTIFYING ZOOSPORE REGULATING FACTORS FROM THE NATURE Zoospores have powerful sensory systems to respond to a wide range of environmental chemical substances. They show both positive and negative chemotaxis toward plants metabolites as well as environmental chemicals. It is very important to choose an appropriate bioassay method to study the motility behavior, viability, and developmental morphological changes of oomycete zoospores. There are four methods for assaying the motility behavior of fungal zoospores namely, 1) capillary method: a glass capillary tube filled with test compounds, placed into zoospore suspension, and the behavior of zoospores toward the capillary is observed. This method is modified (e.g. microchamber method) for getting quantitative results [23,162]; 2) drop method: a quantitative method for studying chemotaxis of zoospores [163, 164]; 3) particle bioassay: mainly a qualitative method for studying chemotaxis of zoospores [21,13,27]; and 4) homogeneous solution method: a qualitative method [82,105]. Methods 1-3 are suitable for studying mainly chemotactic behavior of zoospores, where the homogenous solution method is appropriate for assaying motility halting, encystment, germination or lysis/bursting activities of zoospores. 'Particle bioassay' is a very simple and rapid method, which requires very small amount (less than microgram level) of active substance to test bioactivity toward zoospores. In addition to chemotaxis (negative and positive) study, this method is also useful for qualitative evaluation of the aggregation, attachment, encystment and germination behavior of zoospores especially when gradient of a compound is required for biological activities. Particle bioassay method could easily be used as an effective method for identifying active principles from both host
1113 and nonhost plants by bioassay-directed isolation procedures. Both 'Particle bioassay' and 'homogenous solution' methods are briefly described here. Particle bioassay
One drop of solution of each test chemical dissolved in EtOAc or acetone, and adjusted to an appropriate concentration, is dropped onto a few particles of Chromosorb W AW (175-226 jam) on a watch glass. A tip of filter paper immediately absorbs excess solution and the particles are allowed to evaporate the solvent. It was estimated that each particle holds the amount of compounds equivalent to ca. 4 nl of the test solution [71]. One or two of these particles are carefully dropped into 2 ml of a zoospore suspension (1x10 ml"1) in a small petri dish (3 cm i.d.). Therefore, the particle expose to a high volume of water diffused coated compounds to the surrounding water and develops a gradient of test compound around it. Therefore, the actual concentration of the solution around a particle during the bioassay must be far smaller than the concentration of test solution used to coat it. After the drop of a particle in zoospore suspension, the motility behavior of the zoospores around the particle is observed microscopically up to 60 min after addition of the particle(s). Control particles are treated with solvent alone. Around particles treated with an inactive compounds or solvent alone, the zoospores moved in an unvarying, regular manner and at a constant speed. In contrast, zoospores close to particle(s) treated with an active compound responded in one of the following ways. 1) Attractant activity: relatively large number of zoospores is assembling around the particle(s), moving with increase speed in a complex zigzag or circular manner. There was a clear gradient in zoospore density, which decreased with increasing distance from the particle. 2) Repellent activity: zoospores are repelled by the treated particle(s) and not approaching to the particle(s) quickly become surround by a circular, zoospore-free zone. 3) Stimulant activity: zoospore movement near the particles increase in speed without any variation in zoospore density. 4) Encystment activity: zoospores shed flagella and thus stop their motility, and change into spherical spores called cystospores surrounded by the cell walls. The cystospore is a thick^walled and nonmotile spore that is relatively resistant to environmental stresses and can germinate when specific signal substance(s) are present. 5) Halting activity: motility of zoospores immediately inhibit around the particles. 6) Lytic activity: The halted zoospores become round-shaped and the cellular materials granulate and/or burst. 7) Regeneration activity: The halted zoospores encyst and then regenerate zoospores after a certain period leaving the outer cyst-coats (ghost).
1114 Homogenous solution method
The "homogeneous solution method" is carried out to measure the halting and bursting activities of compounds in the zoospore suspension where each test compound was homogeneously dissolved. To 2 ml of the zoospore suspension into a small Petri dish (3 cm i. d.), an appropriate amount of the deionized water or DMSO solution of test compound was added, and quickly but gently mixed well to give a homogeneous solution. The behavior of zoospores was observed microscopically up to 60 min after addition of the test solution. A control is run using equal volume of solvent (DMSO or deionized water) in place of the test solution. The relative halting or bursting activity in the homogeneous solution method is quantified as follows: Halting or bursting activity (%) = 100 x (B-C) / (A-C) A: Total zoospores counted from the average number of encysted zoospores per microscopic field in 30 s vortexing where swimming zoospores disappeared in a few min and all settled themselves on the bottom of the Petri dish. B: Average number of encysted or burst zoospores in the test solution. C: Average number of encysted or burst zoospores in the control solution (zoospores suspension and solvent). The numbers of A, B and C are counted from at least 5 microscopic fields of each Petri dish and averaged. GENERAL CONCLUSION AND FUTURE PERSPECTIVES Zoospores have very powerful sensory transduction system to respond to the gradient of host-specific chemical signals to target their hosts by chemotaxis. Bioassay-guided chromatographic separation procedures resulted in identification of several host-specific plant signals for phytopathogenic oomycete zoospores. The higher concentration of those host signals at the host surface (source) triggers encystment and germination of accumulated zoospores, which is essential for invasion of pest propagules into the host tissue. It appeared that zoospore perceives host signal by a G-protein-coupled receptor and then translate into responses probably via phosphoinositide/Ca2+ or phosphatidic acid second messengers pathways. Much progress has been made in understanding the homing sequence in root-infecting oomycetes, and the findings indicate several points of potential general significance. The understanding of the components of signal transduction pathway in chemotaxis and
1115
differentiation of zoospores is the most challenging research needed to be done. The first step should be the identification of the receptor protein in zoospores. Molecular biological approach may be suitable for characterizing the involvement of stage-specific enzymes, and lipid metabolism in the pathway. As our knowledge on the mechanism of chemotaxis and differentiation of other motile cells is advanced much in recent years, many known techniques could be used to uncover the facts in oomycete zoospores. Since the components of the signal transduction pathway represent attractive targets for alternative methods of disease control, agriculture may benefit from research, which will deliver such results in the long term. Triggering of developmental transitions in accumulated zoospores on host surface by a host signal would offer some future studies on the mechanism of differentiation of oomycete zoospores. It may also be worthy to survey of some specific inhibitors/antagonists by screening nonhost plant extracts that could mimic or agonise or antagonise the activities of the known host or nonhost triggers. In addition to the host signals, zoospores are also sensitive to a variety of nonhost compounds as well as environmental chemical substances. The nonhost plants appear to possess diverse groups of secondary metabolites to protect themselves from the attack of oomycetes. Bioassay-directed survey of nonhost 'chemical weapons' is certainly an interesting area for future research. Several rhizospheric epiphytes are also exhibited disease suppression activities against oomycetes. Isolation of chemical substances from those microorganisms responsible for antioomycete activities would lead novel targets for designing new bioregulators for controlling the notorious soil-borne oomycete phytopathogens. ABBREVIATIONS BABA = /?-aminobutyric acid BPA = bisphenol A DES = diethylstilbestrol G^>roteins = GTP binding regulatory proteins MALDI-TOF-MS = matrix-assisted laser desorption/ionization time of flight mass spectrometry Py. = Pythium Ph. = Phytophthora SEM = scanning electron microscopy TEM = transmission electron microscopy
1116 ACKNOWLEDGEMENTS We are thankful to Prof. R. Yokosawa, Health Science University of Hokkaido for kindly providing Aphanomyces cochlioides strain AC-5 and Mr. Toshiaki Ito, Laboratory of Electron Microscopy, Hokkaido University for his support in electron microscopy. The first author is thankful to the Japan Society for the Promotion of Science (JSPS) for his Postdoctoral fellowship. The financial support from MEXT (to S. T; Grant-in-Aid for Scientific Research, No. 14206013) is also acknowledged. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] II1] [12]
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BIOPROSPECTING IN THE BERKELEY PIT: BIO ACTIVE METABOLITES FROM ACID MINE WASTE EXTREMOPHILES ANDREA A. STIERLE and DONALD B. STIERLE Department of Chemistry and Geochemistry, Montana Tech of the University of Montana, Butte, Montana ABSTRACT: The Berkeley Pit Lake system in Butte, Montana is part of the largest EPA Superfund site in North America. It includes Berkeley Pit Lake, an abandoned open-pit copper mine, 1300 feet deep and one mile across. During its thirty years of operation the Pit and surrounding deep shaft mines were dewatered through constant pumping. On Earth Day 1982 the pumps were turned off for the last time, and the Pit began to evolve into an acid mine waste lake. As infiltrating ground water continually seeps into the Pit, rich veins of pyrite and other minerals dissolve, generating acid in the process. There are currently 30 billion gallons of water in the Pit, with an inflow rate of 4 million gallons/day. The water is acidic (pH 2.5 - 2.7) and contaminated with high concentrations of metal sulfates including iron, copper, aluminum, cadmium and zinc. Unfortunately, the Pit Lake system sits at the headwaters of the Clark Fork River, a major tributary of the Columbia River. If the water rises another 200 feet, it will reach the critical overflow level. At the current rate of rise, the critical level will be reached in approximately ten years. Although the chemical dynamics and possible remediation strategies of the Pit Lake have been studied for twenty years, the microbial ecology was neglected. With its low pH and high metal content, it was considered too toxic to support life. Since 1995, however, with colleague Grant Mitman, we have isolated over sixty fungi, protists, algae, protozoans and bacteria. Although conditions within the Pit Lake System are toxic for "normal" aquatic biota, these same conditions represent an ideal environment for extremophiles. This hostile environment may also select for new species that produce novel secondary metabolites. It can be a challenge isolating and culturing these extremophiles, but it is the unique challenge of drug discovery to find methods for targeting the bioactive components in these organisms.
INTRODUCTION In 1986 we began our investigation of marine microorganisms, inspired by the pioneering work of D. John Faulkner in this field a
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decade earlier. While Faulkner's earliest ventures into marine microbes involved a seawater sample from an intertidal pool at La Jolla shores, we targeted endosymbionts of marine sponges [1], Our early results were promising, but the logistics of marine research from a land-locked Montana School of Mines proved difficult [2, 3]. We did not abandon our study of aquatic microbes, however. After a brief hiatus searching for a taxol-producing fungus [4] and for endosymbionts of giant redwoods [5-7], we returned to aquatic microorganisms. This time, however, we changed our focus from marine microbes to those that could be isolated from a lake system located less than one mile from our laboratory. Bioprospecting in the Berkeley Pit There were several reasons for selecting this particular research arena. Our search for taxol-producing microbes was drawing to a close and we were ready for a new project. Our group is firmly entrenched in the search for bioactive metabolites from microorganisms, and it was time to study a new population of microbes. Unfortunately, we had no funding available and with limited resources, we could not launch an expedition into uncharted territories in remote, exotic ecosystems. Our budget could not accommodate a trip to Antarctica or to a volcanic lake in Peru. When colleague Bill Chatham discovered green scum growing on a piece of wood floating just below the surface of Berkeley Pit Lake less than one mile from our lab, we knew we had a research site. This is no ordinary lake, however, and its microbial inhabitants reflect the unique nature of the lake. Berkeley Pit Lake is located in Butte, Montana, called the "richest hill on earth" because of the high quality ore bodies that concentrated in this area. While scientists and engineers attempted to remediate the Pit Lake, we initiated a different type of mining venture— mining for microbes. Over the last eight years, we have isolated and studied over 60 fungi and bacteria from the surface waters down to the lake bottom sediments at 720 feet. Last year we were able to access deep Pit Lake sediments as well and have isolated several new microbes not found in the water column.
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We are currently restricting our research to microbes that can be cultured using fairly routine culture conditions. Although many scientists are using molecular biological tools to isolate, amplify and sequence DNA from extreme locations, we need to study the actual microbial culture broths to isolate secondary metabolites. Several Pit microbes have already yielded novel, biologically active secondary metabolites [8-10]. Although the Environmental Protection Agency and Montana residents view the Berkeley Pit as an ecological time bomb, we view it as something more - an evolving and dynamic ecosystem - a classic by-product of the industrial age. Over the next few years our research will focus on the following goals: • •
•
To isolate and identify culturable microorganisms from Berkeley Pit lake water and sediment samples. To grow each Pit microbe under varying physicochemical conditions, and examine the effect on the secondary metabolites produced. To isolate and characterize selected bioactive metabolites produced by selected Pit microbes.
Clearly, this is not a microbial ecology study nor are we assessing the total diversity of microbial life in the Pit Lake. These compounds may or may not be produced in situ under natural conditions. Our concern is that these compounds are produced in our lab under the conditions described and that they can be isolated, characterized and studied for their drug potential. Extremophilic Organisms The earth is rich in hostile environments. These include a wide array of natural systems: deep-sea vents, salt brines, thermal pools, volcanic lakes, and frigid ice fields. Others are anthropogenic, often the result of extractive hard rock, oil or coal mining. Nobel laureate Paul Crutzen suggested that we are currently living in the "anthropocene", an era in which humans and human activities have become a major geological force on the planet [11]. Whether man-made or natural, extreme environments can harbor life forms called extremophiles.
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Bacteriologist Thomas Brock demonstrated the importance of extremophiles in the 1960's when he isolated bacteria from a 70°C thermal pool in Yellowstone National Park. In the forty years following Brock's discovery, scientists have explored ecological niches as varied as deep sea vents and Antarctic ice sheets and have also found unusual life forms in unexpected places [12]. For the natural products chemist, unusual microbes hold the potential of novel chemistry with important medicinal, industrial or agrochemical applications. The rapidly developing tools of molecular biology have focused attention on genomics and proteomics. Despite this trend, however, the study of secondary metabolites, or "secondary metabolomics", is still an important means to the discovery of new chemotherapeutic agents. When populations of extremophiles are discovered, and established in culture when possible, it is the unique challenge of drug discovery to find methods for isolating and characterizing the bioactive components in these organisms. Berkeley Pit Lake is our extreme environment. We are confining our studies to microbial life in the Pit for the simple reason that microbes are the predominant, and perhaps sole inhabitants of this toxic lake. Unlike most rivers and lakes in Montana, Berkeley Pit Lake does not harbor trout, grayling or other blue ribbon fish species. Aside from a single water bug photographed resting on the surface of the Pit Lake, and a flock of snow geese that landed on the water and subsequently died, no evidence of macrobial life exists. The term extremophile was coined by MacElroy in 1974 to describe microorganisms that thrive under conditions that would be considered extreme from a human perspective [13]. Of course, extreme is a relative term. Obligate anaerobic microbes have long been known and are not viewed as extremophiles, yet life without oxygen would certainly be a challenge to most of us. In essence, the term extremophile is used to describe microbes that thrive in environments where most microbes cannot grow or thrive because of extremes in temperature, salinity, pH, or pressure. Extremophiles can be classified according to the environments in which they thrive [14]: Acidophiles thrive in an acidic environment, usually at an optimum pHof2-3. Alkaliphiles thrive in an alkaline environment, usually at a
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minimum pH of 10. Halophiles require a salty environment, with a minimum salt concentration of 0.2 M. Piezophiles thrive at high pressures. Psychrophiles thrive in a cold environment. Thermophiles thrive in a hot environment, with an optimum growth temperature of 45° C or higher. Hyperthermophiles flourish at even higher temperatures, between 80-100°C. Brock's Thermus aquaticus is perhaps the most famous thermophile discovered to date. His initial discovery of viable bacteria thriving in hot springs surprised many scientists because most organims would either die or fail to reproduce in 70°C water. T. aquaticus owes its continued fame to more than just its unusual habitat, however. It owes its survival in part to a special variant of the enzyme DNA polymerase. DNA polymerases catalyze the synthesis of deoxyribonucleic acid in a template-dependent process that results in a faithful copy of the original DNA molecule [15]. Consequently, these enzymes are necessary to propagate, maintain, and manipulate the genetic code of living organisms. Although DNA polymerases are usually not heat labile, the T. aquaticus DNA polymerase variant remains operational at high temperatures. This variant (known as Taq polymerase) is now a key ingredient in the polymerase chain reaction (PCR) technique. Extremophilic Microorganisms Many scientists worldwide are looking for life forms in places too extreme for conventional life, and much of the search is focused on microorganisms. This is not surprising. In the past thirty years, researchers have compiled an increasingly robust map of evolutionary diversification showing that the main diversity of life is microbial, distributed among three primary domains (relatedness groups): Archaea, Bacteria, and Eucarya [16]. During this same period, the number of microbes discovered living in extreme environments has increased dramatically. Springer-Verlag has created a journal -
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Extremophiles - dedicated to the organisms found in extreme environments. A few examples of microbial extremophiles are listed below. This is only a small sample of the unusual microbes that have been found in unexpected ecosystems. Extremophilic microbes of Yellowstone National Park In the fine tradition established by Thomas Brock, Yellowstone National Park continues to be a major research arena for microbiologists and ecologists. David Ward, a microbiologist at Montana State University and former Brock student, has been studying extremophiles in this grand and hostile ecosystem for several years. According to the 1996 Investigators' Annual Reports [17], his is one of 23 research projects studying the Park's tiniest, toughest inhabitants. Although hot springs and geothermal vents are found in several parts of the world, the largest single concentration is in Yellowstone National Park. Thermophilic prokaryotes - bacteria and archaea - abound in these environments. They are often colored, due to the presence of photosynthetic pigments (blue-green of cyanobacteria, red of red algae or purple bacteria or carotenoid pigments (yellows and browns of some archaea). Although most visitors to the nation's oldest National Park are drawn by its outstanding geology and charismatic megafauna, they cannot help but be intrigued by extensive microbial communities that color the hot springs, fumaroles and geyser basins with garish and gorgeous hues of rich browns, greens and yellows. Ward is also collaborating with organic geochemists Geoffrey Eglinton at Bristol University, in Bristol, United Kingdom, and Jan de Leeuw at the Netherlands Institute for Sea Research, Texel, the Netherlands, to study ancient mat-forming organisms, "chemical fossils" considered to be analogs of stromatolites, the predominant fossils of the Precambrian era. He is investigating Chloroflexus mats found in sulfide-rich springs in Yellowstone Park. He believes that these anoxygenic phototrophs might have arisen before cyanobacteria. If true, the mats they formed would predate cyanobacterial mats.
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Ward and his colleagues are using the tools of molecular biology, including 16S rRNA-based molecular methods, to analyze microorganisms that grow in hot spring habitats but resist cultivation in laboratories. Such analytical methods are a good way to explore the remarkable diversity of these microorganisms. The diversity of uncultivated (or unculturable) prokaryotes within microbial communities like the cyanobacterial mats found in the alkaline siliceous Octopus Spring in Yellowstone is remarkable. According to Ward, 31 unique 16S rRNA sequences have been detected in the Octopus Spring mat, none of which match the 16S rRNA sequence of any previously cultivated isolate. Ten years ago, standard cultivation techniques led scientists to believe that a single Synechococcus species made up the mat. Using molecular techniques, however, he has detected at least 11 distinct cyanobacterial 16S rRNA sequences in the mat [18-19]. Thermophiles from Deep-Sea Vents Thermophiles are not unique to the Yellowstone ecosystem. They have also been found in many different environments. To date, over 50 thermophiles have been isolated and identified. At the time of this publication the most extreme thermophilic mircoorganism is the ironreducing archaeon "strain 121". Kashefi and Lovley isolated an ironreducing archaeon from a water sample taken from an active black smoker (an undersea thermal vent which ejects water at very high temperatures and pressures) hydrothermal vent along the Endeavor segment of the Juan de Fuca Ridge, in the Northeast Pacific Ocean. Strain 121 could grow in temperatures of 85-121°C (185-250° F). The former thermophilic record-holder, Pyrolobus fumarii, could not meet the challenge. After an hour at 121° C, only 1 percent of its cells were intact and none appeared viable) [20]. Pyrolobus fumarii can multiply in temperatures up to 113°C, however, which is still a remarkable upper limit for a living organism [21]. It grows on the walls of deep ocean black smokers. The water is rich in minerals, and these (in combination with carbon dioxide) provide the food source for P. fumarii. For this reason, P. fumarii is
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classified as a chemoautotroph: it synthesizes its own food from surrounding chemicals. It was discovered by German scientist Karl Stetter and his colleagues who also discovered the world's smallest microorganism, Nanoarchaeum equitans [22]. Its name translates as "ancient dwarf who rides the fire ball" from its tendency to adhere to the surface of the archaeal microbe Ignicoccus ('fireball'). The discovery of this nanosized hyperthermophilic archaeon has led to the creation of a new phylum, Nanoarchaeota. N. equitans was found in a 120 m deep submarine hydrothermal vent, north of Iceland and thrives in temperatures close to 100°C. With less than 500 kb in its genome, N. equitans represents the smallest archaeal genome sequenced to date and has the smallest genetic code of all living organisms to date. In 1996, Methanococcus jannaschii became the first archaeon to have its genome sequenced. It was isolated in 1983 from the sea floor surface of a Pacific thermal vent "white smoker" off the coast of Baja, California. It is methanogenic (methane producer), thermophilic, strictly anaerobic and autotrophic (uses CO2 as the sole source of cell carbon), and normally lives at about 2400 m below sea level, where the pressure is approximately 230 atmospheres [23]. Extremophiles from Inland Environments David Boone, a Portland State Univrsity microbial ecologist, studies include the microbiology of deep terrestrial subsurfaces. He discovered an iron and sulfate-reducing bacterium, Bacillus infernos, the first anaerobic member of the bacterial genus Bacillus. "The Bacillus from hell", as the name implies, was isolated 2700 m below the land surface. It is thermophilic (60°C), halotolerant (salt concentrations 0.6 M) and slightly alkaliphilic [24]. Deinococcus radiodurans is the most radiation-resistant organism known. It was discovered by Arthur W. Anderson at Oregon Agricultural Experiment Station in Corvallis in 1956 in a can of radiation-sterilized meat. D. radioduransis is resistance to genotoxic chemicals, oxidative damage, dehydration, and high levels of ionizing and ultraviolet radiation. It can withstand exposure to radiation levels
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up to 1.5 million rads (500 rads is lethal to humans). A recombinant strain has been engineered to degrade organopollutants in radioactive, mixed waste environments [25]. George Roadcap and colleagues from the University of Illinois Champaign Urbana found a microbial community that sets a new record for tolerance to alkaline environments. These microbes are primitive and live in contaminated groundwater created by dumping of slag waste from iron ore processing just south of Chicago. These alkalinophiles live at a pH of 12.8 - tolerating conditions 100 times more alkaline than the closest contender - in contaminated groundwater created by dumping of slag waste from iron ore processing just south of Chicago [26]. Extremophiles as a Source of "Extremozymes" The phenomenal impact of Thermits aquaticus excited much interest in the primary metabolism of microbes living on the edge. Comparatively few of the studies of extremophilic organisms target secondary metabolites. Rather, new proteins and other primary metabolites that are stable in a wide range of physical conditions have dominated the research scene. It is not surprising considering the scientific and economic importance of the enzyme Taq polymerase and the polymerase chain reaction. Like Taq polymerase, many of these proteins are key enzymes in important biological pathways and may exhibit biological activity with pharmaceutical or commercial potential. Many scientists hope to discover other proteins that are stable at extremes of temperature, salt concentration, or pH. Biocatalysis can be a useful tool in a number of different applications. Just as Taq polymerase revolutionized PCR, other extremophilic enzymes might also serve as useful catalysts in commercially important reactions. T. aquaticus was a focus of much of the early work. In 1973, Stellwagen reported the isolation of a thermostable enolase from T. aquaticus YT-1. It was remarkably thermostable compared to enolases isolated from either yeasts or rabbit muscle cells. Taq
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enolase, however, did not find the same commercial success or notoriety as Taq polymerase [27]. In the three decades following the "Taq revolution", many diverse extremophiles and their metabolites found there way into commercial products. Lynn Rothschild, a scientist at the NASA Ames Research Center, reported that extremophilic proteins and other biomolecules, as well as whole organisms, are being used in processes as varied as cheese making and the desulfurification of coal [28]. Specifically, thermophilic microbes are yielding a-amylases for the starch hydrolysis for the production of soluble dextrins and corn syrups; xylanases for paper bleaching; lopases and proteases for detergents; alcohol dehydrogenases for chemical synthesis; and proteases for food processing and brewing. Psychrophilic microbes are yielding Upases and proteases for low temperature cheese maturation; proteases, Upases and amylases for detergents; and ice nucleating proteins for ice cream manufacturing and the production of artificial snow at ski resorts. Live psychrophiles are also being used to help remediate oil spills in cold, salty, northern seas. Halophilic microbes are yielding salt tolerant membranes and glycerol for pharmaceutical manufacturing. Acidophilic microbes are being used to oxidize sulfur in coal desulfurication processes and in metal recovery in mining operations. Alkaliphiles are yielding cellulases, proteases, amylases, and Upases that are stable at high pH and can be used effectively in alkaline detergents [28]. More recently, the discovery and commercialization of Laminoacylase from Thermococcus litorali was a product of the LINK project between Chirotech Technology and the University of Exeter. The L-aminoacylase of T. litoralis had broad substrate specificity for the hydrolysis of N-acylated a-amino acids, with respect to both the side chain and the N-acyl group. It is especially useful for the enantiospecific hydrolysis of acyl groups, particularly N-benzoyl groups of a-amino acids. This can be used to advantage in synthetic processes that require the enantiospecific deprotection of racemates [29]. The enzyme activity of the L-aminoacylase of T. litoralis was maximal at 85°C but with a half life (ti/2) limited to 1.7 h. Reduction of the operating temperature to 70°C yields a significant increase in
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t\a to 25 h. The pH optimum was shown to be pH 8 but more than 70% of the maximal activity remained over the range pH 6.5-9.5. Although not all of the commercial applications of this Laminoacylase relied on its thermostability, one application demonstrated a unique advantage that only a thermophilic enzyme could provide. This process requires the resolution of a racemic mixture of an N-acyl-protected amino acid. The protected amino acid is insoluble at room temperature but will dissolve at 50° C. Other available L-aminoacylases would be inactivated at 50° C, but the thermophilic enzyme can easily complete the conversion to deprotected amino acid with 99% enantiomeric excess [29]. Peptides and proteins can also exhibit important pharmaceutical potential and extremophilic microbes have produced some noteworthy examples. One of these in Halocin C8. The halocins are bacteriocinlike (antibacterial) proteins or peptides produced by many species of the family Halobacteriaceae. Halocin C8, excreted by the Halobacterium strain AS7092, is a single 6.3-kDa polypeptide that can be desalted, boiled, frozen, subjected to organic solvents, and stored in culture supernatant at 4°C or in dHzO at -20°C for more than 1 year without losing activity. Halocin C8 is active against a wide spectrum of bacteria including most haloarchaea and even some haloalkaliphilic rods [30].
Extremophiles as a Source of Bioactive Secondary Metabolites
Although the search for novel extremozymes is a hot area of research, the secondary metabolites of these same microbes have attracted little attention. While scientists isolate and characterize new proteins that are stable in extremes of temperature, pH and salinity, others are applying the tools of molecular biology rather than classical culture techniques to study microbial communities. The two techniques give a. very different view of community diversity in part because certain microbes may act like weeds, quickly outgrowing all other, more fastidious members of the community in culture. Many microbes may
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resist cultivation entirely, stubbornly refusing to grow under any of the physicochemical conditions used. It is important to understand the ultimate goal of a research endeavor. If a complete investigation of the microbial diversity of a site is the goal, then the tools of molecular biology are preferable to those of cultivation. But if the goal is the isolation and characterization of bioactive metabolites from these microbes, then cultivation has been our method of choice. It is necessary to generate a fair amount of microbial biomass to isolate adequate quantities of secondary metabolites for thorough testing. Unlike proteins that are usually closely related to the DNA-RNA templates from which they are generated, secondary metabolites are often produced in complex multi-step biosynthetic pathways. Even if the more fastidious microbes resist standard cultivation techniques, the culturable microbes are already proving themselves capable of producing some very interesting new compounds.
Bioactive Metabolites from Deep-Sea Sponges Marine natural products chemists studying deep-water sponges conducted some of the earliest studies of the bioactive secondary metabolites of extremophilic organisms. Scientists at Harbor Branch Oceanographic Institution (HBOI) and several collaborators studied metabolites from sponges collected in the Bahamas by Johnson-SeaLink submersible. Drug discovery has long been an important goal for natural products chemists and the investigation of secondary metabolites in deep-sea sponges was no exception. Scientists used "state of the art" bioassays to guide compound isolation. Of particular interest in the 1980's was the isolation of potential anticancer agents [31]. The discovery and development of anticancer drugs with clinical potential has been the responsibility of the Developmental Therapeutics Program (DTP), Division of Cancer Treatment, National Cancer Institute (NCI). In the mid-1980's, approximately 10,000 compounds/year were selectively acquired and screened against murine (mouse) tumor models in order to discover new, active
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materials. From 1975 to 1985, the in vivo P388 mouse leukemia model was used almost exclusively in the NCI screening program as the initial or primary screen. With few exceptions, agents that showed minimal or no activity in the P388 system were not selected by the NCI for further evaluation in other tumor models or alternative screens. Most of the available clinical anticancer agents are active in the P388 system. Certain human cancer cell lines were also important screening tools, including human colon cancer HCT8 and human alveolar cancer A549 [32]. In 1988 Rinehart (University of Illinois Champaign Urbana) and Gunasekera (Harbor Branch Oceanographic Institution) reported the isolation and characterization of topsentin, bromotopsentin, and dihydrodeoxybromotopsentin, antiviral and antitumor bis(indolyl)imidazoles from Caribbean deep-sea sponges of the family Halichondriidae that were collected at depths of 174 m, 229 m, and 355 m. The researchers used growth inhibition of cancer cell lines P388, HCT8, A549, and others to guide the isolation of these compounds. They also used a plaque-reduction assay against HSV-1 (Herpes Simplex Virus-1) to target potential antiviral agents [33]. In that same year, researchers at HBOI isolated a novel cytotoxic alkaloid from a deep-water marine sponge, Dragmacidon sp., collected at a depth of 148 m. Compound isolation was guided by inhibition of the in vitro growth of P388 murine leukemia cells. The active constituent of the extract was a bis(indole) alkaloid, dragmacidin that was active against P388, A549 (human lung) and HCT8 (human colon) and MDAMB [34]. Gunasekera's lab also isolated a very promising new polyhydroxylated lactone Caribbean marine sponge, Discodermia dissoluta, from a depth of 33 m, using P388 cell line growth inhibition assay. Subsequent work on this compound has shown it to be an antimitotic agent that stabilizes microtubules more effectively than taxol. It is now in the first phase of human clinical trials as a treatment for many forms of solid malignancies such as pancreatic cancer [35]. In the 1990's scientists at Harbor Branch Oceanographic Institution continued to study the secondary metabolites of deep water sponges. They introduced new, highly specific bioassays into their
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search for potential drug candidates. In particular, they were interested in novel phosphatase inhibitors that target calcineurin (CaN) and the caspases, including CPP32 (caspase-3). Calcineurin is a serine-threonine protein phosphatase involved in signal transduction and is recognized as a principal signaling molecule in the regulation of the immune response [36]. Immunosuppressants such as FK506 and cyclosporin A have been shown to exert their effect through inhibition of CaN following their association with binding proteins [37]. The caspases, which include CPP32, are a group of cysteine proteases (also known as interleukin-2 converting enzymes or ICE2) that play a major role in the programmed cell death mechanism known as apoptosis [38]. Inhibitors of caspase-3 (CPP32) have been shown to prevent apoptotic mediated death in a number of cell lines and in various tissues [39, 40]. These findings prompted a search for small molecule inhibitors of CaN and CPP32 that might be expected to have useful pharmacological activity. In 1998 they reported the discovery of discorhabdin P, a new discorhabdin analogue from a deep-water marine sponge of the genus Batzella. The sponge had been collected in August 1994 at a depth of 141 m, from the western Great Bahama Bank, Bahamas. Discorhabdin P inhibited the phosphatase activity of calcineurin and the peptidase activity of CPP32. It also showed in vitro cytotoxicity against P388 and A549 cell lines [41]. Secobatzelline A and secobatzelline B were isolated from a deepwater marine sponge of the genus Batzella. Secobatzellines A and B inhibited the phosphatase activity of calcineurin, and secobatzelline A inhibited the peptidase activity of CPP32. Both compounds showed in vitro cytotoxicity against P388 and A549 cell lines [42]. Bioactive Metabolites from Deep-Water Sediment Microbes Other labs are joining the search for deep-sea organisms in a quest for new bioactive secondary metabolites. A few of these labs are studying the microbial symbionts of macroorganisms or microbes from deep-sea sediments. In 2003, Jongheon Shin's lab at the Natural Products Research Institute, Seoul National University, reported the
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isolation of a new cyclic tetrapeptide from the culture broth of an actinomycete of the genus Nocardiopsis collected from underwater sediment (depth 3000 m) collected at Clarion-Clipperton Fracture Zone Mid-Pacific, during an expedition of the Korea Deep Ocean Study Program in July 1998. No biological activity was attributed to this compound [43]. Phil Crews (University of California, Santa Cruz) and his collaborators have expanded their studies of the secondary metabolites of marine fungi to include isolates of deep-water sediment. In 2004 they reported the isolation and characterization of three previously unknown pentaketides, (+)-formylanserinone B, (-)epoxyserinone A, and (+)-epoxyserinone B, along with two known fungal pigments, anserinones A and B, from a deep water (-4380 ft), marine-derived saltwater fungal culture. These compounds were isolated using two separate cell-based assays. Epoxyserinone A and (+)-epoxyserinone B showed the greatest anti-leukemia selectivity, while all three of the pentaketides exhibited modest activity against the MDA-MB-435 cell line [44].
BERKELEY PIT LAKE - EVOLUTION OF AN EXTREME ENVIRONMENT Many of the most intriguing extremophiles have been isolated from dramatic geologic or climatologic phenomenon. Berkeley Pit Lake, however, is not buried deep in the ocean or nesting in a volcanic caldera. Instead, it is nestled in the Rocky Mountains in Butte, Montana. Since 1870 Butte has been a mining mecca, with 42 miles of vertical shafts and 2700 miles of tunnels honeycombing the terrain in the quest for gold, silver, and ultimately, copper. In 1955 the Berkeley Pit was created and gradually developed into a mile wide, mile high, 1300 foot deep pit that sits in the shadow of the Continental Divide. The Pit and surrounding deep shaft mines many of which are over 3000 feet deep - were dewatered through constant pumping, which ceased abruptly in 1982. Within 2 years, the water level had risen to the base of the Pit, and continued to rise,
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percolating through the mineral rich overburden of the Boulder Batholith [45]. The Berkeley Pit evolved from an open-pit copper mine to an acid mine waste lake in less than twenty years. There are actually many acid lakes worldwide. Some are volcanic crater lakes and include Lake Tiwo Nua Muri Koohi Fah in Flores, Indonesia; Crater Lake in Ruahepu. New Zealand; Yugama Lake in Japan; and Laguna Caliente in Costa Rica. Acid mine pit lakes like the Berkeley Pit Lake and the Phelps Dodge Tyrone Mine in New Mexico owe their existence to anthropogenic activities. Acid Mine Waste Lakes Pit lakes formed in limestone formations gradually evolve into community recreation areas ideal for boating and swimming. Unfortunately, the 342 snow geese that landed in the Berkeley Pit Lake in 1995 and died shortly thereafter as a result, belied any notion that the Berkeley Pit had recreation potential. Yet no single tributary of the Pit Lake can match it in either metal ion or hydrogen ion concentration. A single mineral species - iron pyrite - plays a dominant role in the geology of the area, and is ultimately determining the nature of Berkeley Pit Lake. Pyrite reacts with air and water to produce sulfuric acid. As oxygen concentration decreases with depth, pyrite oxidation and resulting acid generation should also decrease. However, oxidation of pyrite by dissolved ferric iron can take place at a rapid rate in acidic waters, even in the complete absence of oxygen. The rate of ferrous iron oxidation by O2 is known to increase many orders of magnitude in the presence of certain acidophilic bacteria, chiefly Acidithiobacillus ferrooxidans [46]. These coupled processes continually generate sulfuric acid, which further dissolves the mineral rich ore body, releasing high levels of solubilized iron, aluminum, copper, zinc, cadmium, magnesium and a host of other metal cations. After twenty years, the result of this dynamic process is a very large hole filled almost to the critical point, with 30 billion gallons of a mineral rich, acidic solution poised at the headwaters of the Columbia River system.
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Today, Berkeley Pit Lake is part of the largest EPA Superfund site in U.S. Although rain, snow and ground water continue to raise the water level, several factors conspire to prevent dilution of either the metal ion or hydrogen ion concentration of the water. Butte is a high desert with an annual rainfall between 10-12 inches. In essence, the evaporation rate exceeds the input of pure rainwater. Much of the groundwater entering the Pit is already contaminated as it percolates through mine tailings and tunnels. But even if all of the influent waters were pristine, the pyrite walls of the Pit would continually react with air and water to generate sulfuric acid [45]. Characterization of the Berkeley Pit Lake Scientists at the Montana Bureau of Mines and Geology have conducted much of the characterization of the Berkeley Pit. According to their studies, the shallow water of the Pit Lake - the epilimnion - is separated from the deep water - the hypolimnion - by a chemocline, a zone of rapid chemical and physical change. In Berkeley Pit Lake, the chemocline is between 35 to 50 feet below the lake surface. The epilimnion is characterized by a pH of 2.5 and a temperature of 0°C in winter (ice forms on the surface) to 25°C in summer. Dissolved-metal concentrations in the epilimnion include Cu, 140 mg/L and Zn, 540 mg/L. The hypolimnion is characterized by a pH of 2.5 and an annual temperature of 4.5 °C. Dissolved-metal concentrations in the hypolimnion include Cu, 190 mg/L and Zn, 620 mg/L. Iron(II):Iron(III) ratio in the hypolimnion is 2.5 and in the epilimnion 0.36 [47]. Bioprospecting in an EPA Superfund Site The discovery of a relatively rich microbial flora in the rising waters of the Berkeley Pit has provided a new arena for chemical investigation. Until now, the primary concern has been effective remediation of this enormous Superfund site. Ground and surface waters that percolate through Berkeley Pit Lake ultimately enter the
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Clark Fork River, an important tributary of the Columbia River. Acid mine drainage seriously degrades water quality and threatens the existence of animal and plant populations. The average pH is 2.5, an acidity level toxic to most life forms, both macrobial and microbial. Many cations reach levels well above drinking water standards set by the EPA. A sample of Berkeley Pit lake water analyzed by Inductively Coupled Plasma (ICP) has high levels of Fe+2/Fe+3, Al , Cu+2, and many other cations. It is also very rich in sulfates, the predominant anionic species present (8500 ppm). Some typical cation levels in Pit water (24) and EPA drinking water standards for these same cations are shown below in Table 1 [48].
T a b l e 1. Metal cation levels in Berkeley Pit L a k e c o m p a r e d to d r i n k i n g w a t e r s t a n d a r d s Cations Fe +2 & Fe +3 Zn +2 Al +3 Mn + 2 Cu +2 Cd +2 Be +2
Pit Lake Levels 1100 ppm
E P A drinking w a t e r s t a n d a r d s 0.3 ppm
650 290 230 190 2 0.80
5.0 0.002 0.05
1.3 0.005 0.004
Initial Discovery of Pit Lake Microbes Although much attention has been paid to the geochemistry of the Berkeley Pit and its evolving pit lake, little attention was paid to the biological aspects of the water. Although Brock's discovery of bacteria in Yellowstone thermal features was well-known in scientific circles, the rush to explore life in extreme environments gained momentum gradually. Many large-scale extremophile studies were launched in more dramatic environments like Yellowstone Park or deep thermal vents. Mine sites were studied almost exclusively for the presence of microbes that could participate in either oil or mineral recovery or site bioremediation. Many scientists thought the combination of low pH and high metal ion concentration would render the Berkeley Pit Lake too toxic to support life, until analytical chemist Bill Chatham discovered a green scum growing on a piece of
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wood floating just below the water surface almost eight years ago. It looked like a clump of green algae that might be found in any pond in this area, yet he had found it growing in the shallow edge waters of Berkeley Pit. We gave the sample to another colleague, phycologist Dr. Grant Mitman, who identified the organism as the euglenophyte (protist) Euglena mutabilis Schmitz [49]. If protists could survive in Pit water, we thought that bacteria and fungi might be present as well. We took a 100 mL sample of Pit water from Chatham's sample and streaked it onto nutrient agar plates incubated at 25°C. Within a few days smooth yeast colonies and fungal hyphae began to appear. The microbes were isolated and established in pure culture. Three fungi were established in culture from this initial attempt and were identified by Microbial ID, Inc. as Pithomyces sp., Penicillium chrysogenum and the yeast Hansenula anomala [50]. Five years ago we began to focus our studies on the possibility that this unusual body of water might house an equally unusual collection of microorganisms. Initially, we hoped to find sulfatereducing bacteria that might participate in the remediation of the acid mine waters of Berkeley Pit Lake. Although we did not find such a bacterium, we have since isolated over sixty different fungi and bacteria from water samples from the surface down to the basal depth of 720 feet. Last year we were also able to access sediment cores collected by the Montana Bureau of Mines and Geology that yielded several new microbes not found in the water column. There is usually a driving rationale behind any research venture. Why study the secondary metabolites of acid mine waste microbes? For us, the primary reason is that an unusual environment supports unusual microbes that might produce new chemistry. The Berkeley Pit Lake is a rarified environment for biota. Its low pH, and high levels of toxic metals are not conducive to the growth of many organisms. This is a chance to determine how this particular group of microbes flourishes in such an extreme environment. The production of secondary metabolites reflects the biosynthetic processes of a particular microorganism. At the most basic level, these processes help to define a particular organism and can be as characteristic as spore or colony characteristics. For the natural products chemist, the possibility of discovering unusual compounds from this unique
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environment is compelling. For our purposes, the use of specific biological assays is an effective way to follow secondary metabolite production. Finally, the secondary metabolites of microorganisms have tremendous potential for exploitation. Microorganisms are excellent chemists. Their metabolic by-products include a phenomenal number of drugs or drug precursors, agrochemicals, paint additives, biofilms, ionophores, and thermally stable enzymes. Microbes that can withstand extreme pH or high levels of toxic substances may have evolved chemical means of doing so, means which may eventually prove suitable to our needs. Isolation of Berkeley Pit Lake Microbes For our purposes, the most effective method of studying the secondary metabolites of microbes is to establish them in culture. Once a microbe is isolated from an environment, established as a pure culture and encouraged to grow in an appropriate medium, the secondary metabolites can be studied. We have studied microbes from many different environments and have found that the choice of culture conditions can dramatically affect the production of secondary metabolites. For this reason we used a variety of culture media and conditions to encourage optimal production of biologically active secondary metabolites. We use two methods to isolate microbes from Berkeley Pit Lake water: streak-plate method and nutrient enrichment method. In the streak-plate method, we simply streak water from different depths onto sterile nutrient agar plates. Several different DIFCO® media were used for this isolation step: standard and acidified potato dextrose agar (pH 2.5); standard and acidified tryptic soy agar (pH 2.5); mycological agar; and Pit agar (50% filter sterilized pit water, 5 g/L glucose, 5 g/L soytone, 15 g/L agar - pH 2.5). The plates were incubated at 25° C until fungal hyphae or bacterial colonies appeared. Over the next three weeks, each plate was studied daily for evidence of new microbial growth. Fresh hyphae or bacterial colonies were transferred to fresh sterile culture plates until pure isolates were
1143
established. Multiple copies of each isolate were maintained on at least three optimal different media. The nutrient enrichment method involves the addition of carbon and nitrogen sources to the Pit water samples to encourage luxuriant microbial growth. To each 100 mL Pit water sample we added 100 mL of a sterile solution of soytone (5g/L) and glucose (5 g/L) and incubated at 25°C. Each Pit sample was then streaked onto sterile nutrient agar over the next week and individual microbes were established in pure culture as described above. These two methods generated a collection of microbes from the surface waters to the basal sediments. Several of the microbes isolated in this manner were present at all depths. Several species of Penicillium were ubiquitous, as was the yeast Pichia anomala. Some of the microbes grew faster and produced more biomass at low pH and in the presence of minewaste water than they did in more hospitable media. These were the thrivers - microbes suited to the environment in which they were found. Others grew very slowly at low pH - it was clear these microbes merely survived their environment. Most of our subsequent work focused on the thrivers not the mere survivors. We have already isolated several new, bioactive compounds from these robust, acid mine waste bacteria and fungi, several of which will be described later in this chapter. Over the next few years we will continue the search for bioactive metabolites from these microbes. THE SEARCH FOR BIOACTIVE METABOLITES FROM MICROORGANISMS. Fungi and bacteria are not only the cause of infectious diseases: their metabolic products can also cure such infections. In the past fifty years, fungi and bacteria have proven a valuable source of chemotherapeutic agents. The ecological niches of these superb chemists are established by their ability to kill or control fellow microorganisms. Their chemical arsenals have provided many of the important chemotherapeutics used to date. The potent antifungal agent griseofulvin is of fungal origin and the antibiotic streptomycin and the anticancer agent calicheamycin are produced by
1144
actinomycetes [51]. And even though it has not proven its worth from a commercial perspective, the fungus Taxomyces andreanae is a secondary source of the yew-derived anticancer agent taxol [4]. Fungi constitute one of the largest kingdoms of living organisms; conservative estimates suggest over 1.5 million species worldwide [52]. Estimates also indicate, however, that only about 5% of the world's fungi have been identified. Of this number, only about 7% have been studied extensively [52]. Fungi and bacteria are usually studied only if they cause a problem, if they are infectious agents in human, animal, or plant diseases. We have studied many endophytic fungi living within the tissues of plants with no outward manifestations of their presence, i.e. necrotic lesions or chlorosis. They have been overlooked as a fungal group because they do not induce any sign of fungal colonization. They appear to coexist with each other in the nutrient-rich tissues of the host plant. This coexistence may actually be more of a "chemical truce", however: the specific antifungal or antibacterial agents synthesized by each endophyte creating an ecological niche for the surviving fungi. These chemical defense agents represent an important area for the discovery of new, bioactive secondary metabolites. We have isolated several unique, biologically active compounds from endophytic fungi of both yew and redwood trees [4-7]. Endophytic microorganisms are not the only neglected population for natural products investigations. Extreme environments have been all but overlooked in drug discovery ventures. Yet clearly these unique environments support unique microbial life. Our attempts to isolate microorganisms from different depths of Berkeley Pit Lake have yielded over sixty culturable, aerobic fungi and bacteria. Microbial secondary metabolites are particularly desirable for the following reasons: •
Industrial production of drugs, agrochemicals, enzymes, etc., requires reproducible, dependable productivity. If a microbe is the source organism, it can be grown in tank fermentors as needed, producing a virtually inexhaustible supply of a desired natural product [53].
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•
Microorganisms respond favorably to routine culture techniques. Cultivation of macroorganisms (tissue culture) is considerably more challenging, requiring either specialized techniques or months of growth before harvesting is feasible [53]. • Productivity amplification is relatively easy in microorganisms. In the case of penicillin, improved culture conditions and genetic manipulation of producing strains of Penicillium increased drug yield from a few micrograms per milliliter to thousands of micrograms per milliliter. [54, 55]. Genetic manipulation of microorganisms is especially helpful in augmenting product yields. With nonculturable macroorganisms (trees, sponges, etc.), larger collection sizes are the usual option for improved productivity. Recollection can be problematic in delicate ecosystems where rigorous collecting operations can be damaging to the area. Endangered source organisms can be equally difficult to recollect. Changing political climates might reduce or prohibit access to medicinal plants in foreign countries. All of these problems are avoided with microbial fermentation. • Different bioactive compounds can be produced by altering culture conditions. The antibiotic aplasmomycins were produced by Streptomyces griseus SS-20 only after the addition of NaCl to the medium. Directed changes in culture conditions can be explored indefinitely as a means of optimizing biosynthetic pathways that may lead to even more effective analogues [53]. Bioactivity Guided Compound Isolation The ability to grow a particular microbe in a culture medium and ultimately isolate compounds from that culture is only a part of the process. A critical component of drug discovery is the ability to selectively isolate and purify bioactive compounds from a complex slurry of secondary metabolites. Desired compounds often represent significantly less than 1% of an aqueous or organic extract of a
1146
microbial culture. Therefore, natural products chemists use a wide array of bioassays to guide secondary metabolite isolation protocols. Our own work is guided by a series of bioassays that can efficiently screen crude extracts, column fractions and pure compounds for potential antimicrobial, anticancer, and anti-inflammatory activities. The use of multiple, broad spectrum assays increases the chances of finding compounds with good bioactivity. The chromatographic and spectroscopic techniques of compound isolation, purification and characterization are identical despite the biological activity of that compound. Only the assay that guides these processes in the overall "fishing trip" of dug discovery changes. Search for Antimicrobial Agents The search for new chemotherapetic agents is driven by many factors. New selective antibacterial and antifungal agents are necessary to stem the rising tide of infectious diseases. The increasing incidence of drug resistance in common pathogenic bacteria, as well as the growing number of immunocompromised individuals, is spurring an aggressive search for potential antibiotics and antifungal drugs. Diseases like AIDS, many cancer chemotherapies, and immunosuppressive drugs used in transplant patients, often seriously compromise the immune system, with concomitant fungal and bacterial infections that can be as dangerous to patients as their primary disease. The introduction of the drug penicillin in 1941 was hailed as the dawn of a new era in medicine - an era in which infectious disease could be eradicated for all time. This early optimism proved ingenuous. Sixty years later, infectious diseases are on the rise, particularly in immunocompromised individuals [56]. The Centers for Disease Control has reported that as many as 58% of reported AIDS patients have contracted potentially fatal mycoses [56]. The CDC also reported that the U.S. death rate from infectious diseases increased 60% from 1980 to 1992 [57]. Heart disease and cancer are still the leading causes of death in the Western world. While the primary tissue damage associated with these diseases may be the
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direct cause of death in afflicted patients, it is often the secondary microbial infections that ultimately prove fatal [56]. The proliferation of drug-resistant strains of bacteria is also alarming. Drug-resistant strains of Staphylococcus, Pseudomonas, Mycobacterium, and Streptococcus have infected a growing number of patients, and the arsenal of effective antibiotics is finite. Staphylococcus is still responsible for most hospital-acquired infections, killing 60,000 to 80,000 people per year. Thirty years ago, almost 100% of these infections could be cured by penicillin; by 1982, only about 10% of all clinical Staphylococcus cases could be cured by penicillin [58]. Even vancomycin is ineffective against the latest strain of Staphylococcus to surface in the United States and Japan [59]. It is imperative that new antibiotic and antifungal agents be discovered. Specific Assays for Antimicrobial Activity As we began our own search for antimicrobial agents from the secondary metabolites of microbes, we used the standard disk assay against the bacteria Staphylococcus aureus, Haemophilus influenzae, Mycobacterium smegmatis, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Vibrio harveyii, Enterococcus faecalis, and the fungus Candida albicans. The standard disk assay is suitable for qualitative testing of crude extracts and semi-quantitative testing of pure compounds. The test microbe is streaked onto a suitable nutrient agar. Sterile disks impregnated with the extracts, column fractions or pure compounds to be tested are placed on the microbial lawn. The plate is incubated and checked for zones of growth inhibition in 24 hr. We have recently added several new fungal targets into our antimicrobial screening program. In collaboration with Allen Harmsen, Montana State University, we will be looking for agents with activity against Pneumocystis carinii, causative agent of Pneumocystic carinii pneumonia PCP, Aspergillus niger, and additional species of Candida. Early in the AIDS epidemic PCP emerged as the leading cause of AIDS-related fatalities [60]. Indeed, the rise of PCP in previously
1148
healthy gay men was an early indication of the emergence of AIDS. Pneumocystis carinii pneumonia (PCP) is still a major source of morbidity and mortality in immunocompromised patients. Despite current treatment options, it is the most common opportunistic respiratory infection in patients infected with HIV, and P carinii is a pathogen in patients with a history of malignancy, organ transplant, or other immunologic disorders [60]. Although intensive antiviral regimens have reduced the absolute number of infections (PCP is most often found in AIDS patients with CD4 count below 200), the mortality rate is still 10%-20% of infected individuals [60]. P carinii is a unicellular organism of controversial taxonomy: It has the biochemical structure of a fungus but the medication susceptibility of a protozoan. It has been recently renamed Pneumocystis jaroveci, although most researchers adhere to the older species designation. The vast majority of the population is exposed and seroconverted by 2 years of age, presumably via airborne transmission. Thereafter, lifelong latency is established in the pulmonary alveoli. With loss of cellular immunity, Pneumocystis organisms can propagate and fill the alveoli with a foamy exudate, resulting in a profound ventilation-perfusion mismatch [60]. The drug of choice for PCP treatment is high-dose trimethoprimsulfamethoxazole (TMP-SMX). Unfortunately the "SMX" part of the drug is a sulfa preparation that induces side effects in at least 50% of patients. While opportunistic infections like Pneumocystis carinii pneumonia, cryptospiridiosis, and toxoplasmosis seem to bask in the media spotlight among AIDS-defining conditions, a few others have been lost in the shuffle. Nevertheless, these illnesses pose very serious problems for increasing numbers of people. These include aspergillosis and candidiasis [61]. Aspergillosis is an unusual fungal infection cause by Aspergillis niger. It is found in the lungs and sinuses and symptoms include cough, chest pain, shortness of breath, facial pain, fever, and night sweats. Diagnosis can be made by bronchoscopy; by biopsy, a procedure in which a sample of tissue is taken and examined under a microscope; or by taking a culture of the infected area. The treament of choice is intravenous amphotericin B, but researchers are studying oral itraconazole as an alternative
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treatment. Candidiasis is a fungal infection caused by Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. PCP, aspergillosis, and candidiasis remain a serious threat to immunocompromised individuals and are important targets for chemotherapy [61]. Search for New Anticancer Agents The need for new anticancer drugs seems more compelling than ever. Unfortunately, the discoveries of vinblastine, vincristine, tamoxifen and taxol, or even of a taxol-producing fungus, do not end the quest for effective anticancer agents. Cancer is the second leading cause of death in the Western world, and even the most effective chemotherapeutic agents rarely constitute a cure. New drugs must be found to help in the fight against the complex diseases referred to as "cancers". As is true for antibiotics and antifungal agents, some of the most promising cancer chemotherapeutics are microbially derived, including doxorubicin (adriamycin) from Streptomyces peucitus and daunorubicin from S. coerulerubidos; mithramycin from S. plicatus; bleomycin from S. verticillus, asparaginases from Escherichia coli and Erwina castovara [62]. Our own work has also shown that taxol can be derived from Taxomyces andreanae as well as other, unrelated fungi [4]. Until 2002 our search for compounds with potential anticancer activity was restricted to those that were brine shrimp lethal and to serendipity. All of our new compounds are sent to the National Cancer Institute for screening against their battery of human cancer cell lines. Brine shrimp lethality is a reasonable indicator of cytotoxicity, so many cytotoxic compounds exhibit some degree of activity against certain human cancer cell lines. Compounds occasionally exhibit activity against a particular cancer cell line even if they are not cytotoxic. This is not at all unreasonable as anticancer activity can be a function of many different metabolic phenomena. We still routinely test all of our crude extracts, column fractions and pure compounds for brine shrimp lethality. Several studies have shown that most cancer chemotherapeutic agents exhibit brine shrimp
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lethality and that this would be an effective tool to guide isolation and purification of these compounds from their natural sources [63-66]. Unfortunately, cytotoxic agents can have serious side effects. The tools of molecular biology, in particular DNA microarray technology, are providing a new approach to an old problem [67]. With advances in high-density DNA microarray technology, it has become possible to perform "gene-expression profiling" screens on large numbers of genes to evaluate patterns of up-regulation and down-regulation under various conditions [67]. Even simpler ELISA screens (enzyme-linked immunosorbent assays) can show clear quantitative evidence of the relationship between the overexpression or inhibition of certain enzymes and diseases [68]. Such profiling screens may revolutionize diagnosis and treatment of a wide array of diseases. The data acquired through DNA profiling studies associated with specific disease conditions have highlighted the importance of complex biological pathways in both the development and the treatment of diseases. These signal transduction pathways, often involve a cascade of enzymes that transfer information from remote sites in the body to a target receptor. Either the inhibition or the uppromotion of a specific enzyme in one of these pathways may play a key role in the treatment of a specific disease. Many enzymes have been associated with carcinogenesis and metastasis. Elevated levels of certain enzymes may be associated with several different diseases or disorders. The two enzymes that we use in our bioactivity screens, matrix metalloproteinase-3 and caspase-1, have been associated with certain cancers and with certain immune disorders. These enzyme assays will be described relative to both applications. Autoimmune Disorders and Huntington's Disease and a Search for Potential Therapies An autoimmune disorder is a malfunction of the body's immune system, causing the body to attack its own tissues [69]. Normally, the immune system can distinguish what is self from what is not self through the recognition of specific proteins called antigens. Foreign
1151
proteins - which may be on the surface of bacteria, fungi, viruses, pollen, or food molecules - elicit an immune response. Sometimes the immune system malfunctions, interpreting the body's own tissues as foreign and producing abnormal antibodies (called autoantibodies) or immune cells that target and attack particular cells or tissues of the body. This response is called an autoimmune reaction. It results in inflammation and tissue damage. Different cells or tissues are targeted in different autoimmune disorders. There may be a genetic component to autoimmune diseases, but in many cases, the cause is unknown [69]. Drug prophylaxis can still be effective even in the case of genetic disorders. Studies have shown that selectively inhibiting or stimulating certain enzyme pathways may postpone the onset of disorders or lessen the intensity of symptoms [69]. Over 80 autoimmune disorders have been described. Disorders that target the nervous system include multiple sclerosis, myasthenia gravis, autoimmune uveitis and autoimmune neuropathies such as Guillain-Barre. Primary ulcerative colitis, Crohn's Disease, biliary cirrhosis, and autoimmune hepatitis target the gastrointestinal system. Disorders of the blood include autoimmune hemolytic anemia, pernicious anemia and autoimmune thrombocytopenia. Endocrine disorders include Type 1 or immune-mediated diabetes mellitus, Grave's Disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis and autoimmune disease of the adrenal gland [69]. A few key disorders are pertinent to our work with caspase-1 and matrix metalloproteinase-3 enzymes. I am including the following disorders in this discussion: Multiple sclerosis (MS) is an autoimmune disorder that affects the central nervous system, causing loss of coordination and muscle control [69]. Not all scientists agree that MS is an autoimnune disorder. *
Rheumatoid arthritis (RA) occurs when the immune system attacks and destroys the tissues that line bone joints and cartilage. The disease occurs throughout the body, although some joints may be more affected than others [69].
1152
•
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuromuscular disease caused by the destruction of nerve cells in the brain and spinal cord. This causes the loss of nervous control of voluntary muscles, resulting in the degeneration and atrophy of the muscles. Eventually the respiratory muscles are affected which leads to death from an inability to breathe. The familial type of ALS is caused by a genetic defect in superoxide dismutase, an antioxidant enzyme that continuously removes the highly toxic "superoxide" free radical. However, several researchers have proposed that ALS may have an autoimmune basis. Circulating antibodies have been found in the sera of ALS patients. These antibodies, which have been secreted by denervated muscle, inhibit the stimulation of the sprouting of axons, the long arms of neurons which conduct nervous impulses to other neurons throughout the body [70]. Researchers have also found an immunoglobulin that affects the conductance of neuronal voltage-activated calcium channels, which may induce an excessive release of glutamate from nerve endings [70]. Several studies of ALS patients found the presence of antibodies that interact with motor neurons [71-73]. Huntington's disease is an autosomal-dominant progressive neurodegenerative disorder resulting in specific neuronal loss and dysfunction in the striatum and cortex [74]. Although Huntington's disease is not classified as an autoimmune disease, it has several features common to other autoimmune disorders, including elevated levels of key enzymes [74, 75]. The disease is universally fatal, with a mean survival following onset of 15-20 years and, at present, there is no effective treatment [74]. Each of these diverse conditions has been associated with elevated levels of specific enzymes in the blood sera. These enzymes are key components of signal transduction pathways. If elevated enzyme levels are associated with a particular disease or its symptoms, then inhibition of that enzyme might mitigate the severity of the disease. We have turned to signal transduction pathways to bioactive compounds with pharmaceutical potential.
1153
Use of Signal Transduction Enzymes in Drug Discovery The primary event in all signal-transducing pathways is the reception of an external signal by a specific receptor in the cell membrane. This signal reception activates a chain of reactions that will finally result in an intracellular response. Cell surface receptors generally recognize extracellular signals, so called "primary messengers", and multiply them into a cascade of intracellular events using intracellular signal transducers, so called "second messengers". The movement of signals can be simple, like that associated with receptor molecules of the acetylcholine class: receptors that constitute channels which, upon ligand interaction, allow signals to be passed in the form of small ion movement, either into or out of the cell. These ion movements result in changes in the electrical potential of the cells that, in turn, propagates the signal along the cell. More complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylations by tyrosine kinases and/or serine/threonine kinases. Protein phosphorylations change enzyme activities and protein conformations. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells. We selected two enzyme assays associated with specific signal transduction pathways to guide isolation of active metabolites matrix metalloproteinase-3 (MMP-3) and caspase-1 (casp-1). BIOMOL offers a series of assays geared towards drug discovery that use a 96-well format for moderate through-put of small samples. The inclusion of these inhibition assays in our protocols has enhanced our ability to isolate molecules with drug potential. Preliminary evaluation of each microbe in the Pit collection grown in 12 different physicochemical regimes has provided a series of promising leads for further investigation. Extracts that exhibit strong enzyme inhibitory potential will be purified using these same assays as a guide for compound isolation. Eventually, pure compounds that target the specific signal transducer would be isolated and characterized.
1154
Matrix Metalloproteinases (MMPs) Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodeling [76]. Endogenous tissue inhibitors of metalloproteinases (TIMPs) precisely regulate the levels and metabolic activities of MMPs [76, 77]. Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumor growth and metastasis [77]. Tumor cell invasion is considered to be a disregulated physiologic invasion. Investigators have likened the molecular events involved in the process to events like angiogenesis and wound healing. Matrix metalloproteinase activity is a common denominator in these pathologic conditions and in normal responses. Several studies suggest that the inhibition of matrix metalloproteinase activity may prevent tumor cell dissemination [78]. It has been suggested that MMP inhibitors represent a new therapeutic approach to the treatment of advanced cancers [79]. These inhibitors block the activity of MMP's used by tumor cells to break down and remodel tissue matrices during the process of metastasis. This was believed to be their sole role in anticarcinogenesis. However, recent studies have shown that MMP inhibitors can also act to inhibit tumor growth by preventing local invasion and promoting stromal encapsulation and by inhibiting tumor neovascularization. MMP may have the potential to halt tumor progression and it is possible to envision their use as a low toxicity complement to cytotoxic therapies [79]. Research on the therapeutic use of MMP inhibitors for the treatment of cancers has shown promise [80]. Of particular interest has been data showing that MMP-3 (also called stromelysin-1) plays an important role in the promotion of neoplasia in mice and that inhibition of MMP-3 blocked this activity [80]. Further, serum MMP-3 levels showed significant correlation to clinical disease activity in patients with active rheumatoid arthritis [81]. Elevated levels of MMP-3 have also been found in the
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synovium and cartilage of osteoarthritis and rheumatoid arthritis patients. It has been suggested that serum MMP-3 may serve as a consistent synovial-derived marker of RA disease activity and that early changes in MMP-3 levels may be predictive of disease prognosis [81]. MMP-3 inhibitors may also help to mitigate the symptoms of RA and related diseases [81 - 84]. MMP-3 has also been implicated in the occurrence of multiple sclerosis [85, 86]. Caspase-1 Apoptosis or programmed cell death is a series of ordered events in which selected cells die in a non-necrotic fashion as an essential element of developmental organ formation, cellular homeostasis and immune system function [86]. Key enzyme of the apoptosis signaling cascade (including caspases) are promising targets for the pharmacological modulation of cell death and inflammation [87]. Inflammation is a natural defense reaction instigated by tissue damage or the presence of foreign proteins or pathogens, characterized by redness, heat, swelling, and pain. The primary objective of inflammation is to localize and eradicate the irritant and repair the surrounding tissue. For the survival of the host, inflammation is a necessary and beneficial process, but it can be difficult to control [69]. When the body's own immune responses are directed against its own tissues, autoimmune disorders, characterized by prolonged inflammation and subsequent tissue destruction can result [69]. Autoimmune disorders can cause immune-responsive cells to attack the linings of the joints—resulting in rheumatoid arthritis—or trigger immune cells to attack the insulinproducing islet cells of the pancreas leading to insulin-dependent diabetes [69]. Caspase-1 was the first of a novel type of cysteine proteases responsible for converting interleukin-ip to its mature form in monocytes [88]. Mature IL-lp is a key mediator of inflammation. Caspase-1 is believed to be analogous to CED-3, a cell death protein in Caenorhabitis elegans [89]. Scientists studying various autoimmune disorders have found elevated levels of caspase-1 in
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patients with Huntington's disease [74], amyotropic lateral sclerosis [90], multiple sclerosis [68, 91], stroke [92], and rheumatoid arthritis [93]. All of these diseases exhibit certain autoimmune phenomena. Several researchers have been able to demonstrate that caspase-1 inhibitors have shown some promise in delaying the onset of Huntington's disease [74] and amyotropic lateral sclerosis [90], and in mitigating the effects of stroke [92] and multiple sclerosis [68, 91]. It has also been implicated in the physiological production of interferongamma-inducing factor (IGIF). It therefore appears to play a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors might provide a new class of anti-inflammatory drugs with multipotent action [94]. Although elevated levels of caspases are important in inducing cell death in many types of cancer cells, the overexpression of caspase-1 has been observed in pancreatic cancer (adenocarcinoma) and pancreatitis [95, 96]. Researchers found that although caspase-1 plays an important role in the regulation of apoptosis, it is involved in antiapoptotic processes in pancreatic carcinoma, and inhibition of caspase-1 induces cell death in pancreatic carcinoma cells [96].
5-HT2A Receptor Ligands Any small amino acid derivatives isolated from Pit microbes are tested for activity as specific 5-hydroxytryptamine (5-HT) receptor ligands. Many amino acids and small peptides participate in signal transduction processes, including hormonal control and synaptic transmission of nervous impulses. Neurotransmission involves movement of substances between the synapses of adjacent cells, while hormonal transmission occurs over a distance. The hormonal messenger is transmitted through the bloodstream to the effector cells. Neurotransmitters are usually low molecular weight and reasonably polar, so our search for candidates is confined to the aqueous extracts of each microbe. Our collaborator, Keith Parker works with Chinese hamster ovary (CHO) cells expressing the human 5-HT1A receptor (a gift of Dr. John Raymond; South Carolina Medical University) and NIH 3T3 cells expressing the rat 5-HT2A receptor (a gift of Dr. David
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Julius; UCSF). The 5-HT receptors (5-hydroxytryptamine) are serotonin receptors that have been characterized over the past few years [97]. Serotonin has multiple regulatory roles, including neurotransmission. These include regulation of sleep cycles, the sense of satiety, and blood pressure regulation. Multiple subtypes of 5-HT receptors are recognized: antimigraine drugs are believed to interact with the 5-HT1 and 5HT2 subtypes [97].
RESEARCH DESIGN AND METHODS Microbial Fermentation Schemes Once a Pit microbe is established in pure culture it is grown in several different nutrient broths under different physical conditions. Different physicochemical fermentation conditions can greatly affect the metabolic processes of a microorganism. Studying the secondary natural products produced during fermentation is an effective method of monitoring some of these changes. Biological assays and thin layer chromatography are used to compare secondary metabolite production. Each Pit microbe was initially grown in 6 different nutrient broths as both shake (7 day) and still culture (21 day) to assess what growth conditions yielded the maximum biological activity. Each microbe was grown in a 100 mL nutrient broth in 250 mL Erlenmeyer flasks. The media used included: PDB (DIFCO® potato dextrose broth); PDBH+ ( PDB acidified to pH 2.5 by the drop wise addition of sterile 1.0 M H2SO4); Pit II (filter sterilized Berkeley Pit surface water added to an equivolume sterile solution of 5 g/L soytone and 5 g/L glucose); TSB (DIFCO® tryptic soy broth); TSBH+ (TSB acidified to pH 2.5); and Myc (DIFCO® mycological broth). At time of harvest, these small pilot cultures was killed by the addition of 50 mL of methanol. Cultures were homogenized by omnimixing and extracted thoroughly with chloroform (3X). The chloroform layer was reduced in vacuo to an organic soluble extract. The remaining water layer was rotoevaporated to remove any residual
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solvent, lyophilized, then thoroughly extracted with chloroform/ methanol (1:1, v/v). This second organic extract was reduced in vacuo to generate the freeze-dried extract (FDX). The remaining aqueous material was the freeze-dried residue (FDR). The organic extract, freeze-dried extract and freeze-dried residue were tested for brine shrimp lethality, signal transduction enzyme inhibition, and antimicrobial activity. Microbes with promising activity were tagged for further study using the physicochemical conditions that produced maximum biological activity. RESULTS AND DISCUSSION Berkeley Pit Lake Microbes - Preliminary Data In our first water sample from Berkeley Pit Lake surface waters we found three fungi that we isolated and established in pure culture: Penicillium chrysogenum, Pithomyces sp., and the yeast Hansenula anomala (now called Pichia anomala). In this first study, the organisms were grown in multiple media and harvested as described. Each culture was omnimixed and extracted thoroughly with chloroform (3X). The chloroform layer was reduced in vacuo to an organic soluble oil. The water layer was lyophilized, then extracted with chloroform/methanol (1 :l,v/v). The organic extract was reduced in vacuo to generate the freeze-dried extract (FDX). The remaining aqueous material was the freeze-dried residue (FDR). The organic extract, freeze-dried extract and freeze-dried residue were tested for antifungal and antibacterial activity using standard disk assays, and for cytotoxicity using brine shrimp lethality assay. None of the freeze-dried residues exhibited biological activity in this pilot study. At that time, signal transduction assays had not been incorporated into the testing protocol. The biological activities of the extracts were compared. Activity was concentrated in the organic and freeze-dried extracts. Dramatic differences in activity were observed in extracts from the same fungus grown in different media. For instance, the chloroform extract of Penicillium chrysogenum, exhibited 100% brine shrimp lethality when grown in PDB broth, but
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no activity in any other media. The yeast Hansenula anomala (Pichia anomald) exhibited maximal brine shrimp lethality only when grown in acidified PDBH+ broth and TSB broth. Pithomyces sp. exhibited maximal brine shrimp lethality in PDBH+ and no activity in any other nutrient medium. Data from these preliminary fermentation studies was used to direct specific fermentation studies of the most promising microbes under conditions that promote greatest biological activity. For instance, Pithomyces sp. actually grew more profusely in an acidic medium than in a medium with a neutral pH. As indicated in the activity profile, the chloroform extract of the fungus grown for 10 days in PDBH+ demonstrated 100% brine shrimp lethality, while most of the other extracts were relatively benign. Bioassay guided fractionation of the brine shrimp lethal compounds yielded a series of novel isoprenylated tyrosine derivatives, which will be discussed in a later section [8]. These early results generated sufficient interest to warrant a more ambitious collection of Pit microbes. With the help of James Madison, a research scientist with the Montana Bureau of Mines and Geology, we obtained 100 mL samples of Pit water in sterile collection vials from various intervals down to 720 ft. Microbial isolation was conducted as described previously. The same three fungi found in the surface water in 1995 were isolated again in 1998 from different depths throughout the Pit. Several other bacteria and fungi were also isolated. All of these microbes were established in collection in our lab for further investigation. We have begun the process of comparative fermentation of the Berkeley Pit microbial collection. Biological Activity Profiles At this point we have grown approximately 45 different Pit microbes from water samples and sediment samples using 12 different growth conditions (6 nutrient broths, still and shaken). Microbes isolated from Pit sediments were also grown in both still and shake cultures of DIFCO® actinomyces broth. Each extract generated a crude
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chloroform extract, a freeze-dried extract (FDX) and a freeze-dried residue (FDR). Over 1200 crude extracts have already been tested for biological activity and from these data, specific microbes have been grown in larger volumes for investigation of the bioactive components produced in culture. Table 2 shows signal transduction enzyme inhibitory activity relative to known standard inhibitors. For MMP-3, 0.000 is total inhibition and 0.541 is no inhibition; for caspase-1, 0.000 is total inhibition and 0.100 indicates no inhibition. Table 2.
Signal Transduction Enzyme Inhibition Profiles for Microbial Extracsts MMP-3
Caspase-1
0.000
0.000 0.100
extract code
microbe code
broth
extract
0.541
4-144-1A
PS45A-1E
PDBH+
FDX
0.315
0.095
IB
PS45A-1E
Pit II
FDX
0.003
0.032
1C
PS45A-1E
PDB
FDX
0.194
0.093
ID
PS45A-1E
myc
FDX
0.318
0.089
IE
PS45A-1E
TSB
FDX
0.022
0.067
IF
PS45A-1E
TSBH+
FDX
0.157
0.072
1G
PS45A-1E
act
FDX
0.250
0.030
2A
PS45A-2A
PDBH+
FDX
0.014
0.046
2B
PS45A-2A
Pit II
FDX
0.010
0.057
2C
PS45A-2A
PDB
FDX
0.251
0.082
2D
PS45A-2A
myc
FDX
0.383
0.067
2E
PS45A-2A
TSB
FDX
0.140
0.040
2F
PS45A-2A
TSBH+
FDX
0.095
0.099
2G
PS45A-2A
act
FDX
0.279
0.003
3A
PS45A-4
PDBH+
FDX
0.483
0.100
3B
PS45A-4
Pit II
FDX
0.002
0.000
3C
PS45A-4
PDB
FDX
0.364
0.116
3D
PS45A-4
myc
FDX
0.419
0.090
3E
PS45A-4
TSB
FDX
0.051
0.050
3F
PS45A-4
TSBH+
FDX
0.269
0.140
1161 Table 2.
cont'd
3G
PS45A-4
act
FDX
0.027
0.013
4A
PS 7B-1
PDBH+
FDX
no growth
no growth
4B
PS 7B-1
Pit II
FDX
no growth
no growth
4C
PS 7B-1
PDB
FDX
no growth
no growth
4D
PS 7B-1
myc
FDX
0.167
0.080
4E
PS 7B-1
TSB
FDX
0.105
0.058
4F
PS 7B-1
TSBH+
FDX
no growth
no growth
4G
PS 7B-1
Act
FDX
0.214
0.068
5A
PS 7B-2
PDBH+
FDX
0.317
0.082
5B
PS 7B-2
Pit II
FDX
0.014
-0.330
5C
PS 7B-2
PDB
FDX
0.322
0.094
5D
PS 7B-2
myc
FDX
0.445
0.093
5E
PS 7B-2
TSB
FDX
0.225
0.021
5F
PS 7B-2
TSBH+
FDX
0.015
0.061
5G
PS 7B-2
Act
FDX
0.029
-0.025
6A
PS 7B-3
PDBH+
FDX
no growth
no growth
6B
PS 7B-3
PitH
FDX
no growth
no growth
6C
PS 7B-3
PDB
FDX
0.275
0.080
6D
PS 7B-3
myc
FDX
0.266
0.105
6E
PS 7B-3
TSB
FDX
0.015
0.097
6F
PS 7B-3
TSBH+
FDX
no growth
no growth
6G
PS 7B-3
Act
FDX
0.005
0.052
7A
PS 10-A
PDBH+
FDX
0.339
0.070
7B
PS 10-A
Pit II
FDX
0.002
-0.041
7C
PS 10-A
PDB
FDX
0.303
0.096
7D
PS 10-A
myc
FDX
0.315
0.092
7E
PS 10-A
TSB
FDX
0.029
0.092
7F
PS 10-A
TSBH+
FDX
0.080
0.089
7G
PS 10-A
Act
FDX
0.119
0.007
8A
PS 45A-2B
PDBH+
FDX
0.146
0.075
8B
PS 45A-2B
Pitll
FDX
0.032
0.095
8C
PS 45A-2B
PDB
FDX
0.212
0.095
8D
PS 45A-2B
myc
FDX
0.320
0.094
8E
PS 45A-2B
TSB
FDX
0.103
0.005
1162 Table 3.
Antimicrobial and Brine Shrimp Lethality Data AA MS
(zones of inhibition in mm) SA CA EC PA
1A
PS45A-1E
PDBH+
FDX
8
IB
PS45A-1E
Pit II
FDX
9
15
11
EF
BS S.Pn
HI %kill 8
13
9
_
18% 7%
1C
PS45A-1E
PDB
FDX
8
_
10
_
_
ID
PS45A-1E
myc
FDX
10
11
_
10
11
9
15%
_
_
16%
IE
PS45A-1E
TSB
FDX
8
_
_
_
_
_
_
_
_
_
_
_
19% 36%
_
_
29%
-
8%
IF
PS45A-1E
TSBH+
FDX
8
_
1G
PS45A-1E
act
FDX
8
_
_
_
_
_
2A
PS45A-2A
PDBH+
FDX
8
11
_
8
12
9
2B
PS45A-2A
PitH
FDX
10
13
_
11
15
9
8
8
8%
_
_
_
_
_
_
28%
_
2C
PS45A-2A
PDB
FDX
10
_
2D
PS45A-2A
myc
FDX
.
_
25%
2E
PS45A-2A
TSB
FDX
9
_
2F
PS45A-2A
TSBH+
FDX
8
12
_
2G
PS45A-2A
act
FDX
9
9
_
-
-
_ _
18% 11
3A
PS45A-4
PDBH+
FDX
-
3B
PS45A-4
Pit II
FDX
8
_
_
_
3C
PS45A-4
PDB
FDX
8
_
_
3D
PS45A-4
myc
FDX
8
-
13
3E
PS45A-4
TSB
FDX
.
3F
PS45A-4
TSBH+
FDX
10
14
3G
PS45A-4
act
FDX
-
.
4A
PS 7B-1
PDBH+
FDX
4B
PS 7B-1
Pit II
FDX
4C
PS 7B-1
PDB
FDX
4D
PS 7B-1
myc
FDX
10
15
_
17
17
PS 7B-1
TSB
FDX
4F
PS 7B-1
TSBH+
FDX
4G
PS 7B-1
act
FDX
9
5A
PS 7B-2
PDBH+
FDX
_
5B
PS 7B-2
Pit II
FDX
5C
PS 7B-2
PDB
FDX
5D
PS 7B-2
myc
FDX
_
FDX
_
5E
PS 7B-2
TSB
14 8
_
_
-
10% 16% 10%
_
_
_
_
_
_
_
31%
-
8
-
100%
16
4E
_
_
-
12
9
-
100%
14
9
.
-
10%
12
9
-
100%
12
11
-
100%
12
9
25
_
-
_
12
_
42%
_
11
81%
8
100%
-
19% 44% 9% 30%
_
9
77%
1163 Table 3.
cont'd.
5F
PS 7B-2
TSBH+
FDX
_
_
_
5G
PS 7B-2
Act
FDX
_
_
_
6A
PS 7B-3
PDBH+
FDX
6B
PS 7B-3
Pit II
FDX
6C
PS 7B-3
PDB
FDX
_
PS 7B-3
myc
FDX
6E
PS 7B-3
TSB
FDX
6F
PS 7B-3
TSBH+
FDX
6G
PS 7B-3
Act
FDX
7A
PS 10-A
PDBH+
FDX
7B
PS 10-A
PitH
FDX
7C
PS 10-A
PDB
FDX
7D
PS 10-A
myc
FDX
_
_
7E
PS 10-A
TSB
FDX
_
_
_
_
_
g _ 10
17
PS 10-A
TSBH+
FDX
7G
PS 10-A
Act
FDX
_
_
_
_
-
-
-
PDBH+
FDX
Pit II
FDX
13
20
8C
PS 45A-2B
PDB
FDX
_
_
8D
PS 45A-2B
myc
FDX
8E
PS 45A-2B
TSB
FDX
FDX
17 _
15% 40%
_
_
75%
-
10
13
9
-
15%
-
-
-
33%
55%
.
_
_
_
_
-
15
_
-
-
9 _
29%
23% 62%
g
_
-
-
_
31% 30%
10
-
_
42%
33%
-
-
50%
_
_
PS 45A-2B
40%
_
_
_
PS 45A-2B
Act
_
_
_
_
8B
TSBH+
-
_
8A
PS 45A-2B
-
_
_
-
PS 45A-2B
_
_ 14
7F
8G
-
_
_
8F
_
_
6D
FDX
_
_
27% 18%
-
64%
-
-
30%
_
_
23%
The data displayed in Table 2 and 3 provide clear guidelines as to which microbes should be studied, and what conditions should be used for that study. The code name for each microbe reflects its isolation history. For instance, microbe PS45A-4 was isolated from Pit sediment (PS) from 45 cm below the water-sediment interface. Using these data, we prioritized the order in which the microbes would be studied and the broths in which each would be grown. PS45A-4 exhibited strong signal transduction enzyme inhibitory activity against both MMP-3 and caspase-1 when grown in Pit II, TSB and Act broths. It also inhibited the growth of Streptococcus pneumoniae in the latter two broths. This microbe has been regrown
1164
in larger volumes of each of these broths to facilitate the isolation and characterization of the compounds responsible for these activities. These same data profiles have been generated for all of the microbes grown to date. As each microbial extract is fractionated, the appropriate bioassay is used to guide which fractions are resolved into pure compounds and eventually characterized. Three such studies will be described below. 5-HT2A Receptor Ligand from Pithomyces sp One of the first fungi isolated from the Pit Lake, Pithomyces sp. exhibited good brine shrimp lethality when grown in PDBH+ broth (pH 2.7, 11 days shaken) [8]. When the fungal culture was harvested and extracted, most of the activity was confined to the chloroform extract. LH-20 chromatography of this extract followed by RPHPLC gave the three cytotoxic compounds 1-3, and the three inactive sesquiterpenes, 5-7. The structures of all seven compounds, including 4, the methyl ester of amido acid 3, were determined through the use of mass spectrometry, and ID and 2D NMR techniques. The brine shrimp lethality assay was used to guide isolation.of compounds 1-3. Sesquiterpenes 5 - 7 were isolated by "NMR-guided fractionation". We often isolate and characterize compounds with interesting NMR spectra even if they are inactive. Sesquiterpenes are more commonly found in higher plants rather than fungi.
CQ2R
3R=H 4R=Me Fig. (1). Structures of isoprenylated tyrosine derivatives
1165
HO y
CH 3
HO y
CH3 CH 3
OH
CH3
Fig. (2). Structures of sesquiterpenes from Pithomyces sp.
Simple aromatic amino acid derivatives often exhibit neurotransmitter activity. Therefore, compounds 1-4 were evaluated using the 5-HT2A receptor assay. Studies suggest that 5-HT2A (serotonin) receptor antagonists might act as migraine preventatives [98], or as antihypertensive agents [99]. In this assay, we assessed the ability of a compound to displace radiolabeled ketanserin from rat 5HT2A receptor's ligand binding site. Since 100% of the ketanserin is bound in the control setting, smaller numbers indicated positive results as less ketanserin is bound when the test compound is a good displacer. Compounds were tested at 100 uM concentrations [97, 100]. Data from this assay suggested that 2 acts as a 5-HT2A receptor ligand, while the other compounds are only marginally active. Drugs that act as 5-HT2A ligands include ketanserin, methysergide, the tricyclic antidepressant amitryptiline and certain calcium channel and beta blockers [98]. Isolation of Novel Sesquiterpenoid MMP-3 Inhibitors from an Acid Mine Waste Extremophile We also isolated a filamentous fungus from the surface waters of the Berkeley Pit that was identified as a Penicillium sp. [50]. The fungal isolate (PitNA4) was grown in six different broths, including unmodified (pH 5.1) and acidified PDBH+ broth ( pH 2.7) as still cultures for 21 days. The cultures were killed by the addition of methanol the mycelia were removed by gravity filtration and the filtrates were extracted with chloroform. The chloroform extract of
1166
this Penicillium sp. was active in both the MMP-3 and caspase-1 inhibition assays when grown in PDBH+ broth. Both of these assays were used to guide flash silica gel column chromatography and silica gel HPLC that yielded sesquiterpenes 8, 10 and 11 and coumarin analog 12. The structures of all of the compounds, including 9, the acetylation derivate of compound 8 were determined by spectral methods. Compounds 8-11 belonged to the bisabolane family of sesquiterpenes. Bisabolane sesquiterpenes are not typical microbial metabolites. Most compounds of this skeletal class have been isolated from numerous terrestrial plants, a basidiomycete [101], sponges [102-103], octocoral [104, 105], and red algae [106]. To our knowledge there have been two reports of bisabolanes from fungi. The first report in 1989 was of a mycotoxin from Fusarium sambucinum [107]. More recently, mass spectral analysis of the volatile constituents of toxigenic Penicillium roqueforti strains yielded p-bisabolene [108]. The molecular formula and collective NMR data for compound 12 seemed to fit nicely into the structure of the isochromenone, orthosporin, which was previously reported from the fungus Rhynchosporium orthosporum [109]. More careful comparison of the data, however, indicated that 12 fit into a coumarin skeleton, as shown in Fig. (3). There are few 3-alkyl- 6,8-dioxy coumarins known from fungal sources. These include 3-hydroxymethyl-6,8-dimethoxy coumarin from Talaromyces flavus [110].
u Fig. (3).
OH
12R = I 13R-Ac
Bisabolane and coumarin derivatives from PitNA4.
1167
The three sesquiterpenes showed moderate inhibitory activity against both MMP-3 and Caspase-1. Each compound was tested in triplicate at concentrations from 300 uM - 300 nM. All three compounds showed MICso's in the 30 uM range against Caspase-1 and in the 300 nM range against MMP-3. Compound 8 showed the greatest potency and 10 the least inhibitory potential.
Isolation of Berkeleydione and Berkeleytrione Signal transduction enzyme inhibition assays guided the isolation of two novel hybrid polyketide-terpenoid metabolites from a Penicillium sp. growing in the deepest waters (>750 ft) of Berkeley Pit Lake [9]. Their structures were deduced by spectroscopic analysis and confirmed by single crystal x-ray analysis on berkeleydione (13). Both compounds inhibited signal transduction enzymes caspase-1 and matrix metalloproteinase-3. Berkeleydione (13) was also active against non-small cell lung cancer in NCI's 60 cell line anti-tumor screen. o .OH
14 Fig. (4). Structures of berkeleydione and berkeleytrione
The crude organic extracts of a Penicillium sp. isolated from a depth of 885 ft. were active against Staphylococcus aureus and in the brine shrimp lethality screea These extracts were further tested using enzyme inhibition assays for two different signal transduction enzymes - matrix metalloproteinase-3 (MMP-3) and caspase-1 (Casp1). Compounds 13 and 14 were isolated from the chloroform extracts of the broth filtrate of & Penicillium sp. found growing in Berkeley Pit Lake. Berkeleydione (13, 5.5 mg/L) was isolated as a crystalline
1168
solid. High-resolution CIMS (chemical ionization mass spectrometry) established the molecular formula of C26H33O7 (M++H) with 11 units of unsaturation. Extensive ID and 2D NMR data generated three possible structures, but there were inconsistencies with each structure proposed. The quantity and proximity of the many quaternary carbons made an unambiguous structural determination impossible. A single crystal was submitted for X-ray crystallographic analysis (Fig. 5).
Fig, 5
ORTEP drawing of berkeleydione
With the structure in hand we could make the spectral assignments which were largely straightforward based on extensive ID and 2D NMR experiments. Berkeleytrione (14, 3.4 mg/L) was isolated as an amorphorus solid. High-resolution EIMS established the molecular formula of C26H34O7 with 10 units of unsaturation. The structure was determined through spectroscopic methods. Several hybrid sesquiterpene-dimethyl orsellinate metabolites are known from Aspergillus sp [111-114]. All of these are highly oxygenated and have undergone rearrangements. Biosynthetic studies have demonstrated that the precursor of the terpenoid portion is farnesyl pyrophosphate and of the nonterpenoid portion is a bis-Cmethylated polyketide [115]. Berkeleydione (13) and berkeleytrione (14) effectively inhibited both MMP-3 and caspase-1 in the micromolar range. Berkeleydione
1169
(13) was tested in NCI's anti-tumor screen against 60 human cell lines. It showed selective activity towards non-small cell lung cancer NCI-H460 with a Logio GI50 of -6.40. This extreme selectivity is noteworthy in a natural product that has not been derivatized or tailored towards a particular cancer type. Although we are still at an early stage in this overall research endeavor we have already found the microbes of the Berkeley Pit Lake to be a source of new and interesting secondary metabolites. It is not often that scientists have the opportunity to explore such a unique environment and we are fortunate to have easy access this dynamic ecosystem. Based on preliminary data, we expect to find much interesting chemistry in this project. Of equal importance, however, is the learning environment that this research has provided not only our undergraduate students, but also other students in related Pit research projects. Our combined efforts should afford new insights into the acid mine waste phenomenon and the organisms that live in these inhospitable waters. As to the secondary metabolites and their microbial producers - they could be the richest products ever mined from "the richest hill on earth". ABBREVIATIONS DNA PCR Taq pH HBOI P-388 HCT8 A549 HSV-1 CaN CPP32 ICE2 ICP PDB
= = = = = = = = = = = = = =
deoxyribonucleic acid polymerase chain reaction Thermus aquaticus -log(H+) Harbor Branch Oceanographic Institution murine (mouse) leukemia human colon cancer human alveolar cancer Herpes Simplex Virus-1 calcineurin caspase-3 interleukin-2 converting enzymes inductively coupled plasma potato dextrose broth
1170
PDBH+ TSB TSBH+ myc act Pit II PCP TMP-SMX casp-1 MMP-3 TIMPs 5-HT CHO FDX FDR NMR
potato dextrose broth acidified to pH 2.7 tryptic soy broth tryptic soy broth acidified to pH 2.7 mycological broth actinomyces broth Pit Lake water broth Pneumocystis carinii pneumonia trimethoprim-sulfamethoxazole caspase-1 matrix metalloproteinase-3 tissue inhibitors of metalloproteinases 5 -hydroxytryptamine Chinese hamster ovary freeze-dried extract freeze-dried residue nuclear magnetic resonance
ACKNOWLEDGEMENTS We thank our colleagues from the Department of Chemistry, Montana State University: S. Busse for assistance with NMR spectroscopy and L.J. Sears for mass spectral data and J.Madison, Montana Bureau of Mines and Geology, for Pit water samples. We thank the National Science Foundation grant # 9506620 for providing funding for NMR upgrades at the MSU facility and grant #CHE-9977213 for acquisition of an NMR spectrometer; NIH grants GM/OD 54302-02 and NCRR Grant # P20 RR15583 to the NIH-COBRE Center for Structural and Functional Neuroscience for funding the neurotransmitter bioassay work; NIH Grant P20 RR-16455-02 (BRIN Program of the National Center for Research Resources); USGS grant 02HQGR0121, and NIH grant CA24487 (JC) for financial support of this research. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of NIH or the U.S. Government.
1171
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.
ISOFLAVONESAS COMPONENTS
FUNCTIONAL
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*F.R. MARIN1, J.A. PEREZ-ALVAREZ2, C. SOLER-RIVAS1. Departamento de Quimica-Fisica Aplicada (Area de Tecnologia de Alimentos), Facultad de Ciencias, Universidad Autonoma de Madrid, 2804,. Madrid, Spain. 'Departamento de Tecnologia Agroalimentaria (Division de Tecnologia de Alimentos), Escuela Politecnica Superior de Orihuela, Universidad Miguel Hernandez de Elche, 03312, Orihuela (Alicante), Spain. * Author to whom correspondence should be addressed: Francisco R. Mar in (e-mail: [email protected]; Phon.: 3414973585; Fax.: 34914973579) ABSTRACT: Isoflavones constitute a characteristic and very important subclass of flavonoids. Their structures are based on the 3-phenylcromen skeleton that is chemically derived from the 2-phenylchromen skeleton, by an aryl-migration mechanism. Structurally, isoflavonoids can be classified according to the oxidation of the Ci5 skeleton, their complexity and the internal formation of the heterocyclic rings. Isoflavones are mainly found in legumes and particularly in soy, although their presence has also been reported in black beans, green split peas, chickpeas, lima beans, split peas, alfalfa sprouts, sunflower seeds and clover sprouts. Moreover their natural distribution in raw materials, their presence as an ingredient in the composition of several foods, soy products in infant foods, vegetarian formulations, etc. lead to their ubiquitous presence in foodstuffs. Due to their ubiquitous distribution in food and the claimed beneficial health effects of foods containing isoflavones, their distribution in foods and healthy properties have been reviewed. Isoflavones have been proposed to have estrogenic activity and play a putative role in the prevention of climacteric syndrome. Other healthy properties as a possible role in renal disease protection, learning memory behaviour during aging, prevention of some types of cancer, bone metabolism, and thrombogenicity, among others, have been reported. Related to their biological activities, studies at molecular level involving interaction between different types of isoflavones and estrogen receptors, and others, show hierarchies correlating biological activities and chemical structures. Key Words: Isoflavones, nutraceuticals, functional foods.
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INTRODUCTION Flavonoids are a type of compounds omnipresent in the plant kingdom and, unavoidably, form part of our diet, because they constitute up to 2% of the total photosynthesised carbon. Flavonoids are plant secondary metabolites (they have not been reported as naturally-occurring in animals), aromatics and belong to the group of plant phenols [1]. From a purely chemical point of view, flavonoids are characterised by a skeleton of three units, C6-C3-C6, that forms a cyclic structure in most cases [2], In this skeleton two aromatic rings, referred to as A and B (in chalcones), can be distinguished, with an additional third ring (C) in the rest of the flavonoids. This last ring appears as a cyclation of chalcones with a hydroxyl in 6' position (Fig. (1)), while the A and B rings have a different metabolic source. The B ring is formed in the shikimate pathway, while the A ring comes from the condensation of three units of malonyl Co-A [3, 4]. Flavonoids can be classified according to the degree of oxidation of the three-carbon central segment. From lower to higher degree of oxidation, flavonoids are usually classified as catechins, chalcones, flavanones, isoflavones, flavan-3, 4-diols, flavones, aurones, flavonols and anthocyanins (Fig. (2)) [5], Isoflavonoids are a characteristic and very important subclass of flavonoids. Their structures are based on the 3-phenylchromen skeleton that is chemically derived from the 2-phenylchromen skeleton, by an arylmigration mechanism. Structurally, isoflavonoids can be classified according to the oxidation of the C15 skeleton, their complexity and the internal formation of heterocyclic rings [6]. Isoflavonoids are metabolically derived from the flavanones. The central step is the migration from the C2 to the C3 of the aryl block, which constitutes the B ring of the flavanone intermediate. This reaction is catalysed by 2-hydroxyflavone synthase, a cytochrome P450. At the same time, the isoflavones are precursors of a substantial number of compounds involved in the biosynthesis of phytoalexins and pterocarpanes. Epidemiological data report that isoflavones have multi-biological and pharmacological effects in humans, including estrogen agonist and antagonist activity, cell signalling conduction, as well as cell growth and death promoting. The mechanism through which isoflavones exert the above-mentioned functions is not only based on their estrogenic properties, but also on their roles as protein tyrosine kinase inhibitors,
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regulators of gene transcription, modulators of transcription factors and as antioxidants. They may also alter some enzymatic activities [7].
Fig. (1). Basic skeleton of flavonoids: (i) Chalcones, (ii) Phenylbenzopiran-4-one.
HO.
HO.
OH OH
Anthocyanldin
OH 0H
Catechin
Fig. (2). Chemical structures of the various classes of flavonoid.
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CHEMICAL FEATURES OF ISOFLAVONES Isoflavones constitute one of the largest groups of natural flavonoids, with about 364 aglycones (unconjugated forms) reported yet [6]. The most thoroughly investigated and perhaps most yet interesting phytoestrogens are the isoflavones, i.e. daidzein, genistein, formononetin and biochanin A, together with the coumestan coumestrol -a coumarine-like compoundmetabolically derived from the isoflavone daidzein. The isoflavones (daidzein and genistein) exist in four related chemical structures, namely aglycones (daidzein and genistein), the 7-O-glucosides (daidzin and genistin), the 6'-O-acetylglucosides (6'-O-acetyldaidzin and 6'-O-acetylgenistin) and the 6'-O-malonylglucosides (6'-Omalonyldaidzin and 6'-O-malonylgenistin). Other naturally occurring isoflavones are metabolically derived from these. Thus, daidzein is the metabolic precursor of formononetin and genistein of biochanin A. On the other hand, both formononetin and biochanin A are metabolised, after ingestion, to daidzein and genistein, respectively, and subsequently to equol and p-ethylphenol, respectively, as we will see below. In addition, glycitein, an isoflavone similar in structure to daidzein and genistein, has been isolated from plants, but only one report on its estrogenic activity has been found when preparing this review. Similarly to daidzein and genistein, there are four different chemical structures for glycitein: the aglycone glycitein, 7-O-glucoside glycitin, 6'-Oacetylglycitin and 6'-O-malonylglycitin. Other compounds, such as coumestrol, cannot be classified as isoflavones. However, coumestrol is a natural metabolic product of daidzein, together with other chemicals, such as the phytoalexins, phaseollin, dalbergioidin and kievitone. From a structural point of view, the coumestran coumestrol has been described as a free form and as an aglycone, although no reports on glucosylated and/or other natural forms of conjugation have been found. In Fig. (3), which shows some of the above-mentioned chemicals, the structural similarity with natural estrogens can be seen. Distribution of Isoflavones Fransworth and co-workers [8] have reported 94 plants as exhibiting estrogenic activity; not all these plants are edible. Isoflavones are mainly found in legumes and particularly in soy. Soybeans and soy products are a
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particularly a rich source of isoflavones, which may range in dry weight, from 0.2 to 1.6 mg/g. The principal isoflavones found in soy foods are genistein, daidzein and glycitein. Their presence has also been reported in black beans, green split peas, clover sprouts, chickpeas and other legumes [4]. Other isoflavones, such as brochamin and formononetin, have been found in green beans, chickpeas, lima beans, split peas, alfalfa sprouts, sunflower seeds and, particularly, in red clover [9, 10]. The widespread use of soy products in infant foods, vegetarian formulations and as an ingredient in the composition of several foods contributes to its ubiquitous presence in foodstuffs. Soy isoflavones are found in four chemical forms: the unconjugated form, as glucoside (daidzein, genistein and glycitein), acetylglucoside and malonylglucoside, Fig. (3) [10]. Moreover, processing and fermentation of the soybean has been reported to influence the forms isoflavones take. Similarly, after ingestion, isoflavones are modified during gut transit and/or in the liver to give a batch of metabolic by-products, including glucouronic conjugates, such as p-ethylphenol [11]. Reinli & Block [10] reviewed more than 36 research papers in 1996 with the aim of compiling and standardising all the previous information. Due to the variability of the research results, partially due to analytical procedures, isoflavone levels were recalculated on a wet weight basis. Table 1 shows some of the most relevant results reported by Reinli & Block -together with others from different sources- on the ratio of isoflavones present in different foods. However, it has to be borne in mind that substantial variations in the phytoestrogen content may occur as a consequence of the genetic differences of various varieties, environmental conditions, such as location and harvesting year and degree of maturity. BIO AVAILABILITY AND METABOLISM Isoflavones, a special kind of polyphenols, show similar behaviour regarding absorption and metabolism to this family of compounds. Thus, Scalbert et al [12] put forward two primary sources of evidence concerning the absorption of polyphenols: first, indirect evidence for their absorption through the gut barrier as can be seen from the increase in the antioxidant capacity of the plasma following the consumption of polyphenol-rich foods, and second, their recovery in urine after the ingestion of given amounts of a particular polyphenol.
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OH
0
Genistein
OH
Daidzein
0 OCH3 OCH3
Formononetin
Biochanln A
OH
0
Glycitein
OCH3
O-DMA
Coumestrol
OH
Estradiol Fig. (3). Chemical structures of major isoflavones, metabolic derivatives, coumestrol and estradiol.
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Since polyphenols are retained until they, or their metabolic products, are excreted by the urine or faeces, they undergo a number of complex microbial and chemical transformations that may include chemical conjugation, hydrolysis and biliary excretion [12]. The few existing studies in humans show that the quantities of polyphenols found intact in urine vary from one phenolic compound to another. Among flavonoids, recovery is low for some flavonols, such as quercetin and rutin (0.31.4%), but high for other flavonoids, such as catechins, anthocyanins, flavanones and soy isoflavones (3-27%) [13]. Absorption It has been suggested that the polyphenol structure may have a major impact on intestinal absorption. The most widely discussed structural parameters are molecular weight, glycosylation and esterification. As mentioned above, single flavonoids, such as isoflavones, have higher recovery values than polymeric flavonoids, such as tea theaflavins (0.0006%) and other kinds of flavonoids, such as flavanols. However, the recovery values of isoflavones are lower than those reported for phenolic acids [14]. On the other hand, a different pattern of urine recovery has been described for the soy isoflavone structure. For genistein, for example, a urine recovery of approximately 8% has been reported while for daidzein the rate was approximately 3%. This difference may be explained by structural differences. Thus, genistein has a hydroxyl moiety in C-5, while daidzein does not have it, Fig. (4). This slight difference in the hydroxyl substitution patter influences their water solubility. The Merck Index describes that genistein is slightly soluble in hot water, while daidzein is not at all soluble in water, which goes a long way to explaining their differential behaviour in urine.
OH Genistein
0H
Daidzein
Fig. (4). Differences between the isoflavones, genistein and daidzein, which might influence their recovery in urine.
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The glucosylation pattern seems to influence absorption through the gut barrier. Thus, the absorption of quercetin, a flavonol, has been measured in ileostomised volunteers. The absorption of the quercetin glycosides contained in onions was higher (52%) than that of quercetin aglycone (24%) [15]. In contrast, rhamnosides of quercetin are more poorly absorbed than the former glycoside and aglycone structures. It is proposed that the gut absorption of these rhamnosides would require deglycosylation by the colonic microflora, as suggested by their delayed absorption compared with the glycosides [16]. On the other hand, the ready absorption of quercetin glucosides might be due to their hydrolyses either by the lactase phlorizin hydrolase or the cytosolic P-glycosidase in the enterocyte, although this explanation cannot be extended to all classes of flavonoids [11]. The main soy isoflavones (genistein and daidzein) are 7-O-P-Dglucosides, while the aglycone forms (genistein and daizdein) are minor compounds compared with the glycosylated forms. Some authors suggest that after ingestion, the conjugated form of isoflavones is hydrolysed by intestinal (3-glucosidases -lactose phlorizin hydrolase, a membrane-bound enzyme found in the brush-border of the small intestine, which releases the principal bioactive aglycones, daidzein and genistein. These compounds may be absorbed or further metabolised in the distal intestine with the formation of specific metabolites, such as equol, O-DMA and pethylphenol [11]. Regarding the formation of these isoflavone derivatives, it is extremely interesting that some people are unable to produce equol or that they produce it in very low amounts. In fact, studies report that a third part of the general population cannot form equol. This demonstrates that the breakdown of isoflavones by the microflora in the gut determines the recovery of the compounds, and that the excretion to the urine of equol and other isoflavones derivatives, such as O-DMA, is dependent on the different composition of intestinal microflora. To confuse the issue even more, some researchers report that the production of isoflavone derivatives, such as equol, also depends on diet and gender: a high fat/meat content diet increases equol production in women but not in men, which is explained by promotion of the growth or the activity of the bacterial populations responsible for equol production. On the other hand, no age-related differences for isoflavone metabolism have been reported [12, 13]. There are contradictory findings as regards the need for the intestinal modification of isoflavones before they can be absorbed and
1185 Table 1. Food sources and content in major isoflavones. Where concentrations are available they are quoted as extreme values [8,9]. ND: Not Detected. Trace Daidzein Genistein Formononetin Biochanin A Food product Soybeans Black soybeans Green soybeans Soy nuts Tofu Tofu, fermented Soy flour Soy meal, defatted Soy meal, whole Soy flakes Soy flakes, defatted Soy flakes, whole Soy granule Soy protein, textured Soy concentrate Soy milk Alfalfa (buds) Alfalfa sprouts Clover sprouts Mung bean sprouts Soy bean sprouts Tempeh Soy sauce Miso Soy hot dog Soy bacon Tofu yoghurt Soy mozzarella Soy fibre Green beans Large lima beans Red beans Chick-pea Kidney beans Pink beans
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
676-1007 270-699 546 575 29-146 36-117 226-655 575-706
200-1382 277-612 729 729 50-166 40-218 478-1125 683-1000
ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND
706 221-880 419-1166
1000 280-1561 1411-1951
ND ND ND
ND ND ND
744
1320
ND
ND
549 83-568
748 568-707
ND ND
ND ND
43-107 37-74 ND ND ND ND
58-211 41-103 ND ND ND ND
ND ND T 3.4 22.8 T
ND ND T ND 4.4 ND
138-211 137-273 8-14 71-366 34 28 57 11 171 ND ND T ND ND ND
112-230 235-398 5-9 260-524 82 69 94 36 210 ND ND 3 ND ND ND
ND ND ND ND ND ND ND ND ND 1.5 14.8 ND ND 10.5 T
ND ND ND ND ND ND ND ND ND ND ND ND 15.2 ND ND
become available to the organism. Virtually all circulating polyphenols are glucuronidated and/or sulphated and no free aglycones are found in plasma [12, 17, 18], except for particular flavonoids, such as phloretin,
1186
which is present in rat plasma both in conjugated and non-conjugated forms [19] and for diosmetin, which is the circulating form in plasma of its glycosylated forms [20], In general terms and according to quercetin model, O-glucouronides are formed in the gut barrier, as mentioned above and secreted back either to the gut lumen or to the serosal side. These conjugates then reach the liver, where they are further metabolised [21]. However, free flavonoid aglycones are found when pharmacological doses are administered, indicating a possible saturation of the conjugation pathways [22]. Notwithstanding, other authors do not agree with this point of view. Andlauer et al [23], for example, reported that genistein was partly absorbed without a previous cleavage. Consistent with this suggestion is the finding that both aglycones and their glycosides are absorbed very rapidly [24]. Other authors even report that isoflavone aglycones are absorbed faster and in greater amounts than their corresponding glycosides in humans [25], which of course contradicts the above assumption. A different behaviour has been described in non-monogastric animals. Thus, in ruminants, isoflavones are mainly absorbed in the rumen, where the gastrointestinal epithelium is the major site of metabolism and the liver contributing very little to total isoflavones metabolism in these animals [26]. Metabolism The aglycones, along with any other bacterial metabolites derived from them, are absorbed from the intestinal tract and transported via the portal venous system to the liver, where the isoflavones and their metabolites are metabolically modified [7]. In general terms, all polyphenols are extensively metabolised either in tissues after absorption through the gut barrier, or, in the case of the nonabsorbed fraction and the fraction re-excreted in the bile, by the colonic microflora. All polyphenols, including isoflavones, are conjugated to form O-glucouronides, sulphate esters and O-methyl ethers. Lundth [26] proposed that isoflavones and their metabolites are conjugated mainly (95%) with glucuronic acid and, to a lesser extent, as sulphate conjugates. The formation of anionic isoflavone derivatives by conjugation with glucouronides and sulphate groups facilitates their urinary and biliary excretion and explains their rapid elimination. Thus, when the oral intake of soybean products is studied in human, genistein
1187
and daidzein are found in plasma two hours after feeding and reach their highest concentration six hours later. The half-life of genistein and diadzein is estimated to be 8.3 and 5.8 hours, respectively, both showing a high degree of recovery in faeces [12]. These results indicate that isoflavones persist more in the body than other classes of fiavonoids, which are quickly eliminated both in urine and bile after ingestion. In human, a post-prandial peak is observed 1-2 h after ingestion of various flavonols and flavanols. In the case of most fiavonoids absorbed in the small intestine, the plasma concentration then rapidly decreases, the repeated ingestion of polyphenols over time being necessary for the maintenance of high concentrations in plasma [11, 27]. Isoflavones can be detected in many animal tissues. A tissue distribution of daidzein of 40 mg/kg body weight has been reported in rats 15 min after intravenous injection. Daidzein concentration was found to be high in plasma, liver, lung and kidney (about 30 ug/g wet weight) to be moderate in skeletal muscle, spleen and heart (about 15-20 ug/g wet weight) and to be low in brain and testis. After feeding rats with 5, 100 and 500 ug/g genistein, other researchers reported a dose-dependent increase in total genistein concentration in brain, liver, mammary, ovary, prostate, testis, thyroid and uterus, with the highest content being observed in the liver and kidney where it is three to five times more concentrated than in plasma [28, 29, 30]. Polyphenol conjugates may also exert biological activity after deconjugation at the cellular level. Deglucouronidation and demethylation of phenolic estrogen conjugates have been described in the presence of lysosomes isolated from hamster kidney and liver. Similarly, the hydrolysis of estrone sulphates by mammary cancer cell lines has been described [31, 32]. The extent of polyphenol methylation would also affect the biological properties of polyphenols, although this is not important, as major food isoflavones do not have a cathecol pattern, Fig. (3), unlike other bioactive polyphenols such as catechin, quercetin or caffeic acid which are O-methylated "in vivo" [11]. While the last part of the small intestine is where fiavonoids glycosides are hydrolysed, conjugated with glucouronides and absorbed. Those not absorbed, such as aglycones and other fiavonoids, undergo metabolic change in the colon. In general terms, polyphenols, once they reach the colon, are extensively metabolised by the microfiora. The flavonoid glycosides, such as rutin, which do not absorb in the upper part of the intestinal tract can be hydrolysed and the aglycone absorbed. The flavonoid glucouronides excreted in the bile can also be hydrolysed by the
1188
microflora and the resulting aglycone reabsorbed, thus entering an enterohepatic cycle [11, 33, 34]. Further, aglycones are also metabolised to a wide array of low molecular weight aromatic acids, such as phenylvaleric, phenylpropionic, phenylacetic and benzoic acids, that are well absorbed through the colonic barrier. Several authors report that the lowest degree of polyphenols absorption takes place in the small intestine the highest amount reaching the colon, where the tissue is exposed to microbial flavonoids metabolites and the non-absorbed flavonoids from the small intestine [35, 36]. Most studies on the biological properties of polyphenols have been carried out on flavonoids in their native form. However, many of their biological effects observed in animal or clinical studies may be equally explained by their microbial metabolites. As regards isoflavones, little is known so far. On the one hand, as we see below, some isoflavone activities may be explained by their similarity with estrogenic hormones and therefore molecular integrity or minimal modification of the structure must occur to preserve activity. For example, equol (a metabolite of daidzein isoflavones) showed higher affinity for estrogen receptors than did daidzein itself, while daidzein and genistein glucouronides had, respectively, a 10- and 40-fold lower affinity for estrogenic receptors as compared to their aglycones [37, 38]. On the other hand, many other isoflavone activities, see below, are not related with their structural similarity with estrogens and therefore their physiological activity may depend directly on their own structure, or conjugates, or putative products of low molecular weight produced by microflora metabolism, which have not been described yet. Whatever the case, our knowledge on polyphenol bioactivity is reaching a point whereof the results used to explain their activity must be reconsidered. FUNCTIONAL PROPERTIES During recent years, isoflavones have received increasing interest due to their estrogenic and antiestrogenic effects, and have been associated with the reduced incidence of breast and prostate cancer, cardiovascular diseases or osteoporosis, although they also exhibit other favourable effects through an array of different mechanisms. These mechanisms are not only based on their estrogenic properties, but also on their properties as antioxidants, gene transcription modulators and enzyme inhibitors. Thus, we will review their hormonal effects, the influence on cell signalling, on cell proliferation and some of their pharmacological and
1189
therapeutic effects, their putative anti-cancer properties, their claimed ability to prevent cardiovascular diseases and their effect on the immune system being the most relevant. Hormonal Effects The classical definition of phytoestrogens encompasses compounds that exert estrogenic effects on the CNS (Central Nervous System), induce estrus and stimulate growth of the genital tract of female animals [39]. Broadly, the term phytoestrogens may also refer to chemicals that show effects suggestive of estrogenicity, such as binding to estrogen receptors, the induction of specific estrogen-responsive gene products and the stimulation of those receptors, which could encourage breast cell cancer growth. Although, it has been known since 1931 that soybeans contain relatively high concentrations of isoflavones, genistein, being the first isoflavones isolated from soybean by Walter, there was no knowledge that these compounds could show some biological activity in animals [7]. It was approximately 15 years later when these were recognised in playing a role in the infertility syndrome in sheep, in which they were correlated with the animal's hormonal potency [40]. Isoflavones and some metabolic derivatives, such as the coumestran coumestrol, Fig (3), are structurally similar to mammalian endogenous estrogens and may act as estrogen agonists or antagonists, depending on their concentration or the tissue in which they act [41]. In this respect, crucial in demonstrating that phytoestrogens share a common mechanism of action were studies in experimental systems, in which phytoestrogens competed with radiolabelled estradiol to bind to the estrogen receptor and elicited estrogenic responses in estrogen-responsive tissues and cells [42]. Further, it was found that they act mainly by binding to the ERJ3, which was found to be expressed in many tissues, including the hypothalamus, pituitary gland, lung and thymus [43, 44]. Genistein (4',5,7-trihydroxyisoflavone) is the principal, most active isoflavone and shows the highest binding affinity for the estrogen receptor [45]. Its methoxy derivative, biochanin A, does not bind to the estrogenic receptor but is estrogenic in vivo [46]. This curious paradox may be explained, as described previously, by the fact that biochanin A, after ingestion, is metabolised in its chemical precursor, the isoflavone genistein. On the other hand, daidzein (4\7-dihydroxyisoflavone) has a
1190
higher binding affinity for the estrogen receptor than its methoxy derivative, formononetin, although both are weak estrogens in vivo. In 1961, Bickoff proposed that methylation could be the mechanism by which the estrogenic potency of isoflavones is reduced. In addition, the different potency of genistein and daidzein could be due to the presence of the hydroxyl group of genistein [7]. Zava & Duve [47] reported that genistein has estrogenic and ERindependent cell growth inhibitory actions. Over a physiologically relevant concentration range, genistein could serve both as a surrogate estrogen agonist and as a growth regulator. Some researchers have suggested that an optimal pattern of hydroxylation seems to be necessary for a flavonoid to have estrogenic activity. According to this, it has been reported that those flavonoids with hydroxyl moieties at C-4' and C-7 were invariably estrogenic and that an additional hydroxyl group at the C5, such as genistein, increased estrogen activity. Obviously, these structural criteria do not differentiate between the different classes of flavonoid, and include some flavanones, such as naringenin, and/or flavonols, such as quercetin, which obviously are not isoflavones [48, 49], Notwithstanding, the above authors indicated that isoflavones were better ligands, for ER, than flavones; and rings A and C of isoflavones were thought to mimic rings A and B of estrogens [50, 51]. Therefore, two kinds of criteria may be highlighted when putative estrogenic activity is based on its chemical nature. The first criterion is based on the hydroxyl moieties at positions 4' and 7', plus an activity enhancer when C-5 is hydroxyl substituted; and a second criterion as regards B-ring location at C-2 or C-3, this last position having an enhancing effect through its simulation of a natural oestrogen structure. On the other hand, criteria to explain structures that probably do not have estrogenic activity have also been proposed. Thus, more than four hydroxyl substituents, as in the case of the flavonol quercetin, or a 4' methoxylated substituent, such as the flavone diosmin, appear to abolish the estrogenic activity of flavonoids, Fig (5) [49]. The structural dependence of the estrogenic activity on the hydroxylation pattern has been reported by several research teams [48, 49, 50, 51]. Thus, Collins et al [52] examined the agonist/antagonist activity of various flavones and isoflavones, using a yeast estrogen system in which yeast cells were cotransformed with the human ER and two copies of an estrogen response element linked to the lacZ gene. The IC50 (the concentration of the ligand competitor at which the binding of radiolabelled 17p-estradiol to the human ER was reduced to 50%) for
1191
coumestrol, genistein, biochanin A, chrysin (a flavone) and naringenin (a flavanones) were determined to be 0.01, 2.0, 6.0, 33 and 45 uM, respectively, indicating that such isoflavones as genistein and biochanin A bind to the ER up to 5-10 times more strongly than flavones such as chrysin and naringenin.
OH
OH
m
Fig. (5). Structural criteria that enhance the estrogenic activity of flavonoids. I, II: Criteria based on the hydroxyl moiety pattern [47, 48, 49]. Ill, IV: Criteria based on the simulation of a natural estrogen structure [50, 51].
Coumestans, an isoflavone-^netabolic derivative, represents a fully oxidised version of the flavonoid pterocarpans and share the same systematic numbering. The most prominent and potent representative, coumestrol, has higher binding affinity for the estrogen receptor than genistein [53]. The hydroxyl moiety of coumestrol at position 12 corresponds to the hydroxyl moiety at C-4' proposed above as a structural criterion [48, 49]. This higher binding affinity suggests that the internal cyclation corresponding to the isoflavone structure at carbons 6 and 4, and/or the presence of an extra keto group in the coumestrol may improve the affinity for the ER and, therefore, of the estrogenic activity. One last additional criterion may be mentioned: The glycosylation pattern, which has until recently been ignored by researchers, should be taken into consideration. Recent studies have shown, as we will see below, that the aglycone forms show higher affinity for ER than the glycosylated ones.
1192
Besides the affinity of genistein, daidzein, biochanin A, formononetin, coumestrol and some other flavonoids, there is little information regarding other isoflavones, of which more than 300 have been reported to exist [6]. One such example is glycitein, whose estrogenic activity has been reported in one study. The data indicated that glycitein, when fed to female mice at 3 mg/day for 4 days, produced weak estrogenic activity comparable to that of other isoflavones, but much lower than that produced by diethylstilbestrol and 17(3-estradiol [54]. As regards these and those previously reported, a hierarchy of estrogenic activity may be proposed, in which flavone 7, 4'-dihydroxylate has the lowest activity and coumestrol with its internal cyclation the highest, Fig. (6). Table 2 shows the relative potencies of different isoflavones and coumestrol in different models for evaluating estrogenic activity.
Fig. (6). Estrogenic hierarchy, in increasing order, of flavonoid structures. A: Flavone 7, 4' di-OH. Rp H or OH, B: Flavanone 7, 4' di-OH, R,: H or OH. C: Isoflavone 7-OH, R^ H or OH, R2: H or CH3. D: Isoflavanone 7,4' di-OH. E: Isoflavone 5, 7, 4' tri-OH. F: Coumestrol.
When isoflavones are compared to estradiol, their estrogenic effects are seen to be weaker. In the mouse uterine growth assay, genistein and daidzein are roughly 100,000 times less effective than estradiol [43]. Moreover, the circulating forms of isoflavones, such as the glucouronides, showed between 10 and 40-fold lower activity for estrogenic receptors compared with their aglycones, but still showed weakly estrogenic activity at physiological concentration [11, 38].
1193 Table 2. Relative potency of some phytoestrogens in human adenocarcinoma cells [9].
Estradiol Coumestrol Geni stein Equol Daidzein Biochanin A Formononetin
In vitro 1,186 2.40 1.00 0.72 0.16 0.08 0.01
endometrial
In vivo 100,000 35 1.00 0.75 0.46 0.26
Apart from epidemiological evidence then, how is it possible to explain the physiological effect of isoflavones? Although, their estrogenic potency is extremely low compared with animal sterols, their blood concentrations may reach 50-800 ng/mL (0.2-3.2 ug/mL) in adults consuming modest quantities of soy food containing approx. 50 mg/d of isoflavones. Similarly, in response to the consumption of soy foods, blood isoflavone concentrations may be doubled. When soy is consumed on a regular basis, plasma isoflavone levels far exceed normal estradiol concentrations, which both in men and women generally range between 40 and 80 pg/mL [12, 55]. These observations lead to the hypothesis that isoflavones may be biologically active, conferring health benefits that could explain the relatively low incidence of hormone-dependent diseases in countries where soy is a dietary staple. However, the effects of soy consumption on hormonal metabolism have been inconsistent, probably as a result of methodological differences in the studies as regards the characteristics of the subjects, study design, isoflavone form and other factors [7], Soy isoflavones appear to affect the menstrual cycle and the concentrations of reproductive hormones in premenopausal women, by increasing the length of their follicular phase [56]. Moreover, it has been reported that dietary genistein exerts estrogenic effects upon the hypothalamic-pituitary axis in rats, and increases plasma prolactine. Also, genistein and daidzein may suppress glucocorticoid and stimulate androgen production in cultured human adrenal cortical cells [56, 57]. In brief, it is generally accepted that isoflavone consumption exerts various hormonal effects. However, the resulting health benefits are of uncertain clinical significance and further research is necessary to determine
1194
whether the responsible constituents are the isoflavones contained in food or other food constituents. Besides the estrogen agonist effect of isoflavones, an antagonist effect has also been described for them. Thus, isoflavones at concentrations 100-1000 times higher than that of estradiol have been considered capable to compete effectively with endogenous mammalian estrogens and prevent estrogen-stimulated growth in mammals [43]. This estrogen antagonist pattern of isoflavones, by competing with estradiol for the ER, may end up by interfering with the release of gonadotropins and interrupting the feedback-regulating system of the hypothalamuspituitary-gonadal axis. Kuiper et al [43] and Cassidy [58] reported the identification of a novel rat ERp. A subsequent study of the ligand selectivities and the tissue distribution of both ER subtypes a and P have thrown new light on the estrogenic activity of phytoestrogens. Both coumestrol and genistein exhibit a significantly higher affinity for ERP protein than for ERa. On the other hand, ERP is expressed prominently, in testicular tissue, secretory epithelial cells of the prostate, in the vascular system and, apparently, in breast tumour cells. ERp is also found in brain, bone, bladder and vascular epithelia, which have been seen responsive to classical hormone replacement therapy [59, 60, 61]. Although the regulation of the sex hormone receptors at the transcriptional level may not be considered as a hormone-like activity, it is one of the ways to control the hormonal signal. Thus, it has been reported that genistein, at concentrations of 500 ug/g body weight, decreased ERa mRNA expression [62]. In a similar way, it has been described that daidzein can decrease ERp mRNA levels in the hypothalamus of newborn piglets [63]. On the other hand, some authors find that soy isoflavones increase nerve growth factor mRNA and brain derived neurotrophic factor mRNA in rats [64, 65]. Since the discovery of a second estrogen receptor, ERP, it is necessary to re-evaluate the molecular basis for the action of estrogen and its agonists. Structurally ERP is highly homologous to ERa in the DNA binding domain, with more than 95% amino acid identity but only 55% homology in the ligand binding domain [66]. The structural differences lead to different relative binding affinities in ligand binding assays. Compared with ERa, isoflavones have a greater relative binding affinity to ERP, while estradiol binds to ERa and ERP with an equal affinity. Thus, structural biology shows that genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to 17p-
1195
estradiol. However, in the genistein complex, the activation-function does not adopt the distinctive agonist position but instead lies in a similar orientation to that induced by estrogen antagonist, which is consistent with genistein's partial agonist character in ERp [67, 68, 69]. Isoflavones in Hormone Replacement Therapy Estrogen deficiency in peri- and postmenopausal women results in a variety of neurovegetative, physical and somatic symptoms and may contribute to serious diseases within the aged female population. Such estrogen deficiency symptoms and the resulting diseases can be relieved, or their progression slowed down by conventional hormone replacement therapy (HRT) involving 17P-estradiol esters or conjugated estrogens [70]. However, estrogen has been demonstrated to be associated with the increased incidence of breast and endometrial cancer after prolonged treatment. In addition, during HRT, venous thrombo-embolic complications are encountered more frequently than in women not undergoing HRT [71]. Therefore, there is a growing interest in using isoflavones as a potential alternative to the estrogens in hormone replacement therapy. However, in this respect, there is no clear evidence that isoflavones are superior to a placebo when used to alleviate climacteric complaints such as hot flushes [71, 72], although some studies suggest the opposite [73]. Similarly, there is no evidence that isoflavones have a positive effect on the urogenital tract as stabilizers of the acidity of the vaginal milieu and thus preventing ascending infections [74]. On the other hand, isoflavones have been shown to have mild osteoprotective effects on the bones of ovarictemised rats, as well as on the bones of postmenopausal women [75, 76]. Also, a protective effect against cardiovascular risk has been described, as we shall see below [77, 78], whereas any protective effect on mammary cancer and endometrial cancer may be linked to the dosage [79].
Antiproliferative Effects and Regulation of Signalling Most of the support for the anticancer effects of isoflavones comes from epidemiological studies, for which data suggest that a diet rich in
1196
isoflavones provides protection against several forms of cancer, particularly those that are hormone-dependent, such as breast, prostate and lung cancer [7, 80, 81]. In vitro data lend support to the view that isoflavones inhibit cancer cell growth, prostate cancer and MCF-7 human breast cancer cells being the most relevant [82, 83]. Moreover, over 100 in vitro studies have shown that genistein inhibits the growth of a wide range both of hormone-dependent and hormone-independent cancer cell lines, with IC50 values ranging between 5 and 100 umol/L [84]. Some authors suggest that isoflavones may exert cancer-preventive effects by decreasing estrogen synthesis and altering metabolism away from genotoxic metabolites towards inactive metabolites. Thus, it has been found that daily consumption of a soya diet (providing 113-207 mg/day of total isoflavones) reduces circulating levels of 17(3-estradiol by 25%, and of progesterone by 45% compared with the levels recorded for a controlled diet in healthy and regularly menstruating women [85, 86]. It has become apparent that the anticancer mechanisms of isoflavones do not operate exclusively via the estrogen receptor. The intervention of isoflavones in tyrosine kinase activity is one of the ways of interfering cancer development. Enhanced protein tyrosine kinase activity due to the overexpression of receptor and/or protein tyrosine kinases leads to a continuous signalling that results in uncontrolled cell proliferation, which produces cancer growth. Many of the transduction pathways of peptide growth factor signals that have been implicated in certain cancers involve the action of tyrosine kinases. Therefore, a circulating tyrosine kinase inhibitor, such as genistein, may have beneficial effects on the prevention and treatment of cancers [87]. It has been reported that genistein can downregulate the intrinsic protein tyrosine kinase involved in neuroblastoma development [88]. It has also been described that in androgen-independent, human prostate carcinoma DU145 cells, genistein inhibits the transforming growth factor (TGF)-a-caused activation of membrane receptor erBl (a component of RTK family, Receptors of Tyrosine Kinase), before inhibiting downstream cytoplasmic signalling [89]. Indeed, more than 2000 papers supporting the regulation of tyrosine kinase by genistein are available in the scientific literature [90]. Most isoflavone studies on cell proliferation were performed using estrogen-dependent human breast carcinoma MCF-7 cells. The results pointed to a biphasic effect: stimulation of growth at low concentrations and inhibition at high concentrations. Thus, it has been reported that cell growth is stimulated by daidzein and genistein at low concentrations of < 0.25 ug/mL and < 10 ug/mL, respectively, while at higher concentrations
1197
(over 25 ug/mL and 20 ug/mL) cell growth is significantly inhibited in a dosage-dependent fashion [91. 92]. It has also been reported that isoflavones may inhibit cell proliferation through the inhibition of angiogenesis, which is a well-regulated and limited process intimately bound to vessel growth and carcinomas. The generation of new capillaries from pre-existing vessels does not occur in the healthy adult organism except in very few cases. However, pathological angiogenesis occurs during the development of some diseases and particularly in tumours. Well-vascularised tumours expand both locally and by metastasis, whereas avascular tumours do not grow beyond a diameter of 1-2 mm [93, 94]. Genistein is a well-known and potent inhibitor of cell proliferation and in vitro angiogenesis [95, 96], which may suggest that this property is related to its isoflavone structure. However, some studies have shown that some flavonoids, but not isoflavones, are even better antiangiogenic agents than genistein. Fotsis et al [97] found that several flavonoids such as 3',4'-dihidroxiflavone, luteolin and 3-hidroxyflavone were stronger inhibitors of angiogenesis that genistein. Apigenin, eriodyctiol and quercetin showed a similar effect, while luteolin glycoside, fisetin, myricetin, hesperetin and catechins had a less pronounced effect. These results suggest that a nonhydroxylated C ring with oxo function at position 4 and a double bond between C2 and C3 are required for maximal biological activity. Moreover, their glycosylation pattern seems to imply the lack of these properties. Based on these findings, Fotsis et al [97] suggested that this behaviour might be correlated with early events and some enzymatic inhibitors, such as tyrosine kinases [98] and protein kinases [99, 100]. The structural properties highlighted previously would make isoflavones competitive inhibitors with respect to the ATP binding site in a variety of enzymes, a region of considerable homology among kinases [101]. On the other hand, the concentration of genistein required to inhibit angiogenesis in vitro was reported to be higher than the genistein concentration likely to be achieved in vivo. However, it has been found that isoflavones in vitro may also be active at physiological concentrations (< 5-6 umol/L) [38, 102, 103]. In vitro studies have revealed that numerous mechanisms may be involved apart from the tyrosine kinase mechanism. In several cell lines, genistein did not alter tyrosine phosphorylation of the EGF receptor or other tyrosine kinase substrates. On the other hand, some authors have repotted that genistein inhibited the expression of the EGF receptor in the
1198
rat dorsolateral prostate, suggesting that the effect of genistein is via transcriptional processes rather than directly on tyrosine kinase activity [104, 105]. Thus, it has been suggested that the variable effects of isoflavones in estrogen-sensitive tissues may depend on the production of paracrine and autocrine growth factors that cause proliferation of cells not expressing ERa or ER.p [106]. Another anti-cancer mechanism of isoflavones may involve inhibition of key enzymes of estrogen metabolism, such as 3 P-hydroxy steroid dehydrogenase, 17P-hydroxysteroid dehydrogenase, 5a-reductase and aromatase, as a consequence of which the level of active steroid hormones is affected [81, 106, 107]. Isoflavones may also exert their activity either directly on DNA expression, or by protecting it. Thus, isoflavones may either inhibit DNA topoisomerase I and II activity, which is thought to cause DNA damage [108, 109]. The transcription factor p53 has become one of the most important tumour suppressors, and genistein has been shown to induce the upregulation of this protein [110, 111]. Genistein may also inhibit cell growth both by increasing the expression and production of the transforming growth factor (TGF) pi [112]. Some of the anti-cancer effects of isoflavones may result from their modulation of apoptosis. Thus, genistein inhibits the proliferation and differentiation of N2A, JC, SKNSH, MSN and Lan5 neuroblastoma cell lines and induced apoptosis in one (N2A line) [113]. Davis et al [114] reported that genistein induced apoptosis by inactivating NF-KB, in this way promoting cell death. Traganos et al [115] demonstrated that genistein at 5-20 ug/mL produced cell cycle arrest in both the Gl/S and G2/M phases of the human myelogenous leukaemia HL-60 line and the lymphocytic leukaemia MOLT-4 cell line. Further studies reported that genistein up to 60 uM arrested the development of human gastric cells at G2/M. Also, studies conducted in a non-small-cell lung cancer cell line demonstrated that genistein, at 30 uM, induced G2/M arrest through the p21 upregulation and the induction of apoptosis [116, 117]. Cell cycle arrest and the induction of apoptosis could be functionally related to the activation of p53 and/or the inhibition of cell cycle kinase activity, and research results suggest that flavonoids may be more effective in controlling the growth of tumours with certain mutational spectra [118]. A non-cancer related influence of isoflavones on cell signalling was reported by Liu et al. [119], who found that when pregnant sows where fed with daidzein, fetal growth was promoted, sow milk production was
1199
improved and postnatal growth was positively affected. Further, it was demonstrated that when sows were fed with daidzein the expression of IGF-1R gene in the longissimus muscle of new born piglets was markedly enhanced, suggesting that daidzein may influence fetal growth via the upregulation of IGF-1R expression in skeletal muscle [44, 120, 121]. Effect of Isoflavones on Cardiovascular Diseases Many research papers have demonstrated that soy protein inhibits cardiovascular diseases and reduces the risk of atherosclerosis in animals and humans. Indeed, in October 1999, the USFDA acknowledged the health claims regarding the beneficial effects of soy-based foods in a heart-healthy diet, after reviewing research from 27 studies that showed soy protein's value in lowering levels of total cholesterol and low-density lipoprotein [7, 79, 122]. Most authors support the view that the beneficial effects of soy are primarily due to isoflavones and are mediated by many mechanisms [123]. On the other hand, the mechanisms associated with soy's beneficial effects on cardiovascular health are not fully understood and, it remains unclear which components of soy protein contribute to its protective effects. It is possible that soy substances other than isoflavones, such as saponins, phytic acid, or a protein-isoflavone interaction, among others, may be involved in the multifarious process. However, most researchers consider that these positive effects are due to a reduction of plasma LDL (low density lipoprotein) cholesterol and triglyceride concentrations [124, 125], although other mechanisms, such as lowering diastolic blood pressure in women and improving the vascular and endothelial function, have also been proposed [126]. On the other hand, other authors suggest that the benefits of soy protein on cholesterol levels may be mediated through the upregulation of LDLreceptor activity, thus providing a novel mechanism of plasma cholesterol reduction different from currently available diets and hypolipidaemic drugs [127, 128]. Other proposed mechanisms for the effects of isoflavones include the prevention of atheroma formation through their antioxidant ability, and recently, the oxidative theory of atherogenesis has provided another avenue of therapy involving antioxidants [129]. According to this theory, antioxidants should protect lipoproteins against oxidative modification and reduce the biological consequences. Thus, Tikkanen et al [130] showed that the intake of soy protein containing 60 mg of isoflavones per
1200
day might provide protection against the oxidative modification of LDL. The oxidative modification of LDL particles is considered to be a prerequisite for the uptake of LDL by macrophages in the artery wall, which is an initial step in the formation of atheroma. Other Effects Most autoimmune diseases are more common in women than in men and quite frequently occur when estrogen levels change dramatically. The gastrointestinal tract and the immune system have often been overlooked and not considered as targets of estrogen. However, ERp has been found highly expressed in the human thymus and the gastrointestinal tract. Therefore, some of the immuno-modulatory effects of estrogens might be mediated via ERp. In this respect, daidzein has been proven to increase the activation of murine lymphocytes and activate natural T killer cells [38, 102]. Similarly, in vivo, it has been shown that daidzein, at a high dosage, enhances several immunological functions [131]. Theoretically, phytoestrogens could be expected to improve the cognitive function, particularly verbal functioning, as suggested for conventional hormone replacement therapy. As regards cognitive decline and soy isoflavone intake, the scientific data are varied. Thus, some studies suggest that increased tofu in the diet is associated with cognitive decline. On the other hand, animal data indicate the potentially beneficial effects of soy isoflavones in neuroanatomy and simple memory tasks [79]. Finally, there is growing evidence that the dietary intake of phytoestrogens has a beneficial role in chronic renal disease. Data suggest that the consumption of soy-based products rich in isoflavones and flaxseed rich in lignans retards the development and progression of chronic renal disease. In several animal models of renal disease, both soy protein and flaxseed have been shown to limit, or reduce, proteinuria and the renal pathological lesions associated with progressive renal failure. Isoflavones appear to act through various mechanisms that modulate cell growth and proliferation, extracellular matrix synthesis, inflammation, and oxidative stress. However, further investigation is needed to evaluate their long-term effects on the progression of renal disease in patients with chronic renal failure [132],
1201
ARE ISOFLAVONES TOTALLY INNOCUOUS? The excessive consumption of soybean and its products has been considered goitrogenic in humans and animals. Several researchers have reported the induction of goitre in iodine-deficient rats maintained on a soybean diet [133, 134, 135]. In some cases, the extreme intake of soybean has been correlated with cancer. Thus, Kimura et al reported an increase of up to 40% in thyroid carcinoma in rats fed on iodine-deficient defatted soybean [136], Recent studies have highlighted an explanation for this undesirable effect of soy flavonoids. The function of the thyroid is to synthetise thyroid hormones and TPO (Thyroid Peroxidase), which catalyses the iodination of thyrosyl residues on thyroglobulin and the subsequent coupling of iodotyrosyl residues required for iodothyronine hormone formation. In the presence of iodine ions, genistein and daidzein (the major soy flavonoids) block TPO-catalysed tyrosine iodination by acting as alternate substrates, yielding mono-, di- and triodoisoflavones. Genistein can also inhibit the synthesis of thyrosine by using iodinated casein or human goitre thyroglobulin as substrates for the coupling reaction [137], Genistein and genistin have also been known to be strong cytotoxic agents in vitro. This characteristic can be an advantage when target cells are malignant but can be disadvantageous when they are normal cells. Recent experiences carried out with rat myogenic cells (L8) showed that genistein and genistin strongly inhibited in vitro myoblast proliferation and fusion in a dose-dependent manner. Genistein also inhibited protein accretion in myotubes. Decreased protein accretion is largely a result of the cell (myofibrillar) protein synthesis rate, while no adverse effect on protein degradation has been observed. The results suggest that if sufficient circulating concentrations are reached in tissues of animals consuming soy products, genistein potentially affects normal muscle growth and development [138]. On the other hand, it is widely accepted that isoflavones can freely pass the placenta barrier and in humans isoflavone concentrations in the neonate are similar to those in the maternal plasma. It has been reported that isoflavones at concentrations found in a standard, natural-ingredient diet may affect the sexual differentiation of female rats in the uterus [7, 44]. Finally, the healthy effects of isoflavones on some individuals may be negative on others. Thus, as we have seen above, genistein may induce
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the upregulation of p53 protein. The Arg form at codon 72, instead of the Pro, has been associated with an increased risk of human papiloma virus related cancer in humans [139]. This finding could imply an extra risk associated with higher intakes of isoflavones. However, the former theory is not fully accepted, as other researchers argue that there is no relation between the kind of codon and an extra risk [140]. When soy-based products are promoted as healthy foods possessing putative beneficial estrogenic and anticarcinogenic activity, some of these properties due to their isoflavones, the findings mentioned in the previous paragraph should be highlighted, due to the widespread use of these products in infant food formulas and the consumption of soy products by people with vegetarian diets. ABBREVIATIONS CNS EGF ER HRT LDL 0-DMA RTK TGF USFDA UV/V
= Central Nervous System = Epidermal Growth Factor = Estrogen Receptor = Hormone Replacement Therapy = Low Density Lipoprotein = O-Desmethylangolensin - Receptors of Tyrosine Kinase = Transforming Growth Factor = U.S. Food and Drug Administration = Ultraviolet/Visible
ACKNOWLEDGEMENTS The authors would like to thank Prof. Victor Kuri, University of Plymouth, United Kingdom, for his helpful assistance in preparing this manuscript.
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1209 SUBJECT INDEX Abortifacient acivity 978 ofaristolicacid 977,978 Abyssinone II 836 against Mucor mucedo 826 against Staphylococcus aureus 826 Acanthosicyos 430 Acetoxyverruculogen 580 structure of 580 8-Acetoxywithanolide D 1049 antiparasitic activity of 1049 Acetylcholinesterase inhibition 143 by suvanine 143 12-O-Acetyl-16-O-deacetyl-12,16episcalarolbutenolide 151 cytotoxic activity of 151 Acetylcholine esterase 989 inhibition of 989 Achlya bisexual is 1102 Acid mine waste extremophiles 1123 bioactive metabolites from 1123 Acid mine waste lakes 1138 Acidophiles 1126 in acidic environment 1126 Acnistins 1022,1038,1039 structures of 1039 Acremonium luzulae 512 Actones 671 Acylation 576 Acylsaponins 223 from Silene fortunei 223 Adaptogens 456 cucurbitacins as 429,430-435,438, 439-446,447,456 Adenosine biosynthesis 4 Adjuvant activity 239 and HLB value 239 AF-bio binding protein 1071 Aframomum stipulatum 804 against malaria 371 against remota 371 Aging 705 role of oxygen species in 705 Ajuga decumbens 371 in cough 371 in inflammation 371 in respiratory disease 371
Akaloid phthalide 619 sources of 619 Akebia quinata 1103 Alcholic esters 248 Aldehydes 248 Aldol reaction 10,11,13 ofKetoaldehyde 4,10,13 Aldose reductase 1137 inhibition of 137 Alkaliphiles 1126 in an alkaline environment 1126 Alkaloids 182,248,808-814 actinidine 248 against Plasmodium falciparum 182 ibogaine 808 iboxygaine 808 noribogaine 808 tabernanthine 808 Alkaloid phthalide subtype 630 structures of 630 Allanblackia stuhlmannii 705 HIV-inhibitory activity of 705 Allelopathic 835 5-hydroxyisoflavonoids as 835 y4//o-Cerdran-type sesquiterpenes 409-412 sources of 409-412 structures of 410 Altohyrtin A 71 total synthesis of 71 Altohyrtin C 71 first total synthesis of 71 Alzheimer's disease 386,795 Genipine against 384,386 Amaranthus gangeticus metabolites 1095 regulation of development transitions of zoospores by 1095 Amarogentin 261 from Swertia chirata 261 Amaroswerin 261 Amauromine 571 as vasodilator 571 structure of 571 Amides 891,894 from Aristolochia species 891 structures of 894 Amines 85,86 structures of 86 Aminophenol 20 oxidative cyclization of 20
1210 Amtimicrobial property 463 of cucurbitacins 463 Anacardic acids 1084-1086 and related compounds 1084 biological activities of 1085 isolation of 1084 motility inhibitory activity of 1086 Analgesic 446 cucurbitacins as 429,430-435,438, 439-446,447,456 Analgesic actions 654 ofcuanxiong 654 Analgesic activity 156,209,382,444,816 ofiridoids 248,251,252,291, 305-333,340,352,353,365,381 ofglutinol 816 of Kageneckia oblonga A21,AAA ofsaponins 209 of triacetyl disidein 156 Angolesin 834 anti-MRSA activity of 834 Angolesin (oc-methyldeoxybenzion) 834 structure of 834 Anisatin 419 neurotoxic activity of 419 Anisatin-subtype sesquiterpenes 397 from Illicium floridanum 397 from Illicium minwimense 397 Anislactone B 414 chemical conversion of 414 transformation of 416 Anislactone-type sesquiterpenes 395,412419 biosynthetic route of 418 chemistry of 395 from Illicium species 395 neurotoxic activity of 419 neurotrophic activity of 395,419 sources of 412-419 structures of 413 Anti HIV activity 356 of olive leaves 356 Antiangiongenic agents 1197 isoflavones 1197 Antibacterial activity 125,156,185,209, 482,704,805 of azaphilones 481 of hyperibones A-D 704 of Lippia multiflora 805 of manzamines 185
of neomangicol B 156 ofsaponins 209 Antibiotic activity 842 of.£>y//w7«apterocarpans 842 Antibiotic aspirochlorine 519 from Aspergillus flavus 519 from Aspergillus oryzae 519 from Aspergillus tamarii 519 Antibiotics 1107 and conventional 1107 Anticancer activity 825,859 of Aristolochia mollissima 859 of bioactive compounds 825 Anticancer agents 149,827,835 burttinone as 827 isosenegalensin as 835 search for 1149 Anticancer drugs 1134 development of 1134 Anticarcinogenic activity 1202 of soy-based products 1202 Anticarcinogenic effects 214 of Panax ginseng 214 Anticholinergic property 262 of swertiamarin 262 ofsweroside 262 Anti-convulsant 638 3#-Butyphthalide as 638 3.S-Butyphthalide as 638 Anti-dementia 774 huperzine A as 774 Antidote 266 Genticma lufea as 265,266,270 Antifeedant activity 130,140,144,986, 988 against Carassius aurantus 144 of Aristolochia albido 986 of Aristolochia albida metabolites 988 of Cacospongia linteiformis 130, 140,144 of cyclolinteinone 130 of 12-deacetoxyscalaradial 149 of 3-deoxy derivative 130 Antifertility activity 979 of aristolochic acid 979 Antifertility effects 462 of Cucumis sativus 461 of cucurbitacins 429,430-435,438, 439-446,447,456
1211 Anti-free radical activity 354 of 3,4-dihydroxyphenylethyl-4formy lmethy 1-1 -4-hexenoate (3,4-DHPEA) 354 Antifungal activity 128,179,185,198, 209,477,482,524 against Aspergillus fumigatus 524 against Candida albicans 524 against Cryptococcus neoformans 524 of azaphilones 481 of(S)-cucurphenol 198 of forronychomycosis 477 ofhyrtiolide 128 of itraconazole 477 of Kalihinane diterpenoids 179, 180 of manzamines 185 ofsaponins 209 ofterbinafine 477 Anti-glycation activity 764 in fructose-BSA assay 764 Antihelmintic effects 1095 of condensed tannins 1095 Antihepathotoxic activity 356 ofiridoids 356 of secoiridoids 356 Antihepatitis drugs 262 used as tonic in chronic diarrhoea 262 use in asthma 262 use in bronchitis 262 use in dropsy 262 use in dry cough 262 use in gonorrhoea 262 use in phthisis 262 Antihepatotoxic effects 463 of cucurbitacins 463 Anti-HIV bioassay-guided fractionation 676 Anti-inflammatory activity 110,123,130, 139,150,152,185,209,252 of cavernolide 139 of cyclointeinone 130 of horrosalaonenes 152 ofiridoids 252 ofmanoalide 123 of manzamines 185 of marine metabolite 110 ofsaponins 209 on ear oedema in mice 150
Anti-inflammatory agent 135,156,442 cacospongionolides 135 cucurbitacin R 442 23,24-dihydrocucurbitacin B 442 indomethacine 156 Anti-inflammatory compound 139 luffolide 139 Anti-inflammatory effects 775 ofTWG 775 Antiinflammatory properties 129,354 ofluffariellin A 129 of luffariellin B 129 ofoleuropein 354 Luffariellin A 129,354 antiinflammatory properties of 129,354 Luffariellin B 129,354 antiinflammatory properties of 129,354 Oleuropein 354 antiinflammatory properties of 129,354 Antileishmanial activity 357,1048 against Leishmania donovani 317 in vitro 357 in vivo 357 ofiridoids 357 of secoiridoids 357 of steroidal lactones 1048 Antimalarial 774 artemisinin as 774 Antimalarial activity 145,178,180,181, 188,195,196,989 in vitro 178,181,989 of 3-alkoxy-l,2-dioxane 195 of chondrillin 196 of halerosellinic acid 145 of heptyl prodigiosin 188 of marine isonitriles 180 ofmuqubilone 196 Antimalarial agent 139 deoxy-diacarnoate B 139 Antimalarial derivatives 179 isonitrile-containing 179 Antimalarial diterpenoids 178 from Cymbastela hooperi 178 Antimalarial drugs 128,170,171 against Plasmodium falciparum 128 chloroquine 170,171 mefloquine 170,171
1212 pyrimethamine 170,171 Antimalarial lead compounds 169 from marine organisms 169 Antimicrobial activity 112,115,116,131, 135,1147 against Sarcina lutea 112 against Staphylococcus aureus 112 of cacospongionolides 135 of hippospongin A 116 of palauolol 131 specific assays for 1147 Antimicrobial agents 1146 search for 1146 Antimutagenic activity 210,211 in mammalian cells 210 ofaflatoxinBl 211 of kalopanaxsaponin A 211 ofsaponins 210 Anti-OVA antibody levels 238 Antioxidant activity 354,705 in lipid peroxidation 705 ofiridoids 354 of secoiridoids 354 Antiparasitic activity 187 against Plasmodium falciparum 187 Antiplasmodial activity 177,179,181, 841,844 against Plasmodium falciparum 841 by preventing heme detoxification 181 of axisothiocyanate-3 177 of isonitrile derivatives 179 Antiproliferative effects 1195 of isoflavones 1195 Antirrhinoside 250 Antispasmodic activity 129 of hippospongin 129 Anti-spermatogenesis actions 7776 ofTWG 776 Antithrombin 143 suvanine sodium salt as 143 Antitrypanosomial activity 1048 of steroidal lactones 1048 Antitrypsin activity 143 of suvanine sodium salt 143 Antitubercular activity 147 of heteronemin 147 Antitumor activity 147,214
in vivo 147 ofsaponins 214 on sarcoma-180-implanted mice 147 Antitumor compound 175,535 743 (ET-743) 175 Antitumor promoting activity 212 ofsaponins 212 Antiviral (HSV and PVl)/cytotoxic activities 119 of furanoterpene acid 119 Antiviral activity 113,138,209,355 against Herpes simplex (HSV) 113 against Herpes simplex virus type-1 138 against Polio vaccine (PV1) viruses 113 against vesicular stomatitis virus 138 in vitro 113,355 ofiridoids 355 ofsaponins 209 of secoiridoids 355 of variabilin 113 Antiviral drug 157 arabinofuranosy ladenine 15 7 Anthraquinones 498-502 Apama 855 Aphanomyces 1053,1059 root rot diseases caused by 1059 Aphanomyces astaci 1054 Aphanomyces cochlioides 1055,1058 zoospores of 1058 Aphanomyces euteiches 1055 Aphanomyces invadans 1054 Aphanomyces westlandii 1002 Aphanomyces zollingeriana 856,868 as analgesic 856 Apocynin 253 effects on neurophil oxidative burst 253 Apoptosis inducing activity 224 ofsaponins 224 Apoptotic effects 226 of saikosaponin D 226 of Bupleururm falcatum 226 Aporphines 810,882-886 from Aristolochia species 883-886 Aquilegiosides C-F 231 from Aquilegia vulgaris 231
1213 Aranochlors 529 inhibitory activities of 529 against microorganisms 529 Aranochor A 528 from Pseudoarachniotus roseus 528 structure of 529 Arbortristoside A 355,356 against Candida albicans 356 semilko forest 355 Arcinol 710 in vivo 710 cancer chemopreventive activity of 710 Ardeemin 571 structure of 571 Argentilactone 969 mass spectral fragmentation of 969 Aridanin 812,816 against Biomphalaria glabrata 816 from Tetrapleura tetraptera 816 molluscicidal activity of 816 structure of 812 AristofolinA 875 Aristofolin B 875 AristofolinC 875 Aristofolin D 875 Aristolactam 875,876-879,961 biosynthetic pathway of 961 from Aristolochia species 876-879 Aristolactam III 974 Aristolactam taliscanine 980 in Parkinson's diseases 980 Aristolactaml-A/-P-glucoside 879 Aristolactones 961 biosynthesis of 961 Aristolanes 912 from Aristolochia longa 912 Aristolic acid 977,978 abortifacient acivity of 978 anti-estrogenic activity of 977 anti-implantation activity of 977 from Aristolochia indica 978 immunomodualting activity of 979 short term toxicity study of 979 Aristolin 872 Aristolochia 1002 chemotaxonomy of 1002
Aristolochia sp. 855,857-859,862 chemical constituents of 855,862 ethanopharmacology of 860 medicinal uses of 857-859 pharmacology of 855,971-992 Aristolochic acids 864-868,869,959,967 antitumor activity of 972 biosynthesis of 959 diterpenoid esters of 868 mass spectral fragmentation of 967 sesquiester of 868,869 sodium salts of 868 sources of 864-868 Aristolochic acid alkyl esters 868,870, 871 from aristolochia species 870,871 Aristolochic acid I 971 pharmacology of 971 total synthesis of 972 Aristolochia albido 986 antifeedant activity of 986 aristolic acid from 986 Aristorochia alcinous 1000 Aristorochia arcuata 888 13 -oxidodibenzo [oc,y] -quinolizinium alkaloids from 888 Aristolochia argentina 856,875 in arthritis 856 in poisoning 856 in pruritus 856 Aristolochia bracteata 856 as antihelmintic 856 Aristolochia chilensis 856 to cure wounds 856 to treat arthritis and diarrhea 856 Aristolochia clematitis 863 in cancer 863 in leg ulcer 863 in menstrual troubles 863 in tumors 863 Aristolochia constricta 982 antispasmodic activity of 982 Aristolochia contoria 1004 Aristolochia curcubitifolia 868,879 Aristolochia cymbifera 1004 Aristolochia debilis 860 inhibitor of iNOS activity 860 Aristolochia euteiches 1059 Aristolochia euteiches zoospores 1063 Aristolochia fangchi root 998
1214 Aristolochia foveolata 868 Aristolochia galeata 1004 Aristolochia gigantea 1003 Aristolochia heterophylla 868 Aristolochia kaempferi 868 Aristolochia longa 975 against 1-388 lymphocytic leukaemia 975 Aristolochia manshuriensis 1002 Aristolochia mollissima 859 anticancer activity of 859 antimalarial activity of 859 as anti-inflammatory agent 859 as analgesic 859 in abdominal pain 860 in rheumatism 860 in stomach ache 860 Aristolochia ochraceus 570 Aristolochia onoei 1003 Aristolochia papillaris 981 smooth muscle relaxant activity of 981 Aristolochia paucinervis 860 bacteriostatic activity of 860 in skin and soft-tissue infections 860 Aphanomyces raphani 105 5,1059 Aristolochia ringens 1004 Aristolochia rotunda 1003 Aristolochia shimadai 1002 Aristolochia triangularis 860 in skin diseases 860 in wounds 860 uses of 860 Aristophyllide A 872 Aristophyllide B 872 Aristophyllide C 872 Aromadendranes 917 from Aristolochia chilensis 917 Aromatase 1198 Aromatic compounds 515-521 structures of 516-521 Aromatic ring-A withanolides 1032 Aromatic ring-D withanolides 1034-103 8 Arteflene 192 structure of 192 Artemether 189 structure of 189 Artemisia annua 171,189 antimalarial activity of 189 in fever 189
in folk Chinese medicine 171 Artemisinin 189,190 mechanism of action of 190 structure of 189 Artesunate 189 structure of 189 Asalina bioassay 117 activity of cacospongionolide D in 117 Asari herba 996 Asarum 855 Ascochlorin analogs 534 cytotoxic activities of 534 Asmolactones 352 formation of 352 Aspergilloxide 156 from genus Aspergillus 156 Aspergillus amstelodami 572 Aspergillus rubber 573 Aspergillus ustus 481,570 Asperparalines 594-604 bioactivity of 596 biosynthesis study of 596-598 structure of 595-596 synthetic study of 596-600 Aspochracin 550 from Streptomyces sp. 550 Astragalosie I 230 Astragalus sponins 231 Astrophaneura alcinous 999 Aswagandha 1020 from Withania somnifera 1020 medicinal properties of 1020 Atheroma 1200 Atherosclerosis 136,705 role of oxygen species in 705 Aucubajaponica 370 Aucubigenin 386 Aucubin 249,250,279,383,386,387 as antidote 279 as chemopreventive 383 astringent property of 279 effects on collagen synthesis 387 in dysentery 279 in vivo assay 386 liver protective effects of 279 stimulant effects of 279 Auriculatin 837 antimicrobial activity of 837 from Millettia auriculata 837 Aurones 1178
1215 Austocystins 499 from Aspergillus ustus 499 structure of 499 Autoimmune diseases 1200,1150 therapies for 1150 Auxarconjugatins A and B 529 from Auxarthron conjugation 529 structures of 530 Avicins 213 effects on chemically induced mouse skin carcinogenesis 213 Avicins D 213,226 apoptosis inducing activity of 226 Axinella verrucosa 578 Axinella cannabina 176,177 axisonitrile-1 from 176 axisothiocyanate-1 from 176 isonitrile terpenoids from 177 Axisonitrile-3 177 from Acanthella klethra 111 Azaphilone 485,487 from Penicillium sclerotiorum 485 inhibitory activities of 487 structure of 486,487 Azaphilone metabolites 482 production of 482 Azaphilones 481 antibacterial activity of 482 antifungal activity of 482 l-Azaspiro[4.5]decan-8-ones 15 Sorensen and Ciufolini synthesis of 15 Azaspirodecanediones 17 from ./V-methoxyphenylamides 17 synthesis of 17 Azatricyclic compound 34 Wardrop's alternative route to 34 Bacillus subtilis 123,125,1087 activity to oomycete zoospores 1087 Badrinal 256 Battus philenor 1000 Bee venom PLA2 124,143 against Escherichia coli 125 cytotoxic activity against 143 dehydromanoalide effects on 124 inhibitor of 143 Benicasa 430
Benzenoids 945,949,950,952 from Aristolochia species 945 Benzophenone synthase 722 from Centaurium erythraea 722 Benzophenones 721-725,748-761 biological activities of 721 from Clusia 722 from Garcinia 722 from Hypericum 722 isolation of 721,724,748-752 sources of 725-746 structures of 721,725-746 synthesis of 722-724 Benzylisoquinolines 889,890 from Aristolochia species 889 structures of 890 Benzyltetrahydroisoquinolines 962 Berkeleydione 1167 isolation of 1167 structure of 1167 Berkelytrione 1167,1168 formula of 1168 isolation of 1167 Beticolins 1 500 structure of 500 Beticolins 2 500 structure of 500 Beticolins 3 500 structure of 500 Beticolins 4 500 structure of 500 Beticolins 6 500 structure of 500 Beticolins 8 500 structure of 500 Bicarbocyclic sesterpenoids 131 antimicrobial activity of 131 antiproliferative activity of 131 cytotoxic activity of 131 sources of 131 Bicyclo-(3.3.1)-nonane derivatives 693 conformers in 693 tautomers in 693 Bicyclo-[3.3.1 ]-nonane derivatives 682-697 sources of 683 structures of 684 Bicyclogermacranes 906 from Aristolochia elegans 906 BidwillolA 849 structure of 849
1216 Bioactive cinnamate esters 849 from Erythrina 849 Bioactive cinnamoylphenols 833 from Erythrina 833 Bioactive compounds 825 anticancer activity of 825 anti-inflammatory activity of 825 antimicrobial activity of 825 DNA-repair properties of 825 muscle relaxation properties of 825 Bioactive coumastans 849 from Erythrina 849 Bioactive daidzein derivatives 838 from erythrina genus 838 Bioactive flavanones 826,827 naringenin derivatives 827 Bioactive flavonoids 825 from Erythrina species 825 Bioactive isoflav-3-enes 849 from Erythrina species 849 Bioactive isoflavanones 846,847 activity of 847 sources of 846,847 Bioactive isoflavone derivatives 836 activity of 836 from Erythrina species 836 Bioactive isoflavones 839 activity of 839 from Erythrina species 839 Bioactive marine secondary metabolites 158 by aquaculture 158 by cell culture 158,159 by chemical synthesis 158 by cultivation of marine organisms 158,159 Bioactive metabolites 74,95,97,100, 1134,1143 flaccidoxides 95 from deep-sea sponges 1134 from microorganisms 1143 hamiltonins 100 latrunculins 100 nakafurans 97 pyrroloiminoquinones 74 sarcoglane 95 search for 1143 spongiane diterpenes 100 variabilins 97 Bioactive pterocarpans 840
as antiplasmodial compounds 840 as antibacterial compounds 840 as antimicrobal compounds 840 as phospholipase A3 inhibitors 840 from Erythrina species 840 Bioactive saponins 209 cancer related 209 developments in 209 immunomodulatory activity of 209 Bioactive secondary metabolies 1133 extremophiles source of 1133 Bioactive stilbenoids 831 from Erythrina 831 Bioactivemetabolites 81 halichlorensin 81 halitulin 81 Bioactivity 429,591,596 of asperparalines 594-604 of brasiliamides 584-594 of cucurbitacins 429,430-435,438, 439-446,447,456 of dihydrobrasiliamide B 588, 591-592 Bioactivity isoflavans 848 sources of 848 Biosynthesis 251,411,434-435,475, 591-592,958,959,961 of aristolactones 961 of aristolochic acids 864-868, 869,958, 959,967 of brasiliamides 584-594 of dihydrobrasiliamide B 588, 591-592 of griseoflavin 471-479 of illicinolides 411 ofiridoids 248,251,252,291, 305-333,340,352,353,365,381 Biosynthetic origin 250 ofiridoid 250 Biphenyl ether 948 structure of 948 Bisabolanes 903 from Aristolochia acutifolia 903 Bisbenzyltetrahydroisoquinolines 963 biotransformation pathway from 963 Bishomosesterterpenoids 153 from Phyllospongia foliascens 153
1217 Bisindole 569 Sw-pyrroloiminoquinone alkaloids 74 tsitsikammammine A and B 74 Bit is artetans 984 Bobolstemma paniculatum 212 Bonjoch's synthesis 37-40 of ketone enolates 37-40 via palladium-catalyzation of aminotethered vinyl bromides 37-40 Bothrops atrox 860 as insect repellent 860 hermorrhagic effect of 860 Boucheafluminensis 381 as regulator of digestive function 381 anti-inflammatory activity of 381 Bourbonanes 921 from Aristolochia gibertii 921 Bovine endothelial GM7373 140 Brasiliamides 584-594 bioactivity of 591 biosynthesis of 591-592 conformational analysis of 589 discovery of 584 related compound 593 Brasiliamide A 586 structure of 585 Brasiliamide B 586 structure of 586 Brasilianum Batista JV-379 585 convulsive acivity of 585 Brevianamide E 571 structure of 571 Bridgehead azabicyclic iminium ions 35-39 Kibayashi's approach from 35-39 Brine shrimp lethality assay 95 6-Bromoaplysisnopsin 188 structure of 188 5-Bromoochrephilone 486 from Penicillium multicolor 486 6-Bromopenitrem E 582 'H-NMR spectrum of 583 structure of 582 Bronianone 672 structure of 672 Brummond's synthesis 28-32 ofFR.901483 28-32 via tandem cationic aza-Cope
rearrangement-Mannich cyclization reaction 28-32 Bryonia 430 Bursting activities 1114 homogenous solution method for 1114 Burttinone 827 as anticancer agent 827 Butylidencphthalide 638 as anti-angina 638 as anti-platelet / anti-thrombosis 638 in cardiac function modulation 638 in inhibition of learning and memory impairment 638 in sedation and sleep enhancement 638 in smooth muscle relaxation 638 Butyliphthalide 638,648 against cerebral injuries 648 against cerebral ischemia 638 in blood viscosity reduction 638 protective effects of 648 37?-Butyphthalide 638 as anti-convulsant 638 3S-Butyphthalide 638 as anti-convulsant 638 in inhibition of learning and memory impairment 638 protection against cerebral ischemia 638
Ca2+influx-efflux 1097 blockade of 1097 in zoospores 1097 Cacospongia 139 Cacospongia linteiformis 130,140,144 antifeedant activity of 140 cyclolinteinone from 130 lintenone from 144 Cacospongia scalaris 113,146 ircinin from 113 scalarin from 146 Cacospongionolides 135 effects on panel of secretory PLA2 135
1218 Cadinanes 914 from Aristolochia heterophylla 914 Cancer 134,370,371,705,863 Aristolochia clematitis in 863 Catalpa ovata against 370 drugs against 134 Kigelia pinnata in 371 oxygen species role in 705 Cancer cell lines 66,82,94,111,155 cephalostatins against 64-66 eleutherobin against 94 halitulin against 82 idiadione against 111 neomangicols A and B against 155 Cancer cell proliferation 136 Cancer chemopreventive activity 713-716 of polyisoprenylated benzophenone 713-716 Candida albicans 136,198,356,704 arbortristoside A against 355,356 (S)-cucurphenol against 198 in Candida infections 704 sulfircin activity against 136 Candida gilchristi 65 Candida hamiltoni 101 spongiane diterpene lactones from 101 Candida lucanusianus 805 Candida maritimaum 273 Candida nemorosa 680 Candida ovata 371 Candida pulchellum 273 Candida quintense 273 Candida scalaris 147,150 deacetylscalaradial from 150 Candida spicatum 273 Candida tenuiflorum 273 Cantauhum erythrea 272 as stomachic 272 in blood purification 272 in jaundice 272 in sores 272 in wounds 272 Capillary zone electrophoresis 995 Cardanol 1084 isolation of 1084 Cardol 1084 isolation of 1084
Carnosoflogein A 458 from Hems ley a carnosiflora 458 Carrageenan-induced inflammation 775 in vivo 775 Carvacrol 810,815 structure of 810 Caryophyllaceae saponins 224 Caryophyllanes 910 from Aristolochia argentina 910 Caspase-1 1155 Catalpa ovata 370 against inflammation 370 against cancer 370 Catalpol 249,386 Catechins 1178 Catenaria anguillulae 1057 Catnip tea 289 in chicken pox 289 in colic fevers 289 in headache 289 in insomnia 289 in measles 289 in nervousness 289 Cavernosolide 133 structure of 133 Cayaponsides B 451 strucure of 451 Celastrol 795 antioxidant activity of 795 anti-inflammatory activity of 795 Celastrol type compounds 782 immunosuppressive activity of 782 anti-inflammatory activity of 782 Celosia cristata 1096 Centaurium erythrea 269,273 for languid digestion with heart burn 273 for muscular rheumatism 273 Cephalostatins 64-66 against cancer cell lines 66 as cell growth inhibitors 64 cytostatic properties of 66 from Cephalodiscus gilchristi 65 from Ritterela tokioka 65 trisdecacyclic pyrazine structure of 65 Cephalostatin 1 65 structure of 65 Cephalostatin 2 65 structure of 65
1219 Cephalostatin 3 65 structure of 65 Cephalostatin 4 65 structure of 65 Cepholostatins 5 66 structure of 66 Cephalostatin 6 65 structure of 65 Cephalostatin 7 67 structure of 67 Cephalostatin 8 67 structure of 67 Cephalostatin 9 67 structure of 67 Cephalostatin 10 68 structure of 68 Cephalostatin 11 68 structure of 68 Cephalostatin 12 68 structure of 68 Cephalostatin 13 68 structure of 68 Cephalostatin 14 68 in vitro 68 Cephalostatin 15 68 in vitro 68 Cephalostatin 16 69 structure of 69 Cephalostatin 17 69 structure of 69 Cepharadione A 980 antimicrobial activity of 980 CepharanoneB 989 antimalarial activity of 989 in vitro 989 Chaetoviridines A 491 as monoaminooxidase inhibitor 491 effect on growth of Penicillium oryzae 491 inhibitory effect on monoaminooxidase 491 structure of 491 Chaetoviridine B 491 structure of 491 Chaetoviridine C 491 structure of 491 Chaetoviridine D 491 structure of 491
Chaga's disease 988 eupomatenoid-1 988 hearinAin 988 Chalcones 830 activity of 830 from Erythrina sp. 830 Chamigranes 919 from Aristolochia debilis 919 Chamone I 765 Chemical components 791 bioactivity of 791 from TW plant 791 Chemoreceptors 1071 of flagellar membrane 1071 Chemotaxis 1065 Chemotropism 1070 of hyphal germlings 1070 Chicken ovalbumin (OVA) 239 in mice 239 Chinese hamster ovary (CHO) cells 210 DNA damage in 210 Chinese-herbs nephropathy 990-993 Chlorflavonin 523 structure of 523 Chlorinated anthraquinones 501 structures of 501 Chlorinated benzophenone antibiotic 538 pestalone 538 Chlorine containing polyketides 527 chlorocarolide A and B 527 Chloroform extract 442 anti-inflammatory activity of 442 from Cayaponia tayuya 442 Chlorofusin 493 structure of 493 Chloroorselinic acid A 489 from Emehcella falconensis 489 structure of 4889 Chloroorselinic acid B 489 from Emericella falconensis 489 structure of 489 Chloroorselinic acid C 489 from Emericella falconensis 489 structure of 489 Chlorophylls 893,895 structures of 895 Chlovalicin 530 structure of 530 Chondrillin 197 structure of 197 Chramaloside A 217
1220 Chromatographic techniques 1088 Chromeno-coumarin calanolide A 702 from Calophyllum langiferum var. Austrocoriaceum 702 inhibitor of HIV-1 reverse transcriptase 702 Chromodoris inornata 143 Chronic inflammatory conditions 134 Chronic inflammatory disorders 134 drugs against 134 Ciclopentane derivatives 323 origin of 323 structures of 323 Ciona intestinalis 85 C/s-jasminoside 350 Citrullus colocyhthis 431 as purgative 431 Ciufolini synthesis 22-25 via oxidative cyclization 22-25 of oxazolinetethered phenol 22-25 Classical hormone replacement therapy 1194 (-)-Claussequinon 1082,1083 13 C-NMR data of 1082 structure of 1083 Clavelina lepadiformis 85,185 Cnidilide 638 in blood viscosity reduction 638 in inhibition of learning and memory impairment 638 Cochliobolus lunata 481 Cochliophilin A 1056,1061 from Chenopodium album 1061 from Aphanomyces cochlioides zoospores 1061 structure of 1056 Colon adenocarcinoma 975 Columbin 984 anti-snake venom activity of 984 Communesins 574-579 biosynthetic pathway of 576,577 discovery of 574 identification of 574 insecticidal activity of 576 molecular formula of 574,575 related compounds 577-579 structures of 574,575 Conopharyngine 809 structure of 809 Convulsive activity 550 against silkworms 550
Convulsive compounds 579-593 Coscinoderma mathewsi 143 suvanine from 143 Cacospongionolide B 132 from Fasciospongia cavernosa 132,142 Cacospongionolides E inhibition 135 of human synovial PLA2 135 Cytoxicity 134 of cacospongionolide B 134 Coumarin 521-526,813,817 from Tabernanthe tetraptera 817 Coumestrol 1191 affinity for estrogen receptor 1191 COX-2 expression 139 cavernolide effects on 139 COX-2 inhibitors 447 cucurbitacins as 447 Cuanxiong 654 analgesic actions of 654 for asthma 654 for stroke 654 Cucumis sativus 461 antifertility effects of 462 antimicrobial properties of 463 toxicity of 462 Cucurbita ficifolia 432-434 chemistry of 432-434 hypoglycemic effects of 432 Cucurbitacin 7?-diglucoside 456 against stress-induced alterations 456 Cucurbitacin-glycosides 452 structure of 452 Cucurbitacin glyscosides 254 structure of 254 Cucurbitacins 429,430-435,437,438, 439-446,447,456,462 agonist activity of 459 analytical separation of 435,437 anticancer effects of 447 antifertility effects of 462 as adaptogens 456 as analgesic 446 as anti-inflammatory agents 439-446 as ecdysteroid antagonists 431 bioactivity of 429 biological property of 439 biosynthesis of 434-435 chemistry of 432-434
1221 cytotoxic effects of 447 detection of 435 effects on immune system 456 effects on insects 457 effects on plant parasites 457 extraction of 436 from protostane 435 gibberellin-antagonistic activity of 430 identification of 435 isolation of 435 occurrence of 429 pharmacological activity of 431 purification of 437 structural elucidation of 438 toxicity of 462 Cucurbitacins E 447 from Conobea scoparoides 448 Cucurbitacins WG, 430 from Wilbrandia 430 Cucurbitacins WG2 430 biological significance of 430 gibberellin-antagonistic activity of 430 from Wilbrandia 430 Cucurbitane 433 structure of 433 (S)-Cucurphenol 198 against Candida albicans 198 antifungal activity of 198 Cudraphenones A-D 711 against HGF 711 against HSC-2 711 against HSC-2 cells 711 Curcurbitacins B 440 structure of 440 Curcurbitacins E 440 structure of 440 Cyclic peroxides 81 from Plakortisaff simplex 81 structures of 81 Cyclization 566,723 Cycloartane type triterpene saponins 231 from Astragalus peregrinus 231 Cyclochlorotine 502 from Penicillium islandicum 502 Cycloechinulin 570 structure of 570 Cycloparvifloralone-type sesquiterpenes 407-409 structures of 407-409
Cycloperoxide-containing antimalarial agents 191 Cycloperoxides 189 CyclosporinA 232 Cyclotryprostatin A 581 structure of 581 Cyclotryprostatin B 581 structure of 581 Cyclowithanolides 1023 Cymbastela hooperi 180 marine isonitriles from 180 Cystodytes delechiajei 85 Cystospores 1068,1069 germination of 1068,1069 Cytosolic PLA2 113 variabilin inhibitor of 113 Cytotoxicity 121,143 against human leukaemia MOLT4 cells 121 against human myeloid K562 cells 121 in Artemia salina bioassay 1443 of rhopaloic acid A 121 Cytotoxicity activities 115,120,125,131, 136,138,145 against central nervous system carcinoma XF49 8 116 against human tumor cell lines 131 against human tumor cell lines 138 against NSLC-N6 cells 145 against ovarian carcinoma SKOV-3 116 against skin carcinoma SK-MEL-2 116 in Artemia salina bioassay 120 of 1,2-dioxanes 138 of hipposulfates B 136 ofircinin-2 116 ofluffariolides 125 of mycaperoxides A/B 138 of petrosaspongiolide L 145 ofsarcotinG 116 ofsarcotinH 116 on KB cells 145 Cytotoxic depsipeptides 82 geodiamoli de TA 82 hemiasterlin de TA 82 jaspamide de TA 82
1222 Daidzein 1056,1183 structure of 1056,1183 Dalbergia odorifera 1082 isoflavonoidal from 1082 repellent activity of 1082 structure elucidation of 1082 Danggui 613,654 in treatment of female irregular menstruation 613 use in pain 654 De novo DNA synthesis 231 De novo purine nucleotide biosynthetic pathway 4 2-Deacetoxy-21 -acetoxy scalarin 148 cytotoxic activity of 148 from Japanese H. erecta 148 12-O-Deacetyl-12-ep/-scalarin 146 12-Deacetylhyrtial 151 from H. erecta 151 Deacylsaponins 236 Debilicacid 868 from Aristolochia debilis 868 from Aristolochia longa 868 Debenzylation 11 of diphosphate ester 11 Decarboxylation 959 Decatromicins A 506 against Staphylococcus aureus 506 structure of 506 Decatromicins B 506 structure of 506 Deglucouronidation 1187 of phenolic estrogen conjugates 1187 Dehydration 936 6,7-Dehydrofevucordin A 434-435 biosynthesis of 434-435 structure of 434 (-)-Dehydrogriseofulvin 475 microbial transformation of 475 reduction of 475 Dehydrogriseofulvin 479 from Peltaspermum martinsii 479 Dehydro-luffariellolide diacid 127 structure of 127 Dehydrooxoperezinone 980 from Aristolochia manshuriensis 980 Deinococcus radiodurans 1130
(+)-DemethylaminoFR901483 32-35 Wardrop's formal synthesis of 32-55 Denitroaristolochic acids 873-875 from Aristolochia species 873-875 Densitometric method 996 25-Deoxycacospongionolide B 133 5-Deoxyisoflavones 838 daidzein precursor for 838 Deoxyloganic acid 340 esterification of 340 oxidation of 340 22-Deoxy-variabilin 114 against Bacillus subtilis 114 against Candida albicans 114 from Thorecta sp. 114 Depsides 507-510 structures of 508-510 Depsidones 507-510 Depurative 288 Lamium labiatae as 288 Desaturation 566 DesmethylaminoFR901483 32-35 formal synthesis of 32-35 (±)-Desmethylamino FR901483 9-12,33 synthesis of 9-12,33 Detection 993-999 of aristolochic acids 993-999 Deutzioside 249 Diacamus levii 128 e«Mnuqubilin from 128 Diastereoselective spirocyclization 18 TV-acylnitrenium ion-promoted 18 Dibenzylphosphate 21 hydrogenolysis of 21 Didemmaones B 73 from ectynonanchora flabellate 73 Didemnum listerianum 85 Didemnum sp. 185 20,24-Dihomoscalaranes 154 against KB cells 154 Dihydrobrasiliamide B 588,591-592 bioactivity of 591 biosynthesis of 591-592 (-)-Dihydrocubebin 943 from Aristolochia pubescens 943 Dihydrooxyresveratrol 831 structure of 831 Dihydroplakortin 193 from Plasm odium falciparum 193 structure of 193
1223 Dilemmaones A-C 73 from Cram be chelastra 73 from Ectynonanchoraflabellate 73 Dimeric secoiridoid glucosides 317 2,2-Dimethyl-l,3-dioxan-5-one 26 condensation of 26 7,12-Dimethylbenz[a]anthracene 212 1,2-Dioleoylphosphatidic acid 1079 Dioscin 225 from Polygonatum zanlanscianenens 225 Dioxin 26 retro Diels-Alder cycloaddition of 26 Diphosphate ester 11 debenzylation of 11 Disidein 156 from Dysdidea pallescens 156 stereochemistry of 156 Diterpenoids 777-781,811,927-937 from Aristolochia species 928-935 fromTW 777-781 structures of 780 D-limonene 1110 mycelial growth inhibition by 1110 DNA topoisomerase 1 1198 DNA-damaging agent 188 lissoclinotoxin A 188 £>-Pinitol 1001 defensive role of 1001 against herbivores 1001 Dragmacidon 593 structure of 593 Dragmacidon A 593 structure of 593 Dragmacidon B 593 structure of 593 Drugs 1144 industrial production of 1144 Dunalia brachyacantha 1048 Dysentery 279 aucubin in 249,250,279,383,386, 387 Dysidea avara 158 against skin disorder 158 Dysidea sp. 114 against protein phosphatase enzyme 114 isopalinurin from 114
Dysideapalaunic acid 137 effects on aldose reductase 137 stereochemistry of 137 Ecdysteroid antagonists 459 structures of 459 ECE inhibition 77 of Pachastrella extract 77 Elemanes 904 from Agrostophyllum brevipes 904 Eleutherobin 94 against cancer cell lines 94 Eleuthosides 92-94 from Eleutherobia aurea 92-94 structures of 92-94 Emericellafalconensis 481 Encystment activity 1113 of zoospores 113 Enterohepatic cycle 1188 £«/-kurospongin 120 cytotoxicity of 120 Enzymatic inhibitors 1197 Enzymatic reduction 137 of glucose 137 EPA-Superfund site 1139 bioprospecting in 1139 £f>/-acetylscalarolide 147 cytotoxic activity of 147 Epikingiside 342 biosynthetic pathway of 342 £p/-kingiside derivatives 329 origin of 329 structures of 329 Epikingiside derivatives 344 routes to 344 7-Epinemorosone 689 structure of 689 12-ep;-Scalarin 146 Epoxidation 576 28,29-Epoxyplukenetione A 699 structure of 699 2,3-Epoxysqualene 434 formation of 434 from squalene 434 Epstien-Barr virus early antigen (EBVEA) 451 inhibitory effects on 451
1224 Erdin 479 from Aspergillus terreus 479 from Penicillium sp. 479 Erycristagallin 842 anti-inflammatory activity of 842 Eryepogin F 832 as anti-methicillin 832 Erypostyrene 833,834 against 13 MRSA strains 833 anti-candidal activity of 833 antimicrobial activity of 833 anti-MRSA activity of 833 inhibitory effect of 834 structure of 833 Erythrina abyssinica 822 against malaria 822 against syphilis 822 Erythrina americana 822 in insomnia 822 Erythrina fusca 822 in fever 822 in malaria 822 Erythrina glauca 842 anti-HIV activity of 842 Erythrina indica 822,835 against human KB cells 835 in fever 822 in malaria 822 in vitro 835 Erythrina pterocarpans 842 antibiotic activity of 842 Erythrina species 822-824 as narcrotic 822 as purgative 822 ethnomedical application of 823-824 role in thrombosis 822 Erythrina variegata 827 against inflammation 827 Eryvariestyrene 833,834 against Staphylococcus aureus 833 against Salmonella gallinarium 833 from Erythrina variegata 833 inhibitory effect of 834 structure of 833 Escherichia coli 125 Bee venom PLA2 against 124 Esterification 348 with iridane 348
Estradiol 1182 structure of 1182 Estrogen deficiency 1195 symptoms of 1195 Estrogenic 1202 soy-based products 1202 Estrogenic activity 1190,1191 offlavonoids 1190 Etherification 712 9-Ethoxy aristolactam 882 from Aristolochia mollissima 882 9-Ethoxyaristolactone 895 Eudesmanes 911 from Aristolochia acutifolia 911 Euglena mutabilis 1141 Euglypha 855 Excdeconolides A 1026 from Exodecosus maritimus 1026 Exodeconolides B 1026 from Exodecosus maritimus 1026 Exodeconolides C 1026 from Exodecosus maritimus 1026 Exremophilic microorganisms 1127 Extremophiles 1130 from inland environments 1130 Extremophilic microbes 1128 of yellowstone national park 1128 Extremophilic organisms 1125 Extremozymes 1131 source of 1131 Falconensine E 490 from Emericella falconensis 490 structure of 490 Falconensine K 490 from Emericella falconensis 490 structure of 490 Falconensine L 490 from Emericella falconensis 490 structure of 490 Falconensine M 490 from Emericella falconensis 490 structure of 490 Farnesanes 903 from A. argentina 903 Fascaplysinopis reticulate 127 iso-dehydroluffariellolide from 127 Fasciospongia 1139
1225 Fasciospongia cavernosa 132,139,142 cacospongionolide B from 132 cavemosolide from 142 F a s c i o s p o n g i a sp. I l l Feeding activity 1045 against Tribolium castaneum 1045 of jaborosalactone P 1045 FellutanineD 571 structure of 571 t-Ferulloyl 254 structure of 254 Fever 374,381,822 Erythrina fusca in 822 Erythrina indica in 822,835 Gardenia jasminoides in 374 Logotis brevituba in 381 Fevillea cordifolia 438 cucurbitacins from 439 Fibrosing interstitial nephropathy 991 Flaccidoxides 95,96 structure of 95,96 Flavones 813,817,1179 structure of 1179 Flavonoids 671,944,945,947,1062,1178 from Aristolochia species 945,946 repellent activity of 1062 structures of 947 Fluorescence microscopy 1094 Fluroindolocarbazoles 498 structure of 498 Foliaspongin 153 antiinflammatory activity of 153 Fontanesia secoiridoids 341 biosynthetic pathway to 341 Fontoanesia 340,345 secoiridoids from 340 Formamide-containing sesquiterpenoids 176 axamide-1 176 axamide-2 176 axamide-3 176 axisothiocyanate-3 176 Formononetin 1185 sources of 1185 Forsythia iridoids 340 biosynthetic pathway to 340 (-)-FR901483 8,12-15,21,24 Ciufolini's synthesis of 24 Snider's synthesis of 14
Sorensens synthesis of 21 synthesis of 12-15 total synthesis of 8 (±)-FR901483 25 via amidoacrolein cycloaddition 25 FR901483 3,4,6,7,8-15,19,22,25,29,31, 35-40 biosynthesis of 4 Bonjoch's synthesis of 37-40 Brummond's formal synthesis of 31 Brummond's retrosynthetic analysis of 29 Ciufolini synthesis of 15,22 construction of 6 framework of 6 from Cladobotrym sp. 3 Funk's retrosynthetic analysis of 25 immunosuppressive activity of 3 in vitro 3 Kibayashi's synthesis of 35-39 Snider's synthesis of 8-15 Sorensen synthesis of 15 structure of 3 FR901483 skeleton 32 synthetic approaches to 32 FR901483 synthesis 5-8 main features of 6 Fraxinus excelsior 342 Fromonoetin 1083 structure of 1083 biological activity of 1083 Fumitremorgin B 580 structure of 580 Fungi 471,481,483 Halogen containing compounds from 471,481 isochromophilones II against 483 Fungal antibiotics 518 from Pterula species 518 Fungal metabolite 512 from Fusarium sp. 512 Fungal origin 549 of bioactive alkaloids 549 Fungicides 1107 Funk's synthesis 25-28 via amidoacrolein cycloaddition
1226 25 of(±)-FR901483 25-28 Furnish diketopiperazine 569 cyclization to 569 Furospinosulin-1 110 from Ircinia spinosula 110 Fusarium heterosporum 155 neomangicols A-C from 155 Galium aparine 290,291 as antiscorbutic 291 as apeient 291 as diuretic 291 as refrigerant 291 iridoid glycosides from 291 Galium atuntsiensis 269 Galium cambogia 765 against gastric ulcers 765 Galium campestris 270 Galium depressa 270 Galium gelida 270 Galium hombroniana 672 Galium karroa 266,267 in improving appetite 267 in leucoderma yunani 267 in stimulating gastric secretion 267 in syphilis 267 Galium kurroa 270 Galium linearis 268,270 iridoids from 268 Galium lufea 265,266,270 against snake bites 266 amarogentin from 265 as antidote 266 as antirabies agent 266 gentianine from 265 gentiopicroside from 265 in dyspepsia 266 in gastric inflammation 266 in hepatic/gall bladder disease 266 in liver disorders 266 in stomach ailments 266 sweroside from 265 swertiamarin from 265 Galium macrophylla 270 Galium manshurica 269 Galium punctata 270 Galium purpurea 270
Galium pyrenaica 270 Galium rigescens 269,270 Galium rubiaceae 290 Galium septemfida 270 Galium tessmanii 804 Galium tibetica 267,270 in bacterial infections 267 in constipation 267 in hepatitis 267 secoiridoid glycosides from 267 Gallium triflora 269 Galtonia 220 fom Candicans geltonia 220 Gangliosides GM2 235 Garcinol 704-708,764 anti-MRSA activity of 704 antioxidant mechanism of 708 antioxidative activity of 705 chelating activity of 706 in Fenton reaction system 706 radical scavenging activity of 705 Garciosaphenone A 705 anti-HIV activity of 705 antimicrobial activity of 705 Gardeniajasminoides 374 in inflammation 374 in hypertension 374 in fever 374 Gardenoside 250,386 against Alzheimer's disease 386 Gastric inflammation 266 Galium lufea in 265,266,270 Gastric ulcer 707 oxygen species role in 707 ge/w-Methyls 689 13 C-chemical shifts of 689 Genipine 384,386 against Alzheimer's disease 386 chemopreventive activity of 384 effects on glutathion-transferase 386 hepatotoxic activity of 386 Geniposide 384,386,387 anti-asthamatic property of 387 antioxidant activity of 384 antitumoral property of 384 chemopreventive property of 384 effects on collagen synthesis 387 effects on mieloperoxidase (MPO) 384
1227 Genistein 834,1056,1182 derivatives of 834 structure of 1056,1182 Gentian 270 iridoids from 270 secoiridoid glycosides from 270 Gentiana chyrayta 261 Gentianae radix 265 used in folk medicine 265 Gentiopicrin 261 Gentiopicroside 248,261,262,266 anti-inflammatory activity of 266 fungicidal activity of 266 Genus didiscus 198 Genus erythrina 821,822,825 bioactive non-alkaloidal constituents from 821 bioactive compounds from 825 in female infertility 822 in gonorrhoea 822 in stomach pain 822 pharmacological information of 822 Genus Illicium 395 Genus iridomirmex 247 Genus Sarcotragus 119 Genus Spongia 139 Geodiamolide 83 against P-388 cancer cells 83 structure of 83 Geodin 479 from Penicillium sp. 479 Geranylfarnesol 110 from cochliobolus 110 Germination 1070 hyphal growth after 1070 Germination inhibiting activity 988 of(-)-(3-bisabolene 988 Giant redwoods 1124 endosymbionts of 1124 Gibberellin-antagonistic activity 430 of cucurbitacins 429,430-435,438, 439-446,447,456 Gillusdin 479 from Aspergillus terreus 479 Gilmaniella humicola 531 Gilmaniellin 532 from Gilmaniella humicola 532 Ginkgo fruits 1084 motility inhibitory factor from
1084 zoospore lytic factors from 1084 Ginkgo metabolites 1087 motility inhibitory/zoospore lytic properties of 1087 Ginsenoside Rg3 224 vinblastine efflux inhibition 224 Ginsenosides Rbl 232 Gintiana lutea 265 uses in folk medicine 265 Gisan-depsidone biosynthetic pathway 510 Glaziovine 882 from Aristolochia chilensis 882 Gleditsioside E 227 against MCF-7 cell lines 227 against HL-60 cell lines 227 Glucanase inhibitor proteins (GIPs) 1106 Gluconeogenesis inhibitor 540 Glucosidase-catalysed hydrolysis 350 Glucosylation 1184 (3-Glucuronidase inhibitors 817 8-hydroxytricetin 7-glucuronide 817 isovitexin 817 Glutinol 816 analgesic activity of 816 anti-inflammatory effects of 816 Glycosides 226 apoptosis inducing activity of 226 Glycyrrhizin 230 antitumor properties of 230 characterization of 1071 effects on macrophage-derived NO production 230 in signal transduction pathways 1072 structure of 230 Gratiola officinalis 430,432-435 Griseoflavin 471-479 anti-inflammatory properties of 477 as fungistatic 477 biosynthesis of 475 biological activity of 476 chlorine-containing antibiotic 471 determination of 476 fermentation conditions of 471,472 for onychomycosis 477
1228 for treatment of dermatophytoses 477 from Penicillium griseofulvum 471 from Penicillium janczewskii 474 from microorganisms 472 from Penicillium urticae 474 isolation of 471,475 large-scale prodution of 473 purification of 475 side effects of 478 spatial arrangement of 478 to Treat tinea capitis 477 used in dermatophyte onychomycosis 477 vasodilatory effects of 477 Gonorrhoea 262,822 antihepatitis drugs in 262 Genus Erythrina in 821,822,825 Guaianes 916 from Aristolochia linkiuensis 916 Guibourtia tessmanii 804 Guttiferones A-E 702,712 cytopathic effects of 702 in A2780 human ovarian cell 712 in vitro 702 Gypenosides 226 apoptosis inducing activity of 226 Gypsetin 571 structure of 571 Halichondria sp. 131 palauolide from 131 Haliclona tulearensis 81 Haliotis rufescens 147 Halistanol disulfate B 76,77 effect on ECE 77 structure of 77 Halisulfates 9 136 Halisulfates 2 131 antimicrobial activity of 131 from Halichondria sp. 131 Halitulin 82 against cancer cell lines 82 cytoxicity of 82 structure of 82 Halogen containing compounds 471,481 from fungi 471,481 Halophiles 1127 salty environment for 1127
Hamiltonins A-D 100 from Chromodoris hamiltoni 100 structures of 100 Hansenula anomala (Pichia anomala) 1159 Harpagoside 249 Harpagophytum erecta 151 Hyrtialfrom 151 Harpagophytum procubens 374 a-Hederin 223 HeLa cells 225 proliferation of 225 HelicusinA 488 from Talaromyces helicus 488 HelicusinB 488 from Talaromyces helicus 488 HelicusinC 488 from Talaromyces helicus 488 HelicusinD 488 from Talaromyces helicus 488 Hemiasterline 83 against P-388 cancer cells 83 structure of 83 Hepatitis 267,381 Galium tibetica in 267,270 Logotis brevituba in 381 Hepatoprotective activity 262 of gentiopicroside 262 ofsweroside 262 Heptylprodigiosin 188 in vitro 188 structure of 188 Heteronema erecta 147 heteronemin from 147 Hexanorcucurbitacin I 434 structure of 434 High blood pressure 3 81 Logotis brevituba in 381 Hippospongia sp. 116,117 hippospongins A-C from 116 inhibitory effects on human Rasconverting enzyme 117 sesterterpenoid acid from 117 HIV-1 replication 833 inhibitors of 833 15-HLO 135 halisulfate 1 inhibitor of 135 Homobaldrinal 256 Homofascaplysin A 187 from Hyrtios erecta 187
1229 from Plasmodium falciparum strains 187 Homofascaplysin A 187 structure of 187 Hormonal effects 1189 of isoflavones 1177,1180,1181, 1185 Hormone replacement therapy 1195 isoflavones role in 1195 Host-specific attractants 1056,1059 structures of 1056 Host-specific chemical signals 1057 Host-specific chemoattractants 1066 Host-specific plant signal 1061-1064 in host recognition 1061 in germination of pest propagules 1061 Host-specific signals 1058 evidence of 1058 for zoospore chemotaxis 1058 5-HT2A receptor ligand 1156,1164 from Pithomyces sp. 1164 Human 122-lipoxygenase(12-HLO) 135 inhibitor of 135 Human bronchopulmonary non-small-celllung carcinoma cell line 141 petrosaspongiolides A-J against 141 Human cdc25A protein phosphatase 137 dysidiolide inhibitor of 137 Human colon tumor (HCT-116) cytotoxicity 76 of pyrroloiminoquinones 76 Human KB cells 836 cytotoxicity against 836 Human synovial PLA2 135,139,141,142 cacospongionolides E inhibition of 135 cavernolide effects on 139 effects on 142 Human tumor cell lines 136 Humualnes 908 from Aristolochia birostris 908 Huntington's disease 1150 therapies for 1150 Hydrogenolysis 21 of dibenzylphosphate 21 10-Hydroxyoleoside 249 8-Hydroxygeraniol 250 5-Hydroxyisoflavonoids 835 as allelopathic 835
Hydroxylation 347,566 ofoleosides 347 8-Hydroxymanzamine A 182 structure of 182 5P-Hydroxynicandrin B 1029 structure of 1029 8-Hydroxytricetin 7-glucuronide 813 structure of 813 28-Hydroxywithanolide E 1024 structure of 1025 20-Hydroxywithanolide glycosides 1027 from Dunalia brachyacantha 1027 19-Hydroxywithanolides 1032 Hymenoxin 813,817 against human tumour tissues 817 cytotoxic effects of 817 in vitro 817 structure of 813 Hymonymic Chinese crude drugs 998 Hyperibones A-D 704 antibacterial activity of 704 Hypertension 374 Gardenia jasminoides in 374 Hypertensive stroke-prone rat 652 Hyphal growth 1070 after germination 1070 Hypoglycemic activity 262,356 of gentiopicroside 262 ofiridoids 356 of secoiridoids 356 ofsweroside 262 of swertiamarin 262 Hypoglycemic effects 540 in vivo 540 Hypolipidemic active metabolites 512 ascofuranone 512 ascofuranol 512 Hyrtios erecta 127,146,147 hyrtiolide from 127 Hyrtios erectus 143 hyrtiosal from 143 Hyrtios sponge 71 Hyrtiosal 154 from Hyrtios erecta 154
Iberis amara 461 Iberis umbellate 431 antagonist activity of 431
1230 Ibogaine 814 anti-addictive properties of 814 Ichthyotoxic effects 153,154,157 against Gambussia affinis 153 Ichthyotoxicity 117,143,144 in Artemia salina assay 144 in fish lethality assay 117 to Gambussia affinis 117,143 Idiadione 111 in Artemia salina bioassay 111 in cancer cell lines 111 IFN-Y
238
in vitro production of 238 IL-2 238 in vitro production of 238 IlicicolinD 512 from Cylindrocladium ilicicola 512 Illicinolides 411 biosynthesis of 411 Illicium anisatum 396 toxic substance from 396 Illicum vernum 396 fruits of 396 Immune system 456 cucurbitacins effects on 429, 430-435,438,439-446,447,456 Immunoadjuvant activity 209 ofiridoids 356 ofsaponins 209 of secoiridoids 356 saponins as 233 Immunomodualting activity 209,979 of aristolic acid 977,978 of bioactive saponins 209 Immunomodulatory activity 229,979 of arisolochic acid I 979 ofsaponins 229 Immunomodulatory constituents 573 from Microascus tardifaciens 573 Immunomodulatory effects 233 of Albizzia adianthifolia 233 Immunosuppressants 1136 Immunosuppressive activity 3,774,782 of celastrol type compounds 782 ofFR901483 3,4,6,7,8-15,19,22, 25,29,31,35-40 of Tripterigyum wilfordii 114 IndicamineB 849 strucutre of 849
Indoles 493-498 from Penicillium crustosum 493 Indole alkaloids 809 structures of 809 Indole-3-carbaldehyde 1056 structure of 1056 Inflammatory activity 116 of Thorecta horridus 116 in vivo 116 Influence type A virus 355 Inorolides A 155 cytotoxicity of 155 Insecticidal activity 262,550 against silkworms 550 ofchirata 262 Insecticidal compounds 550 Insecticidal okaramines 582 discovery of 582 Integrin-mediated cell adhesion 441 inhibition 441 Interleukin (IL-2) transcriptional activation 793 inhibitor of 793 Iochroma coccineum 1025 Ipecoside 249 Ircinia 139 Ircinia fasciculate 115 fasciculatin sulphates from 115 palinurin from 115 Ircinia oros 119 Ircinia variabilis 115 fasciculatin sulphates from 115 palinurin from 115 Ircinin 112 anti-inflammatory activity of 112 Ircinin-1 111 from Ircinia oros 111 Ircinin-2 111 in mouse ear edema 111 Iridodial 365 structure of 365 Iridodialogentiobioside 249 Iridoid bearing plants 247 chemical and biological aspects of 247 of temperate region 247 Iridoids 248,251,252,291,305-333,340, 352,353,365,381 analgesic activity of 3 82 antiinflammatory activity of 353,382
1231 antitumoral activity of 383 biological activities of 252,352 biosynthesis of 251,365 cardiovascular activity of 352 characterisation of 251 chemopreventive activity of 383 extraction of 251 from Oleaceae 305 hypothetical biosynthetic pathway to 340 in myxopyreae 340 pharmacological activities of 252,365 structure of 248,291,305-333 Iridoid containing drugs 252 Iridolactones 366 from actinidia polygama 366 from Nepeta cataria 366 Iridomyrmecin 247 defensive mechanisms of 247 Insect repellent 860 Bothrops atrox as 860 Insecticidal activity 576 of communesins 574-579 Insomnia 289,822 catnip tea in 289 Erythrina Americana in 822 Intramolecular aldol reaction 4 ofketoaldehyde 4,10,13 Iridoids 268,355-357 antileishmanial activity of 357, 1048 antiviral activity of 113,138,209, 355 from Grevillea linearis 268,270 hypoglycemic activity of 262,356 Iridomyrmex ants 365 Ishwaranes 920 from Aristolochia argentina 920 Islanditoxin 502 from Penicillium islandicum 502 Isoaristolactone 923 from Aristolochia versicolar 923 Isochromophilone IX 485 from Penicillium sp. 485 GABA-containing metabolite 485 Isochromophilones I 482 anti-HIV activity of 483 120CD4 binding inhibitors 482 from Penicillium multicolor 482 Isochromophilones II 483
against Aspergillus niger 483 against Bacillus subtilis 483 against Candida albicans 483 against Escherichia coli 483 against fungi 483 against Micrococcus luteus 483 against Piricularia oryzae 483 Isochromophilones III-VI 483 against Acholeplasma taidlawii 484 against Aspergillus niger 484 against Bacillus subtilis 484 against Bacteroides fragillis 484 against Candida albicans 484 against Escherichia coli 484 against Micrococcus luteus 484 against Mucor racemosus 484 against Myeobacterium smegmatis 484 against Pseudomonas aeruginosa 484 against Pyricularia oryzae 484 against Saccharomyces sake 484 against Staphylococcus aureus 484 against Xanthomonas oryzae 484 from Penicillium multicolor FO-3216 483 inhibitors of ACAT 484 Isochromophilones VII-VIII 44 against Bacillus subtilis 485 against Micrococcus luteus 485 against Myeobacterium smegmatis 485 against Pyricularia oryzae 485 antimicrobial activity of 485 from Penicillium sp. 484 in vitro 484 Isocoumarins 521-526 Isoflavones 1177,1180,1181,1185 absorption of 1183 antagonist effects of 1194 antiproliferative effects of 1195 as functional food components 1177 bioavalability of 1181 chemical features of 1180 distribution of 1180 effect on cardiovascular diseases 1199
1232 estrogen agonist effect of 1194 food sources of 1185 functional properties of 1188 hormonal effects of 1189 metabolism of 1181,1186 osteoprotective effects of 1195 structures of 1187 Msoferulloyl 254 structure of 254 Isoflavonoids 834,1178,1083 chemotaxis of 1083 from Erythrina species 834 Isogarcinol 714 effects on human leukemia cell lines 714 Isogarcinol-xanthochymol mixture 710 apoptosis-inducing effects of 710 against human leukemia 710 Isonitriles 176 and analogues 176 Isoprenylated tyrosine derivatives 1164 structures of 1164 Isoquinolines 890,892 from Aristolochia arcuata 890 from Aristolochia elegans 890 from Aristolochia gehrtii 890 from Aristolochia species 892 Isosenegalensin 835 as anticancer agent 835 Isoxanthochymol 705 anti-HIV activity of 705 Ixocarpalactone A 1027 from Physalis philadelphica 1027 (-)-Jaboromagellonine 1029 from Jaborosa magellanica 1029 structure of 1030 Jaborosalactol 1023 Jaborosalactone 1033 from Jaborosa araucana 1033 Jaborosalactone 8 1024 from Jaborosa leucotricha 1024 Jaborosalactone O 1032 from Jaborosa leucotricha 1032 Jaborosalactone P 1045 feeding activity of 1045 Jaborosalactone R 1030 from Jaborosa sativa 1030 structure of 1030
Jasmineae 346 biosynthetic pathway in 346 Jasminum lanceolarium 350 Jasminum hemsleyi 309 jashemsloside A from 309 jashemsloside C from 309 jashemsloside D from 309 Jasminoside 350 esterification of 350 Jasmoaldehydes 352 formation of 352 Jasmolactones 332 origin of 332 structures of 333 Jaspamide 83 against P-388 cancer cells 83 structure of 83 Jaspamide TA 82 cytotoxic depsipeptides 82 from Hemiastrella minor 82 Jaspiferals E-F 145 against L1210 145 cytotoxicity of 145 Jaspis johnstoni 520 Jaspolinaloside 347 from Jasminum. polyanthum 2>A1 Jenisseenssosides C,D 227,,232 apoptosis inducing activity of 227 jurkat cells proliferation effects of 232 Jesterone 1109 as mycelial growth inhibitor 1109 Jurkat cells proliferation 232 effects of Jenisseenssosides C,D 227,232 Jujuboside A 238 structure of 238 Jujuboside B 238 structure of 238 Jujuboside Bl 238 structure of 238 Jujuboside C 238 structure of 238 Jujubosides 238 immunological adjuvant activity of 238 Kageneckia oblonga A31,AAA analgesic activity of 444 anti-inflammatory activity of 444
1233 antipyretic activity of 444 as antioxidant 444 cucurbitacins from 437 Kaikosaponin III 210 from Pueraria thunbergiana 210 antimutagenicity activity of 211 Kaitocephalin 527 structure of 527 from Eupenicillium shearii 527 Kalihinane diterpenoids 179,180 antifungal activity of 179 antihelmintic activity of 179 from A canthella sp. 180 KalihinolA 179 against mouse mammary tumor 179 cytotoxicity activity of 179 KalihinolA 180 against Plasmodium falciparum 180 Kalopanaxsaponin A 222 Kampo medicines 998 Ketoaldehyde 4,10,13 aldol reaction of 13 intramolecular aldol reaction of 4 preparation of 14 synthesis of 10 Ketone enolates 38 cyclization of 38 Kibayashi's synthesis 35-39 ofFR901483 3,4,6,7,8-15,19,22, 25,29,31,35-40 Kigelia pinnata 371 in cancer 371 in skin infections 371 Kohamaic acids A and B 137 against P388 cells 137 Kolanone 705,765 against Bacillus subtilis 705 against Staphylococcus aureus 705 antimicrobial activity of 705 Lactonization 350 Lagenidium giganteum 1054 Lamiosie 250 Lamium labiatae 288 against hemorrhage 288 antiinflammatory properties of 288
as antispasmodic 288 as astringent 288 as depurative 288 as haemostatic 288 in bowel movment 288 in menorrhagia 288 Lannea 1088 bioassay-guided fractionation of 1088 Lannea coromandelica 1088,1089 characterization of 1089 MALDI-TOF-MS of 1089 motility inhibitory activity of 1088 zoosporicidal polyflavonoids from 1088 Lannea extracts 1088 physicochemical properties of 1088 Lannea tannins 1094 lysis of zoospores by 1094 Lamium amplaxicaule linn 288 Latrunculin A and B 101 from Chromodoris hamiltoni 101 LC-MS methods 476 LC-MS-MS methods 476 LC-PDA detection method 997 Learning and memory impairment 638 butylidencphthalide in inhibition of 638 35-butyphthalide in inhibition of 638 cnidilide in inhibition of 638 Leg ulcer 863 Aristolochia clematitis in 863 Leishamania spp. 1048 LepadinD 185 structure of 185 Lepadin E 185 structure of 185 Lepiochlorin 180 526 an antibacterial lactol 526 Leukemia 710 against human isogarcinolxanthochymol mixture 710 Levistolide B 638 in blood viscosity reduction 638 Lewis lung carcinoma cells 223 Life-cycle development 1056 ofoomycetes 1056
1234 Lignans 937,939-944 from Aristolochia species 939-944 structures of 941 Ligstral-type secoiridoids 350 Ligusticum acuminatum 613 Ligusticum acutilobum 613 Ligusticum chuanxiong 613 Ligusticum frondosa 152 homoscalarens from 152 Ligusticum jeholense 613 Ligusticum jeholense var. tenuisectum 613 Ligusticum mutellina 613 Ligusticum offwinalc 613 L igusticum porteri 613 Ligusticum sinense 613 Ligusticum sinense cv. chaxiong 613 Ligusticum tenuissimum 613 Ligusticum variabilis 124 dehydromanoalide from 124 Ligusticum wallichii 613 Liguistilide 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Ligustroside 356 against Escherichia coli 356 Limestone formations 1138 Linear sesterterpenoids 110 Lintenolide 141 cytotoxicity of 141 against a tumor cells 141 Lippia multiflora 805 antibacterial activity of 805 pediculocidal activity of 805 5-Lipoxygenase 112 ircinin inhibitor of 112 Liquiritigenin derivatives 826,828 activity of 826 sources of 828 Lissoclin disulfoxide 87 structure of 87 Lissoclinotoxin A 188 against Plasmodium falciparum 188 structure of 188 Liver disorders 266 Gentiana lufea in 265,266,270 Liver protective 279 effect of aucubin 249,250,279, 383,386,387
7-ep/-Loganic acid 341 oxidation of 341 Loganin 356 hepatotoxic activity of 356 Logotis brevituba 381 in fever 381 in hepatitis 381 in high blood pressure 381 Lonicera (caprifoleaceae) 273 antitussive activity of 274 as antipyretic 274 as sedative 274 bis-iridoids from 273 coumarin glycosides from 274 hypotensive activity of 274 iridoids from 273 sulfur containing monoterpenoids from 273 triterpeoids saponins from 274 Lonicera angustifolia 275 use in gastric troubles 275 Lonicera caerulea 277 caeruloside A from 277 caeruloside B from 277 epivogeloside from 277 ketologanin from 277 loganin from 277 secologanin from 277 sweroside from 277 Lonicera japonica 21A caffeoylquinates (CQs) from 274 effects on HIV 274 for rashes 274 for skin ailments 274 Lonicera implexa 371 Lonicerapericlymenum 275 as antispasmodic 275 as diuretic 275 biosidic ester iridoid glucoside from 275 secoiridoid glucosides from 275 use in respiratory tract 275 /-Triptolide 787 synthesis of 787 via /-dehydroabietic acid 787 Luff a operculata 430 Luffalactone 132 from Luffariella variabilis 132 Luffariella geometric 133 Luffariella variabilis 133 Luffariolide F 125
1235 Luffariolide G 125 Lung adenocarcinoma 975 Luteusin C 488 from Talaromyces luteus 488 structure of 488 Luteusin D 488 from Talaromyces luteus 488 structure of 488 Luteusin E 488 from Talaromyces luteus 488 structure of 488 Lymphocyte proliferation 231 action on 231 Lymphoproliferative activity 233 Maclurin 672 structure of 672 Macrocycles 502-506 Macrocystic pyrifera 147 Macrophage activation 230 role in host defense mechanisms 230 Madolis 909 from Aristolochia mollissima 909 Magnoflorine 882 from Aristolochia 882 Majonoside-R2 212 from panax vietnamiensis 212 Majucin-subtype sesquiterpenes 401-404 Malaria 169,170,822 by Plasmodium falciparum 169 by Plasmodium ovale 169 by Plasmodium malariae 169 Erythrina abyssinica against 822 Erythrina fusca in 822 Erythrina indica in 822,835 symptoms of 170 Malaria infection 170 quinine for 170 Mammalian sex hormones 1099,1100 activities of 1100 repellent activity of 1099 Mammary tumor cell line MCF7 140 spongianolides A-E inhibitory effects on 140 Manoalide 123,141 isolation of 123 Manzamine A 182 against Plasmodium falciparum 183
in vitro activity of 183 structure of 182 Manzamine alkaloids 185 against malarial parasite 185 Manzamine F 183 structure of 183 Marcfortine B 604 structure of 604 Marine natural products research 63 in southern Africa 63 Marupone 672 structure of 672 Measles 289 catnip tea in 289 Medicarpin 1083 as phytoalexin 1083 (i)-Medicarpin 1083 structure of 1083 Medico-magic plants 803,806,707 bioactive natural compounds from 803 of Bantu area 803 use of 806,807 Melanin synthesis inhibitor 463 cucurbitacins 463 MelledonalC 511 MelledonalD 511 from Armillaria spp. 511 Menodora robust a 350 Menorrhagia 288 Lamium labiatae in 288 Merrilactone A 417 synthesis of 417 Methanococcus jannachii 1130 7-Methoxyaristolochic acid I 863 6-Methoxybenzoxazolinone 818 from Scoparia dulcis 818 pharmacological activity of 818 Methyl protoneodioscin 214,218 antitumor activity of 214 Methyl-3-epinuapapuanoate 198 against Plasmodium berghei 198 in vivo 198 Methyl-3-epinuapapuanoate 198 structure of 198 24-Methylscalaranes 152 from Dictyoceratida sp. 152 from Halichondria sp. 152 8-O-Methylsclerotiorinamine 487 from Penicillium multicolor 487 structure of 488
1236 Microbial fermentation schemes 1157 Microbial products 549 as antibiotics 549 as enzyme-inhibitors 549 as fungicides 549 as herbicides 549 Micrococcus luteus 125 Mikrolin 532 structure of 532 Millecrols 98 structure of 98 Millecrones 98 against bacillus subtilus 98 against staphylococcus aureas 98 structures of 98 Mimosa 1093 zoosporicidal activity of 1093 Mitochondrial ATP synthase inhibitor 118 oligomycin 118 Miwanensin-type sesquiterpenes 400 MMP inhibitors 1154 therapeutic use of 1154 Modolin type sesquiterpenoids 964 biogenetic sequences of 964 Molluscicidal activity 123 ofmanoalide 123 against Biomphalaria glabrata 123 Monocarbocyclic sesterterpenoids 123 from genus Luffariella 123 Monodora myristica 807,815 antibacterial activity of 807 in vitro 807 Monomeric secoirided glucosides 310 origin of 311 structures of 311 Monordens C-E 539 from Humicola sp. 539 Monoterpenoids 810,896-901,897 from Aristolochia species 897-901 Monotropein 250 Morinda citrifolia 376 Morphine withdrawal 142,983 effect of constrictosine in 983 role of petrosapongiolide M in 142 Motility inhibitors 1078,1079 biological activity of 1079 function of 1079 structure elucidation of 1078
Motility inhibitory activity 1086 of anacardic acids 1084-1086 Motility inhibitory factor 1084 from Ginkgo fruits 1084 Mouse lymphocytic leukemia 975 MPP+ induced apoptosis 990 inhibition of 990 Multiple sclerosis 1151 Muqubilone 197 Muqubilone (or aikupikoxide A) 123 against herpes simplex virus type 123 antiviral activity of 123 from Diacarnus erythraeanus 123 in vitro 123 Murine allogenic bone marrow transplantation 775 Musanga cecropiodes 805,807 analgesic effects of 807 Mycobacterium smegmatis 838 Mycobacterium tuberculosis 263 activity against 263 Mycorrhizinol 532 structure of 532 Mycorrhizins 523 biosynthesis of 523 Myxopyreae 345 biosynthetic pathway in 345 N. arbortristis 305 arborside A from 305 arborside B from 305 arborside C from 305 arbortristoside A from 305 arbortristoside B from 305 arbortristoside C from 305 arbortristoside D from 305 arbortristoside E from 305 nyctanthoside from 305 Narcotic 822 Erythrina species as 822-824 Naja naja PLA2 enzymes 141 Naja nigricollis 984 Nakafurans 97 structures of 97 Nannizzia gypsea var. incurvata 571 Naringenin derivatives 828 activity of 828 sources of 828
1237 Natural phthalides 624-637 biological activities of 637 classification of 624-637 Natural products 1103 bioactivites of 1103 Natural semi-synthetic quillajasaponins 235 Nectandrin-B 942 from Aristolochia chilensis 942 Nematocidal halogenated dihydoisocoumarins 522 from Lachnum papyraceum 522 Nemorosone 688,704,705,716 against human cervix crcinoma 716 against human larynx carcinoma 716 antimicrobial activity of 705 from Clusia grandiflora 704 from Clusia rosea 688 Nemorosone II 765 Neoanistatin 419 neurotoxic activity of 419 Neobavaisoflavone 839 antifungal properties of 839 from Aspergillus fumigatus 839 from Psoralea corylifolia 839 Neoechinulins D and E 572 «eo-Kauluamine 184 structure of 184 (6Z)-Neomanoalide 123 (6£)-Neomanolide 123 Nepatalactone 247 form Nepta cataria 247 Nepeta (labiateae) 289 as diaphoretic 289 as refrigerant 289 as soporific 289 Nepetariaside 249 Neurodegenerative disorder 1152 Neurological disorders 354 Neurotrophic acivity 396 of Illicium jiadifengpi 396 of Illicium merrillianum 396 of Illicium minwanense 396 of Illicium tashiroi 396 Nicotinamide 1095,1096 halting activity of 1096 motility-inhibiting activity of 1096 structure of 1095
Nigella sativa 223 antitumor activity of 223 Nigerazine A 593 structure of 593 Nigerazine B 593 structure of 593 Nigragillin 593 structure of 593 Nitric oxide (NO) 131 Nitrogen-tethered phenol derivatives 15-19 iodine oxidation of 15-19 Nitrone rac-9 9 1,3-dipolar cycloaddition of 9 Nitrongen-tethered phenols 16 spirocyclization of 16 yV-methoxyamide 17 cyclization of 17 NO synthase 650 Nomofungin 578,579 structure of 579 Non-glucosidic secoiridoids 332 origin of 332 structures of 333 Non-alkaloid phthalide 613-619 sources of 613-619 Non-alkaloid phthalide subtype 630 structures of 630 Non-alkaloidal constitutents 822 of Erythrina 822 Non-competitive GAB A antagonist 419 Non-glycosidic secoiridoids 350 Nonhost extracts 1075 Nonsteroidal anti-inflammatory drugs 713 in colonic tumor 713 in colon cancer 713 «or-cucurbitacins WGi 446 chemical structures of 446 «or-cucurbitacins WG2 446 chemical structures of 446 Norscalarals A-C 151 against tumor cell lines 151 from Cacospongia scalaris 151 23-Norscalarane petrosaspongiolide K 152 against NSCLC-N6 cells 152 18-Norwithanolides 1025 Novel bis-anthrones 501 from Anaptychia obscurata 501
1238 Novel sesquiterpenoid MMP-3 inhibitors 1165 from acid mine waste extremophile 1165 isolation of 1165 NPK fertilizers 260 application of 260 iV-fr-ara-Feruloyltyramine 1079 as natural stimulant 1079 iV-/rara-ferulyl-4-0-methyl-dopamine 1059 from Chenopodium album 1059 attractant activity of 1059 Nuclear DNA 1094 fragmentation of 1094 Ochrocarpinones A-C 711 against A2780 ovarian cancer cells 711 from Ochrocarpos punctatus 711 Oil-soluble artemether 190 in treatment of severe malaria 190 Okaramine A 551-573 absolute confirguration of 562 biosynthetic pathway of 564-567 discovery of 551 'H-NMR spectrum of 551 insecticidal activity of 563-564 molecular formula of 551 related compounds 570-573 Okaramines N and J 567-570 synthetic study of 567-570 Oleaceae 303 iridoids from 303 secoiridoids from 303 Oleacin inhibitor 352 of converting enzyme 352 Oleae 346 biosynthetic pathways in 346 Oleanolic acid 263 Olefinic bond 677 epoxidation of 677 Oleosides 347 hydroxylation of 347,566 Oleoside derivatives 343 biosynthetic pathway to 343 Oleoside biosynthesis 343 role of 7-ketologanic acid role in 343
Oleoside-type glucosides 346 biosynthesis of 346 Oleuropein 354-356 against RSV 356 antioxidant activity of 355 hypoglyscemic activity of 356 scavenging effects of 354 Olive oils 355 against reactive oxygen species 355 Oliverine 810,814 from Pimelea suaveolens 814 microfilaricidal activity of 814 structure of 810 Onjisaponins A 239 mucosal adjuvant activities of 239 Onjisaponins E 239 mucosal adjuvant activities of 239 Onjisaponins F 239 mucosal adjuvant activities of 239 Onjisaponins G 239 mucosal adjuvant activities of 239 OocydinA 1109 as mycelial growth inhibitor 1109 structure of 1109 Oomycetes 1070,1075 chemotropic responses of 1070 resistance against 1075 Oomycete phytopathogens 1053 bioactive secondary metabolites related to 1053 life-cycle development of 1053 Oomycete species 1111 host-specifity of 1111 Oomycete zoospores 1064 attractant for 1064 Ophelia chirata Grisebach 261 Organic acids 671 Ornoside 356 against Escherichia coli 356 against Staphylococcus aureus 356 1,2,34-Oxamanzamine A 184 structure of 184 Oxidation 341,936,1025 of 7-epMoganic acid 341 Oxidation products 710 ofgarcinol 719 mechanism of formation of 710 Oxidative decarboxylation 962 Oxidative ozonolysis 87
1239 19-Oxygenatedscalaranes 148 12-Oxygenated withanolides 1029 from Datura ferox 1029 Oxyresveratrol 831 depigmenting effects of 832 in tyrosinase inhibition 831 structure of 831 P-388 lymphocyte leukemia cell line screen 65 Pacchastrella sp. 77 effects on endothelin enzyme (ECE) 77 Paederia scandends 376 Palauolide 131 from Halichondria sp. 131 Palinurin 115 from Ircinia fasciculate 115 Panax saponins 215-219 structures of 215-219 Pancreatic cancer 1135 Papilio xuthus 1000 Para influence type 3 virus 356 Paraherquamide family 602 structures of 602 Paralytic activity 550 against silkworms 550 Paralytic compounds 594-604 Parkinson's diseases 980 aristolactam taliscanine in 980 Particle bioassay 1112 p-Coumaric acid 978 interceptive activity of 978 PDEs phosphodiesterases 650 Penicillium chrysogenum 1158 Penicillium expansion 550 insecticidal activity of 550 Penicillium fellutanum 571 Penicillium multicolor 486 5-bromoochrephilone from 486 Penicillium roqueforti 1166 Penicillium simplicissimum 550 insecticidal activity of 550 Penicillium urticae A1A transformation of 474 Penitrems 494 from Aspergillus species 494 from Penicillium 494 structure of 494 PenitremA 494,582-584
against Bombyx mori 494 against Heliothis zea 494 against Spodoptera frugiperda 494 convulsive effect induced by 584 from Penicillium crustosum 494 identification of 582 insecticidal activities of 494 related compound 583 structure of 495,582 Penitrem C 494 against Bombyx mori 494 against Heliothis zea 494 against Spodoptera frugiperda 494 as convulsive 494 from Penicillium crustosum 494 insecticidal activities of 494 structure of 494 Penitrem F 494 against Bombyx mori 494 against Heliothis zea 494 against Spodoptera frugiperda 494 as convulsant 494 from Penicillium crustosum 494 insecticidal activities of 494 structure of 495 Pentacarbocyclic sesterpenoids 156 Pentaglycoside 232 immunostimulant activity of 232 Pentobarbital-induced sleep 640 Pericosines A 537 from Periconia byssoides 537 Perophoramidine 578,579 from Perophora namei 578 structure of 579 Peroxyplakoric acids A3 195 structure of 195 Peroxyplakoric acids methyl esters 196 synthetic analogues of 196 Petrosaspongia 13 9 Petrosaspongiolide K 145 Petrosaspongiolide P 141,142 effects of 141 Petrosaspongiolides M-R 141 PG27 775 immunosuppressive activity of 775 Phychium cinnamomi 1057
1240 Phychium infestcms 1053 late-blight disease by 1053 Phychium sojae 1055,1058 zoospores of 1058 Pharmacology 855,971-993 of Aristolochia sp. 855,857-859, 862,971-993 Phenanthrenes 894,895,896 from Aristolochia species 894 structures of 895,896 Phenol 20 synthesis of 20 Phenolic amides 16 oxidative O-spirocyclization of 16 Phenolic oxazolines 17 oxidative spirocyclization of 17 Phenolic sulfonamides 19 oxidative spirocyclization of 19 Phleodictynes 186 structure of 186 Phomalactone 1108 by Nigrospora sphaerica 1108 Phomopsin A 505 from Phomopsis leptostromiformis 506 structure of 506 Phorbazoles 80 immunomodulatory activity of 80 structure of 80 PhorbazoleC 80 first total synthesis of 80 Phospholipase C activity 1072 Phospholipase injection 985 in edema 985 Phthalide 612, 611,613-624,640,642, 647,649,652,702 actions on central nervous system 611 against cerebral ischemia 646-647 against seizure 640 anti-spasmodic potencies of 651 as anti-angina 611,642 as anti-hypertensive agents 651 as anti-platelet 611,641 as anti-smooth muscle proliferation 611 as anti-thrombosis 611,641 biological activities of 611,702 chemical structure of 612 chemistry of 611 classification of 612
for food flavorings 611 in cardiac function 611 in cardiac function modulation 642 in dietary supplements 611 in fungi 611 in herbal remedies 611 in inhibition of smooth muscle cell proliferation 643 in smooth muscle relaxation 649 naturally occurring 611 sources of 612-624 vasodilatory actions of 651 vasorelaxing actions of 652 Phthalide derivatives 643 anti-proliferative activity of 643 Phthalide dimer type 620-622,634 sources of 620-622 structures of 634 Phthalide isoquinoline type 622-624,637 sources of 622-624 structures of 637 Phyllactones A 154 against KB cells 154 Phyllogenone B 157 from Phyllospongia foliascens 157 against P388 cells 157 Phylum coelentrata 88-96 bioactive metabolites from 88-96 Phylum hemichordata 64-78 bioactive metabolites from 64-70 Phylum mollusca 96-102 bioactive metabolites from 96-102 Phylum porifera 70-85 bioactive metabolites from 70-85 Physalins 1022 Physalis peruviana 1024 Phythium 1053 Phythium graminicola 1057 zoospores from 1057 Phytoestrogens 1099,1193 in human endometrial adenocarcinoma cells 1193 in vitro 1193 in vivo 1193 relative potency of 1193 repellent activity of 1099 Phytomedicines 996 used as slimming regimens 996
1241 Phytopathogenic oomycete zoospores 1058 Phytophthora 1053,1064 attractant for 1064 Phytophthora spp. 1105 Picfelterracin VI 443 structure of 443 Picracin 450 from Picrorhiza scrophulariaeflora 450 Picroliv 252,385,387 anti-allergic property of 387 anticholestatic activity of 252 antioxidant activity of 385 as chemopreventive agent 385 cholerectic activity of 252 hepatoprotective activity of 252, 385 injury protective activity of 385 Picrorhizia kurroa 252,253,378 in liver disorders 252 picroside-IV from 253 Picrorhizia scrophulariflora 252 in liver disorders 252 Picrosides I 252 hepatoprotective effects of 252 Piericidins 550 from Sreptomyces sp. 550 Pinicoloform 527 antibiotic activity of 527 cytotoxic activity of 527 Piperazinomycin 593 structure of 593 Piperolactam 981 antiplatelet aggregation activity of 981 PIT lake microbes 1140 initial discovery of 1140 PIT microbes 1159 biological activity profiles of 1159 PLA2 134 cacospongionolide B inhibitor of 134 PLA2 activity 986 in vitro inhibition of 986 PLA2 enzymes 986 TFVPL-lb 986 TFV-PL-la 986 PLA2-induced mouse paw oedema 838 effects of E. addisonae EtOAc
extract on 838 effects of warangalone on 838 PlakortideE 193 structure of 193 PlakortideF 194 antimalarial activity of 194 structure of 194 PlakortideK 194 antimalarial activity of 194 structure of 194 PlakortideL 194 structure of 194 PlakortideO 194,195 against Plasmodium falciparum 194 Plakortide P 194 against Plasmodium falciparum 194 Plakortin 193,194 against human colon carcinoma 194 against mouse lymphoma cells 194 structure of 193 Plakortis halichondraides 192 plakortin from 192 Plakortis sp. 196 against Plasmodium falciparum 196 Plantago altissima 280 Plantago asiatica 280 Plantago atrata 280 Plantago berghei infection 196 Plantago cornuti 280 Plantago lanceolata 280 in asthma 280 in cough 280 in inflammed surfaces 280 in pulmonary diseases 280 iridoids from 280 Plantago lundborgi 280 Plantago major 277,279 3,4-dihydroxyaucubin from 278 as diuretic 279 gardoside from 278 geniposidic acid from 278 in dysentery 279 in griping pain 279 in wound healing 279 majorside from 278 use in diarrhea 279
1242 use in eye wash 279 Plant ago major 374 in cancer 374 in skin/respiratory ailments 374 Plantago media 280 Plantago ovata 280 Plantago patagonica 280 Plantago renformis 280 Plasmodium falciparum 128,171 cytotoxicity against 128 Plasmopara viticola 1057 Plukenetione A 699 structure of 699 Plukenetione B 699 structure of 699 Plukenetione C 699 structure of 699 Plycitone 88 effects on retroviral reverse transcriptase enzymes 88 Pneumocystisjaroveci 1148 Polyalthie suaveolens 804,808 Polycitrins 87-88 structure of 88 Polyflavonoid tannins 1090,1092 against zoospores 1092 lytic activities of 1092 motility inhibition by 1092 structural units (A-D) of 1090 Polygalaceae saponins 228 Polyisoprenylated benzophenone derivatives 671 -673,702,703,705-716 against HIV-l 672 anti-cancer activity of 713-716 anti-HIV activity of 702-703 antimicrobial activity of 672, 703-705 antioxidant activity of 705-710 biological activity of 671,702 chemistry of 671 cytotoxicity of 710-713 from Allanblackia 672,673 from Clusia 672 from Garcinia 672 from Hypericum genera 672,673 from Moronobea 672 from Ochrocarpus 672 from Symphonia genera 672,673 from Tovomiptosis 672 from Tovomita genera 672,673
from Vismia genera 672,673 occurrence of 673 Polyketide derivatives 192 Polymerization 1089 Polyphenols 1186 metabolism of 1186 Polyprenylated benzophenones 671 Polyvinylidine difluoride (PVDF) 1071 Pomadasys commersonni 102 diterpenes from 102 Portulaca oleracea 1077 zoospore motility inhibitors from 1077 Preferential chemotaxis 1057,1058 toward hosts 1057,1058 Prenylation 723 Prenyldaidzein 838 against Staphylococcus aureus 838 seco-Prezizaane 395,411,419 biosynthesis of 411 chemistry of 395 from Illicium species 395 neurotoxic activity of 419 neurotrophic activity of 395 neurotrophic activity of 420 seco-Prezizaane-type sesquiterpenes 397 Prianos sp. 128 Promomonilicin 510 antimicrobial spectrum of 510 Propolone A 705 antimicrobial activity of 705 Prostaglandin (PGE2) 131 Prostaglandins 90 biosynthesis of 90 Protein kinase C (PKC) 140 spongianolides A-E inhibitory effects on 140 Protein synthesis inhibitors 113 Protoberberines 882,886,887 structues of 886 from Aristolochia species 887 Protodioscin 225 inhibitory effects on human leukemia HL-60 cells 225 Protojujuboside 238 Protoneodioscin 218 Protopanaxadiol saponin M1 229 antimetastatic property of 229 Protopines 888 structures of 888
1243 Prunetin 1056,1058 from pea seedlings 1058 structure of 1056 Pruritus 856 A. argenuna in 856 PS45A-4 1163 against caspase-1 1163 Pseudoanisating 420 insecticidal activity of 420 neurotrophic activity of 420-425 Pseudoarachniotus roseus 528 aranochor A from 528 Pseudoanisatin-type sesquiterpenes 397,398 structures of 398 Pseudomajucin-subtype sesquiterpenes 404-407 Pseudomajucin-type sesquiterpenes 404-407 structures of 405 Psoriasis 136 12-HLOrolein 135 Pterocarpans 843,844 anti-MRSA activity of 843 effect on rabbit platelet aggregation 844 from the Genus Erythrina 843 Purgative 822,431 Citrullus colocynthis as 431 Erythrina species as 822-824 Pythium aphanidermatum 1057 zoospores from 1057 Pythium arrhenomanes 1057 Pythium insidiosum 1054 Pyrite 1138 oxidation of 1138 Pyrroloinenoquinones 75 structure of 75 QS-21 234 adjuvant activity of 234 Quebracho 1093 zoosporicidal activity of 1093 Quebracho tannin 1089 Quillaja saponaria 234,236 aduvant activity of 234 Quillajasaponins 234 aduvant activity of 234
Racemichydroxylaminerac-10 9 condensation of 9 synthesis of 9 Racemic triptolide 787,788 total synthesis of 787,788 Radix (Angelicae sinensis) 611,655 estrogenic effects of 655 for treatment of cerebroand cardio-vascular disease 611 in female irregular menstruation 611 use in obstructive pulmonary diseases 655 Ram semmal vesicle (RSV) microsome 992 Rebeccamycin 497 as indolocarbazole antitumor agent 497 from Saccharothrix aerocolonigenes 497 Receptor independent apoptosis 69 of leukaemia cells 69 Recombinant human synovial PLA2 133 inhibitory effect on 133 Regeneration activity 1113 of zoospores encyst 1113 Rehmannia glutinosa 379 Respiratory disease 371 Ajuga decumbens in 371 Respiratory syncytial virus 355 Rhabdastrella (jaspis) stellifera 144 jaspiiferals C-F from 144 Rhabdastrella globostellata 144 aurorals from 144 Rhizobium-legume interaction 1058 Rhizoma chauanxiong 611 in female irregular menstruation 611 for treatment of cerebroand cardio-vascular disease 611 Rhopaloic acids A-C 121 from Rhopaloeides 121 minimum inhibitory concentration of 121 Rhopaloic acids A-E 121 from Hippospongia sp. 121 Rietone 90 from Alcyonium fauri 90 in NCI's CEM-SS cell line screen 90 structure of 90
1244 Ring-A aromtatic withanolides 1022 Ring-D aromatic withanolides 1022 RNA synthesis inhibitors 113 Rozella allomyces 1057 zoospores of 1057 Rubrorotiorin 492 in Pyrenula hirayamae 492 structure of 492 Saophularia buergeniana 379 Saikosaponin-D 226 apoptotic effects of 226 structure of 226 Salmahyrtisol A 154 against cancer cells 154 Salmahyrtisol B 147 against human gastric carcinoma 147 Salpichrolides E 1036 degradative pathway for 1036 Salpichrolides J 1036 biosynthetic pathway for 1037 Sampsoniones A-J 700 from Clusia plukenetii 700 structures of 701 Saponification 568 SaponinQS-7 236 Saponins 209,214,230,231,817,1075, 1104 as immunostimulants 230 against microbial pathogens 1104 antitumor activity of 214 antiviral activity of 113,138,209, 355 cyototoxic activity of 215 effects on luteinising hormonereleasing hormone 817 effects on (LHRH)-induced LH release 817 immunostimulant activity of 231 in vitro 214 in vivo 214 macrophage activation by 230 Saprolegnia spp. 1054 Saprosma scortechinii 376 Sarcoglane 95,96 structure of 95,96 SarcotinsN 120 against human tumour cell lines 120
cytotoxicity of 120 from sarcotragus 120 Sarcotins O 120 against human tumour cell lines 120 cytotoxicity of 120 Sativolides 1022,1030 18-epz-Scalaradial 150 cytotoxicity by 150 Scalaradial 150 effects on human neutrophils 150 Sclerotiorin 492 from Pyrenula japonica 492 from Penicillium sclerotiorum 492 structure of 492 Scopadulcic acid B 811,815 in vitro 815 structure of 811 Scopadulciol 811 structure of 811 Scopadulin 812 structure of 812 Scoparia dulcis 804 Scoparic acid A 811 structure of 811 Scopoletin 813 structure of 813 Staphyloccus aureus 123,126,128 luffariolides H against 126 luffariolides J against 126 Scrophularia auriculata 379 Scrophularia deserti 380 as cardiotonic 380 as diuretic 380 in cancer 380 in fever 380 in hypoglycemia 380 Scrophulariafrutescens 380 in inflammation 380 Scrophularia ningpoensis 380 Scrophularia scordonia 3 80 in inflammation 380 Scrophularia punicea 264 swertiapunimarin from 264 Secoiridoids 248,305-333,335-353, 355-357 antiinflammatory activity of 353 antileishmanial activity of 357, 1048
1245 antiviral activity of 113,138,209, 355 biological activities of 352 biosynthesis of 335-352 cardiovascular activity of 352 from Oleaceae 305 hypoglycemic activity of 262,356 structures of 305-333 Secoiridoid 5-hydroxy derivatives 261 Secoiridoid glucosides 317 Secologanin 249 condensation of 249 Secologanoside 342 biosynthetic pathway of 342 Secologanoside derivatives 331 origin of 332 structures of 333 Secomanoalide 124 isomer of manoalide 124 Secoxyloganin 344 routes to 344 Securiosides A 227 apoptosis inducing activity of 227 Sedanenolide 638 as anti-convulsant 638 as anti-platelet 638 anti-thrombosis activity of 638 Senkyunolide A 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide H 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide I 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide J 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide P 638 in blood viscosity reduction 638 Sensitized tumor necrosis factor 794 Sequoyitol 1000 Serotonin 5-HT2c 188 Sertiamarin 262 anticholinergic properties of 262
Sesquiterpenes 98 structure of 98 Sesquiterpenoids 176,784,902-927 from Aristolochia species 903-927 from Axinnella cannabina 176 from Triterygium wilfordii plant 784 structures of 784 Sesterstatins 1-3 148 against P388 cells 148 cytotoxic activity of 148 from Hyrtios erecta 148 Sesterterpenes 511 against CACO-2 (human colon carcinoma) 512 from genus Fusarium 511 neomangicols A and B 511 structure of 512 Sesterterpenoids 109 from marine 109 Sialytransferase inhibitor 210 of soyasaponin I 210 Sigmoidins A 828 against gram-positive bacteria 828 against gram-negative bacteria 828 from Erythrina sigmoidea 828 Sigmosceptrellin A 197 against Plasmodium falciparum 197 structure of 197 Sigmosceptrellin B 197 structure of 197 Signal transduction enzymes 1153 in drug discovery 1153 use of 1153 Signal transduction pathways 1072 glycyrrhizin in 230 Silyloxydiene 26 Diels-Alder cycloaddition of 26 Simple Benzophenone derivatives (SBDs) 673 Simple polysoprenylated benzophenones 674 from Clusiaceae 674 sources of 674 structures of 676,679,681 Simplex herpes type 1 virus (HSV-1) 355 Skin inflammation 442 subchronic model of 442
1246 Smooth muscle relaxation 650 mechanisms of action of 650 effects of butylidenephthalide on 650 Snider's synthesis 8-15 ofFR901483 8-15 via 1,3-dipolar cycloaddition 8-15 via intramolecular aldol reaction 8-15 Sodwanones 78-79 from Axinella weltneri 78-79 structures of 78-79 Sodwanones G-I 80 cytotoxicity of 80 against cancer cell lines 80 Soil-borne zoosporogenic phytopathogens 1087 Soluble guanylate cyclase 650 Songarosaponin C 232 from Vaerbascum songahcum 232 immunosuppressive activity of 232 Sorbitol cycle 137 Sorensen's synthesis 19-22 of amino-teterahedphenol 19-22 via oxidative cyclization 19-22 Southern African coastline 62 bio-geography of 62 Southern African marine ascidians 85-88 bioactive metabolites from 85-88 Southern African marine invertebrates 61 bioactive natural products from 61 Southern African marine mollusks 96-102 bioactive metabolites from 96-102 Southern African marine soft corals 88-96 bioactive metabolites from 88-96 Southern African marine sponges 70-85 bioactive metabolites from 70-85 Southern African marine worms 64-78 bioactive metabolites from 64-70 Soy isoflavones 1194 effects on nerve growth factor mRNA 1194 Soyasaponins 239 adjuvant activity of 239 Soyasaponin A1 239 adjuvant activity of 239 Soyasaponin A2 239 adjuvant activity of 239
Soybeans 1189 source of isoflavones 1189 Spectrofluorimetric methods 476 Spermicidal activity 795 in vitro 795 Spiranoid 1034 Spiranoid withanolides 1032 from Jaborosa odonelliana 1032 from Jaborosa runcinata 1032 structures of 1033 Spirostanol saponins 225 apoptosis inducing activities of 225 Spiroxins 536 mechanism of action of 536 against ovarian carcinoma 536 Spongia idia 111 Spongia sp. 139 spongianolides A-F from 139 Spongiane diterpenes 100 from Chromodoris hamiltoni 100 Spongidines A-D 146 against human synovial PLA2 146 Spongionolide A 140 total synthesis of 140 stereochemistry of 140 Spongiostatins 70-85 antineoplastic mechanism of action of 72 from Spirastrella spinispirulifer 70 Spongiostatin 1 71,72 effect on glutamate-induced polymerization of tubulin 72 total synthesis of 71 Spongiostatin 1-9 73 cytoxicity in NCI's 60 cell line screen 73 inhibitor of tubulin polymerization 73 Sponins 210 chemopreventive activities of 210 as cytotoxic agents 210 as antitumor agents 210 Sporidesmins 495 structure of 496 Stachytarpheta cayennensis 381 Staphylococcus aureus 1048 STAT6 activation inhibitor 525 TMC-264 525
1247 Stephacidins 603 structures of 603 Stephania tetrandra 990 Steroids 949,953,954 from Aristolochia species 953,954 structures of 953 Steroid saponins 220-221 Stroke 654 cuanxiong for 654 Store-operated calcium channel 650 Streat-plate method 1142 Streptococcus erythaeanus 128 Streptomyces pyogenes 123 Strictosidin 249 Strobilurus renacellus 512 Structure-activity relationships of 1061 Suberitenones A 154 from Suberites sp. 154 Subtrifloralactones A-L 1028 Subtriflora-y-lactones 1022 Swamp cancer 1054 Swern oxidation 26 Sweroside 248,262 hepatoprotective activity of 262 hypoglycemic activity of 262,356 Swertia 261 iridoids from 261 secoiridoids from 261 Swertia alata 263 belidifolia from 263 oleanolic acid from 263 Swertia chronic 262 use in chronic fever 262 Swertia japonica 264 swertiaside from 264 sunburiside-II from 264 Swertia nervosa 263 augustiamarin from 263 sweroside from 263 swertiamarin from 263 Swertiamarin 261,266 Swinholide 83 Swinholide A 84 against cancer cell lines 84 structure of 84 Synovial phospholipase A2 112 Synthesis 10,12-15,722-724 of benzophenones 721-725, 748-761 of(-)-FR901483 8,12-15,21,24 ofketoaldehyde 4,10,13
Synthetic isonitrile derivatives 181 Syphilis 267,822 Erythrina abyssinica against 822 Gentiana karroa in 266,267 Syringa jasminum 346 oleoside 11-methyl ester from 346 Tabernanthe iboga 804 aphrodisiac property of 804 hallucinogenic property of 804 Tabernanthe litoralis 1132 L-aminoacylase of 1132 Tabernanthe tetraptera 807 antibacterial activity of 807 Tabernanthine 814 bradycardisant activity of 814 Talaromyces sp. 481 TAN1251 alkaloids 3,5,40,44 Snider's synthesis of 44 structures of 5 synthesis of 3,40 TAN1251A 4,5,42,46 as muscarinic antagonists 5 Kawahara-Nagumo retrosynthetic analysis of 42 synthesis of 46 (+)-TAN1251A 41-43 Kawahara-Nagumo synthesis of 43 (-)-TAN1251A 46,49-50,52-55 from proline derivative 54-55 Honda's approach to 52-53 Honda's synthesis of 53 Kawahara-Nagumo approach to 54-55 Proline-based approach to 54-55 Snider's synthesis of 46 Wardrop's synthesis of 49-50 TAN1251A-D 5 from Penicillium thomii 89 TAN1251B 5,48 as muscarinic antagonists 5 synthesis of 48 (+)-TAN1251B 48 Snider's synthesis of 48 TAN1251C 4 (±)-TAN1251C 45,51-52 Ciufolini's synthesis of 51-52 Snider's synthesis of 45 TAN1251D 4
1248 (±)-TAN1251D 47 Snider's synthesis of 47 TAN1251A 18 synthesis of 18 Tashironins 411 biosynthesis of 411 Tasnemoxides A-C 129 against cancer cell lines 129 Taxol 794 anti-tumor activity of 794 Taxol-producing microbes 1124 TCM herbs 653 rhizoma chuanxiong 653 rhizoma ligustici 653 sources of 653 Terpenes 510-515 from Armillaria spp. 510-515 Terpene derivatives 197 antimalarial activity of 197 Terpenoids 810,815-816 diterpenoids 815 from Scoparia dulcis 815 in blennorhagia 815 in hypertension 815 in stomach disorders 815 Tetracarbocyclic sesterterpenoids 146 Tetracycline 704 for Staphylococcus aureus 704 Tetrahered vinyl halides 38 cyclization of 38 Tetralones 948 structures of 948 Tetraprenyltoluquinols 94-95 structures of 94-95 Tetraterpenoids 937 from Aristolochia species 937 structures of 938 Thl immune response 233 in production of cytotoxic lymphocytes 233 Thl response 234 against intracellular infection agents 234 Thelpin 479 from Thelepus setosus 479 Theonella swinhoei 83 Theopalauamide 83 from Theonella swinhoei 83 Theopalauamide 84,85 in standard paper disk assay 84
inhibitory effect of 84 structure of 85 Thermophiles 1129 from deep-sea vents 1129 Thermus aquations 1127 Thomitrem 493 Thorecta sp. I l l Thorectandra excavatus
133
Thorectandrol E 132 Thorectandrols A-D 132 Thrombin receptor antagonist 77 from Halichondria cf. moorei 11 Thyroid peroxidase 1201 H-Thymidine incorporation 232 in jurkat T cells 232 TNF-oc production 139 cavernolide effects on 139 Tohottea 855 Topoisomerase I inhibitors 498 topopyrones A 498 topopyrones B 498 Topopyrones 498 against gram-positive bacteria 499 against herpes virus 498 in vitro 498 structure of 499 Tosylamide 23 jV-acylation of 23 Total synthesis 8,71,971,972 of altohyrtin A 71 of aristolochic acid I 971 of(-)-FR901483 8,12-15,21,24 Toxocara canis 1095 Toxoplasma gondii 128 Traditional Chinese medicinal herbs 652 phthalide-containing 652 rraw-geranylnerolidol 110 biosynthetic routes to 1034 from Cochliobolus 110 from Ceroplastse albolineatu 110 Trechnolides 1030,1034 from Jaborosa laciniata 1030 from Jaborosa magellanica 1030 Trechonolide A 1033 Tricarbocyclic sesterpenoids 139 Tricyclic intermediate 55 synthesis of 55 Tricyclic skeleton 36 Kibayashi's synthesis of 36
1249 Trim eric secoiridoid glucosides 317-319 origin of 318 structures of 319 Trimusculus costatus 102 16,19,20-Trioxygenetated withanolides 1028 Tripartite tubular hairs (TTHs) 1093 Triprenylhydroquinones 98 structure of 98 Triprenylquinones 98 structure of 98 Triptolide 784,787-791,796 characterization of 784 enantioselective total synthesis of 789-790 isolation of 784 source of 796 structural modification of 784 synthesis of 787-791 Triterpene saponins 214,232 cytotoxic activity of 214 from caryophyllaceae 232 in vitro of 214 Triterpenoids 671,783,816,937 from Triterygium wilfordii 783 structures of 783 Triterpenoid tripterine 795 antiinflammatory activity of 795 immunosupprressive activity of 795
from Xenia macrospiculata 89 structures of 89-90 Tubemioside II 212 in cancer prevention 212 Tubular necrosis 991 Tumors 863 Aristolochia clematitis in 863 Tumor necrosis factor-oc (TNF-a) 134 Tyrosine kinase inhibitor 1196 genistcin 1196 Unsaturated amino alcohols 85-87 against A549 (non-small cell lung) 87 against LOX (melanoma) 87 against OVCAR-3 (ovarion) human tumor cell lines 87 against SNB-19 (CNS) 87 structures of 86 Untenospongin A 120 from hippospongia 120 Untenospongins A and O 120 coronary vasodilating activity of 120 from Hippospongia 120 Urothelial carcinoma 991 Ustilago violaceae 128 hytiolide against 128
Triterygium wilfordii lTi,ll^,116-lM bioactivities of 774 bioactive compounds from 773 chemical components of 776-784 clinical use of 776 diterpenoids from 776 extracts of 774 side effects of 776 Triterygium wilfordii plant 781 alkaloids from 781 Trypanosoma cruzi 1048 Tryprostatin A 581 structure of 581 Tryprostatin B 581 structure of 581 Tryptamine hydrochloride 1063 attractant activity of 1063 Tsitsikamma favus 74 Tsitsixenicins 89-90 from Capnella thyrsoidea 89
Valdivones 91,92 anti-inflammatory properties of 91 from Alcyonium valdivae 91 structures of 91,92 Valepotriates 256,366 Valerenic acid 255,258 by thin-layer chromatography 258 spasmolytic effects of 255 Valerian 259 film-coated tablets of 259 Valeriana edulis 257,258 acevaltrate from 257 isovaleroxyhydroxydidrovaltrate from 257 in vitro 258 mutagenic properties of 258 valtrate from 257 Valerianajatamansi 255,257-259,260 acevaltrate from 257
1250 essential oils from 258 isovaltrate from 257 lanarin isovalerate from 259 4-methoxy-8-pentyl-1 -naphtholic acid from 259 use in ayurvedic system of medicine 255 volatile oil of 260 Valeriana offwinalis 255,257-259 acetoxyvalerenic acid from 257 actinidine from 259 essential oils from 258 extraction of 259 hyroxyvalerenic acid from 257 isoferulic acid g-aminobutyric acid from 259 kanakoside C from 257 kanakoside A from 257 kanokoside D from 257 tinctures prepared from 259 valerenic acid from 257 use as mild sedative 255 Valtrate 249 Variabilin 97,112,113 anti-inflammatory activity of 97,113 anti-microbial properties of 97 anti-tumor properties of 97 antiviral activity of 113,138,209, 355 from Isotericola variabilis 112 icthyotoxic properties of 97 structures of 97 Variabilin inhibitor 113 of cytosolic PLA2 113 Vasodilatory effects 477 ofgriseoflavin 471-479 Vassobia lorentzii 1025 Verbascum 286,287 against influenza 286 antifungal activity of 287 anti-inflammatory property of 286 antitussive activity of 286 antiviral activity of 286 as astringent 286 as diuretic 286 as emollient 286 as heart stimulant 286 as sedative 286 in allergies 286 in asthma 287
in bronchitis 287 in chicken embryos 286 in chronic hard cough 287 in congestion 286 in fever 286 in migraine 286 in pulmonary complaints 287 in tuberculosis 287 in tumor formation 286 in whopping cough 286 Verbena 284,285 as laxative 285 as rubefavient 284 as tonic 284 hypotensive effects of 285 in asthma 284 in dysmenorrhea 284 in gallstones 285 in healing of wounds 284 in insomnia 284 in nervous coughing 284 in rheumatism 284 Verbena officinalis 284 as antispasmodic 284 as diaphoretic 284 as nerve tonic 284 as relaxant 284 as sedative 284 Veronica (scrophulariaceae) 287 Veronica anagallis -aquatica 287 anti-scorbutic properties of 287 use in bladder troubles 287 Veronica arvensis 287 as diaphoretic 287 as diuretic 287 expectorant properties of 287 Veronica beccubunga 287 iridoids from 287 Verbascum thapus 286 coumarin from 286 flavonoids form 286 iridoid glycosides from 286 oligosaccharides from 286 polysaccharides from 286 Verruculogen 579,580 identification of 579 related compounds 580 structure of 579 Verticillium hemipterigenum 534 Vertihemipterin A 534
1251 Vinorelbine 794 anti-tumor activity of 794 Vipera russelli 985 Vismiaguianones A-E 711 against cancer cell line 711 Vismiaguianone B 711 moderate DNA strand-scission activity of 711 Vitex 281 effects on the pituitary gland 281 in amenorrhea 281 in dysmenorrhea 281 in endometriosis 281 in menorrhagia 281 in menstrual complaints 281 in premenstrual syndrome 281 in treatment of menopause 281 Vitex agnus castus 283,284 against anxiety earlybirth 284 antifungal activity of 284 as diuretic 284 in digestive problems 284 in hyperprolactinemia 284 iridoids from 283 treatment of premenstrual problems by 284 Vitex negunda 281-283 analgesic activity of 283 antibacterial activity of 283 antifungal activity of 283 antihistamine activity of 283 anti-inflammatory activity of 283 antioxidant activity of 283 as antihelmintic 283 as expectorant 283 diuretic properties of 283 hepatoprotective activity of 283 in catarrhal fever 282 in headache 282 in rheumatism 283 in skin infections 283 in swelling of joints 282 pain suppressing activity of 283 use in dyspepsia 283 Vitex trifoliate 283 Vitex verbenaceae 280 Voacangine 809 structure of 809 Volatile organohalogen 526 l-chloro-5-heptadecyne 526 Voltage-operated calcium channel 650
Wardrop's formal synthesis of 32-55 Ct)-demethylamino FR901483 32-35 Water-soluble artesunate 190 in treatment of severe malaria 190 WithaferinA 1019,1020 from Acnistus arborescens 1020 from Withania somnifera 1019 structure of 1020 Withajardins 1022,1038,1039 structures of 1039 Withametelins 1022 Withanolides 1019-1023,1046-1045, 1048 antifeedant properties of 1040-1045 as anticancer compounds 1020 as anti-feedant compounds 1020 as anti-inflammatory compounds 1020 as antitumor compounds 1020 as cytotoxic compounds 1020 bactericidal activities of 1048 biological activity of 1019,1040 cancer chemopreventive activity 1046-1048 chemistry of 1019,1023 classification of 1021-1023 from South American solanaceae 1019 immunomodulating activity of 1020
in Ajuga parviflora 1020 in Cassia siamea 1020 insecticidal properties of 1040-1045 occurrence of 1020 phytotoxic activity of 1048 trypanocidal/leishmanicidal activity of 1048 Withaphysalins 1022 Xanthobaccin A 1107 structure of 1107 Xanthochymol 704,755,760,761-764 antibacterial activity of 761,762 antifungal activity of 761,762 anti-MRSA activity of 704
1252 antioxidant activity of 761,762 antiviral activity of 761,762 biological activities of 761 I3 C-NMR data for 761 COSY correlations for 760 cytotoxic activity of 761,763 DEPT data for 761 'H-NMR data for 761 'H-spectrum of 755 HMBC data for 761 molluscicidal activity of 761,763 trypanocidal activity of 761,764 Xanthones 671 Xanthone-O-glucosides 263 as anti-convulsant 263 as cardiovascular stimulant 263 as CNS depressant 263 Xenicane diterpenes 93 against A-549 human lung carcinoma 93 against HT-29 human colon carcinoma cell lines 93 against MEL-28 human melanoma 93 against P-388 mouse leukaemia 93 Xerophenone A 699 structure of 699 Xerophenone B 699 structure of 699 Yingzhaosu A 192 structure of 192 Z- Butylidenephthalide 638 as anti-angina 638 Zahavins 92-94 Zerynthia polyxena 1000 Z-Ligustilide 638 as anti-platelet aggregation/ anti-thrombosis 638 Zoospores 1060,1066,1071,1074 bioassay-guided chromatographic techniques for 1074 developmental transitions of 1066 differentiation of 1066 metabolites affecting motility of 1074 receptors in 1071
responses of 1060 viability of 1074 Zoospore regulation 1112 bioassay methods for 1112 Zoospore lytic factors 1084 from Ginkgo fruits 1084 Zoosporicidial activity 1093 of polyflavonoid tannins 1093 Zuihonin B 941 from Aristolochine arcuata 941
