studies in Natural Products Chemistry Volume 28 Bioactive Natural Products (Part I)
studies in Natural Products Chemistry edited by Atta-ur-Rahman
Vol. 1 Vol. 2 Vol. 3 Vol. 4 Vol. 5 Vol. 6 Vol. 7 Vol. 8 Vol. 9 Vol. 10 Vol. 11 Vol. 12 Vol. 13 Vol. 14 Vol. 15 Vol. 16 Vol. 17 Vol. 18 Vol. 19 Vol. 20 Vol. 21 Vol. 22 Vol. 23 Vol. 24 Vol. 25 Vol. 26 Vol. 27 Vol. 28
Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidaton (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 )
studies in natural Products Chemistry Volume 28 Bioactive natural Products (FM I)
Edited by
Atta-ur-Rahman H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistm
2003
ELSEVIER Amsterdam - Boston - Heidelberg - London - New York - Oxford - Paris San Diego - San Francisco - Singapore - Sydney - Tol
ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands © 2003 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail:
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FOREWORD "Studies in Natural Products Chemistry" has become the world's leading series of volumes in the field of isolation, structure elucidation, biological activity and synthesis of natural products, with comprehensive reviews written by leading experts. Volume 1 of the series was published in March 1988 and during the last 15 years 27 volumes have been published. From Volume 21 onwards, the series has been devoted to bioactive natural products. Natural products present a huge source of organic substances with differing structural features. The very large number of terrestrial and marine natural products found in differing environmental conditions with correspondingly different biosynthetic patterns gives medicinal chemists access to millions of substances with differing bioactivity profiles. This represents a genuine treasure chest for discovering new medicinal agents against a variety of diseases. The series on bioactive natural products should therefore be of considerable interest not only to natural product chemists but also to medicinal chemists, pharmacologists, and synthetic organic chemists working in academia and industry. I hope that the present volume will be received with the same excitement and enthusiasm as the previous volumes of his encyclopaedic series. I would like to express my thanks to Mr. Shakeel Ahmad for his assistance in the preparation of the index. I am also grateful to Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.
Atta-ur-Rahman Ph.D. (Cantab.), Sc.D. (Cantab.) Chairman, Higher Education Commission Government of Pakistan January, 2003
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PREFACE In various segments of the scientific community, natural products research comes in and out of favor In a cyclic manner as a function of time. Irrespective of this rhythmic pattern, the true and absolute value of natural products research is immutable, in the context of both applied and basic investigations.
Clearly, therapeutic modalities
throughout the world rely heavily on natural product drugs and formulations. Similarly, although acknowledgements may be less overt or absent, many aspects of basic research programs are intimately related to natural products.
In essence, natural
products play an integral and ongoing role in promoting numerous aspects of scientific advancement. Obviously, for progress to be realized on a widespread basis, general dissemination of contemporary information is necessary. Incredibly, we now see the 28th volume in the series Studies in Natural Product Ctiemistry edited by Professor Atta-ur-Rahman. The significance of this indelible effort cannot be overestimated. More specifically, in the same impeccable manner as the former volumes, we are again presented with cuttingedge contributions of great importance. The first paper presents over 100 compounds obtained from Broussonetia spp., and discusses biological activities. This is followed by similar contributions dealing with the genus Licania and Ginkgo biloba. Additional papers describe in detail a number of interesting and important natural compounds or structural classes: retinoids, tetramic acid metabolites, isoprenylated flavonoids, plant polyphenols, crocin, marcfortine and paraherquamide, acarlcides, podolactones, triterpene glycosides and sulfur-containing marine compounds. An additional paper focuses on the antitumor activities of lipids, and a final contribution deals with natural product amelioration of cancer chemotherapy-Induced adverse reactions. These astute summaries are provided by well-respected authors from seven different countries. Assembly of the volume is a notable achievement; the work as a whole nicely illustrates the types of critical discoveries that emanate from the interface of chemistry and biology. Volume 28 can stand as a proud member of this great family of useful reference books.
John M. Pezzuto Professor and Dean Schools of Pharmacy, Nursing and Health Sciences Purdue University
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CONTENTS Foreword
v
Preface
vii
Contributors
xi
Bioactive compounds from the genus Broussonetia DONGHO LEE and A. DOUGLAS KINGHORN
3
Chemical and biological studies on Licania genus ALESSANDRA BRACA, ANNA RITA BILIA, JEANNETTE MENDEZ, COSIMO PIZZA, IVANO MORELLI and NUNZIATINA DE TOMMASI
35
Recent progress in retinoid chemistry ALAIN R. VALLA, DOMINQUE L. CARTIER and ROGER LABIA
69
Bioactive tetramic acid metabolites EMILIO L. GHISALBERTI
109
Chemistry and biological activities oi Ginkgo biloba K. SASAKI, K. WADA and M. HAGA
165
Chemistry and biological activities of isoprenylated flavonoids from medicinal plants (moraceous plants and Glycyrrhiza species) TARO NOMURA, TOSfflO FUKAI and YOSfflO HANO
199
Plant polyphenols: Structure, occurrence and bioactivity PIERGIORGIO PIETTA, MARKUS MINOGGIO and LORENZO BRAMATI
257
Promising pharmacological actions of crocin in Crocus sativus on the central nervous system SHINJI SOEDA, TAKASHIOCHIAI, HIROSHI SfflMENO, fflROSHI SAITO KAZUO ABE, MINORU SUGIURA, HIROYUKI TANAKA, FUTOSHI TAURA, SATOSHI MORIMOTO and YUKIHIRO SHOYAMA
313
Synthesis and modification of marcfortine and paraherquamide class of anthelmintics BYUNG H.LEE
331
Acaricides of natural origin, personal experiences and review of literature (1990-2001) GUIDOFLAMINI
381
Podolactones: A group of biologically active norditerpenoids ALEJANDRO F. BARRERO, JOSE F. QUILEZ DEL MORAL and M. MAR HERRADOR
453
Antitumoral activity of lipids A studies in animal models and cancer patients DANIELE REISSER, NOLWENN GAUTHIER, ALENA PANCE and JEAN-FRANCOIS JEANNIN
517
Prevention of cancer chemotherapy drug-induced adverse reaction, antitumor and antimetastatic activities by natural products YOSHIYUKIKIMURA
559
Biologically active triterpene glycosidesfromsea cucumbers (holothuroidea, echinodermata) HUGO D. CHLUDIL, ANA P. MURRAY, ALICIA M. SELDES and MARTA S. MAIER
587
Sulfur-containing natural products from marine invertebrates MICHELE R. PRINSEP
617
Subject Index
753
CONTRIBUTORS
Kazuo Abe
Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan
Alejandro F. Barrero
Department of Organic Chemistry, Institute of Biotechnology, University of Granada, Avda., Fuentenueva, 18071, Granada, Spain
Anna Rita Bilia
Dipartimento di Scienze Farmaceutiche, Universita di Firenze, Via Gino Capponi 9, 55100 Firenze, Italy
Alessandra Braca
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Via Bonanno 33, 56126, Pisa, Italy
Lorenzo Bramati
ITB-CNR, Via F. Ui Cervi, 93 - 20090 (MI), Italy
Dominique L. Cartier
FRE 2125 CMIS, 6 rue de TUniversite 29000 Quinq)er, France
Hugo D. Chludil
Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2, Ciudad Universitaria, (1428) Buenos Aires, Argentina
Guido Flamini
Dipartimento di Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy
Toshio Fukai
School of Pharmaceutical Sciences, Toho University, 2-21 Miyama, Fimabashi, Chiba 274-8510, Japan
Nolwenn Gauthier
Cancer Immunotherapy Research Laboratory, Ecole Pratique des Hautes Etudes, INSERM U517, Faculty of Medicine, 7 Bd Jeanne d'Arc, 21079 Dijon, France
Emilio L. Ghisalberti
Department of Chemistry, University Australia, Nedlands, 6009 W.A., Australia
M. Haga
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Yoshio Hano
School of Pharmaceutical Sciences, Toho University, 2-21 Miyama, Funabashi, Chiba 274-8510, Japan
of
The
Western
M. Mar Herrador
Department of Organic Chemistry, Institute of Biotechnology, University of Granada, Avda., Fuentenueva, 18071, Granada, Spain
Jean-Francois Jeannin
Cancer Immunotherapy Research Laboratory, Ecole Pratique des Hautes Etudes, INSERM U517, Faculty of Medicine, 7 Bd Jeanne d'Arc, 21079 Dijon, France
Yoshiyuki Kimura
Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan
A. Douglas Kinghom
Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA
Roger Labia
FRE 2125 CNRS, 6 rue de I'Universite 29000 Quimper, France
Byung H. Lee
Preclinical Development, Pharmacia Animal Health, 7000 Portage Road, Kalamazoo, MI 49001, USA
Dongho Lee
Chemistry and Life Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709, USA
Marta S. Maier
Jeannette Mendez
Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2, Ciudad Universitaria, (1428) Buenos Aires, Argentina Grupo de Productos Naturales, Centro de Quimica Organica, Escuela de Quimica, Facultad de Ciencias, Universidad Central de Venezuela, Apartado de Correos 47102, Caracas 1020-A, Venezuela
Markus Minoggio
ITB-CNR, Via F. Hi Cervi, 93 - 20090 (MI), Italy
Jose F. Quilez Del Moral
Department of Organic Chemistry, Institute of Biotechnology, University of Granada, Avda., Fuentenueva, 18071, Granada, Spain
Ivano Morelli
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Via Bonanno 33, 56126, Pisa, Italy
Satoshi Morimoto
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Ana P. Murray
Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2, Ciudad Universitaria, (1428) Buenos Aires, Argentina
Taro Nomura
School of Pharmaceutical Sciences, Toho University, 2-21 Miyama, Funabashi, Chiba 274-8510, Japan
Takashi Ochiai
Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
Alena Pance
Cancer Immunotherapy Research Laboratory, Ecole Pratique des Hautes Etudes, INSERM U517, Faculty of Medicine, 7 Bd Jeanne d'Arc, 21079 Dijon, France
Piergiorgio Pietta
ITB-CNR, Via F. Ui Cervi, 93 - 20090 (MI), Italy
Cosimo Pizza
Dipartimento di Scienze Farmaceutiche, Universita di Salemo, Via Ponte Don Melillo, 84084 Fisciano, Salemo, Italy
Michele R. Prinsep
Department of Chemistry, University of Waikato, Private Bag 3105, Hamilton, New Zealand
Daniele Reisser
Cancer Immunotherapy Research Laboratory, Ecole Pratique des Hautes Etudes, INSERM U517, Faculty of Medicine, 7 Bd Jeanne d'Arc, 21079 Dijon, France
Hiroshi Saito
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
K. Sasaki
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Alicia M. Seldes
Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2, Ciudad Universitaria, (1428) Buenos Aires, Argentina
Hiroshi Shimeno
Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
Yukihiro Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Shinji Soeda
Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
Minoru Sugiura
Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan
The
Hiroyuki Tanaka
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Futoshi Taura
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Nunziatina De Tommasi
Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy
Alain R. Valla
VA R&D Pepiniere d'entreprises, 140 Bd de Creac'h Gween 29561 Quimper, France
K. Wada
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Bioactive Natural Products
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AUa-ur>Rahinan (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
BIOACTIVE COMPOUNDS FROM THE GENUS BROUSSONETIA DONGHO LEE^ and A. DOUGLAS KINGHORN* Program for Collaborative Research in the Pharmaceutical Sciences and Department ofMedicinal Chemistry and Pharmacognosy, College of Pharmacy, University ofIllinois at Chicago, Chicago, Illinois 60612, USA, ABSTRACT: The genus Broussonetia of the Moraceae (mulberry family) is of both ethnomedical and industrial interest. Of the approximately 30 species in this genus, only three have been subjected to previous phytochemical investigation, namely, B. kazinoki, B. papyriferay and B, zeylanica. From over 100 compounds isolated from these species, the major secondary metabolites reported thus far are alkaloids of the pyrrolidine type and several types of flavonoids. Some of these compounds have exhibited various biological activities, such antioxidative, aromatase inhibitory, cytotoxic, glycosidase inhibitory, and platelet aggregation inhibitory effects. The biologically active constituents of the species in the genus Broussonetia are discussed in detail.
INTRODUCTION The genus Broussonetia L*Her. ex Vent, of the Moraceae (mulberry family) is represented by lactiferous trees or shrubs. Broussonetia comprises about 30 species and is distributed throughout various regions of the w^orld including Africa, East Asia, and North America [1,2]. Thus far, only three species of the genus Broussonetia have been studied for their secondary metabolites, namely, B, kazinoki, B. papyrifera, and B. zeylanica. Broussonetia kazinoki Siebold & Zucc. is a deciduous tree growing to 4.5 m that flowers in August. It occurs in mainland China, Japan, and Korea [1]. The plant requires well-drained soil but can grow in Address correspondence to this author at Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, U.S.A. E-mail:
[email protected]. ^Current address: Chemistry and Life Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709, U.S.A.
nutritionally poor soil [3]. Preparations made from B. kazinoki have been used as a tonic to increase vision and sexual potency, and to treat boils, eczema, infant colic, and leukorrhea [4,5]. Various extracts of 5. kazinoki have exhibited antifungal, antiinflammatory, antioxidant, and antispasmodic activities [6-10]. Broussonetia papyrifera (L.) L'Her. ex Vent, is a deciduous tree growing up to 15 m that is commonly called the paper mulberry. It is native to East Asia, then later introduced and naturalized in the United States. It flowers from August to September, and the seeds ripen from September to November [1,11]. The plant prefers light and well-drained soil and' is easily cultivated in a warm sunny position in any soil of reasonable quality [3]. Fibers from the bark are used in making paper, cloth, and rope. These fibers can be produced by beating strips of bark on a flat surface with a wooden mallet [12]. 5. papyrifera has been used for cancer, dyspepsia, and pregnancy [13]. In mainland China, the fruits of 5. papyrifera have been employed for impotency and ophthalmic disorders [4,14], Also, the leaf juice of 5. papyrifera is diaphoretic and laxative and the stembark is hemostatic [4]. Antifungal and antioxidant activities of the extracts ofB. papyrifera were reported [6,7,9]. Broussonetia zeylanica (Thwait.) Comer is endemic to Sri Lanka and its tough bark-fibers were used to make string [15]. Several types of bioactive compounds have been reported from the genus Broussonetia including glycosidase inhibitory alkaloids and aromatase inhibitory or cytotoxic flavonoids. This chapter reviews the biologically active constituents from the genus Broussonetia reported by the end of 2001. BIOACTIVE COMPOUNDS FROM BROUSSONETIA KAZINOKI The bioactive secondary metabolites reported from Broussonetia kazinoki can be classified into major two groups, alkaloids and flavonoids (Table 1), Fig. (1). The Kusano group at Osaka University of Pharmaceutical Sciences in Japan reported over 20 pyrrolidine alkaloids, broussonetines A-H, K-M, 0-T, V-X, and Mi, and broussonetinines A and B, four pyrrolidinyl piperidine alkaloids, broussonetines I, J, Ji, and J2, two pyrroline alkaloids, broussonetines U and Ui, and one pyrrolizidine alkaloid, broussonetine N, from hot water extracts of 5. kazinoki [16-24]. As shown in Table 1, some of these alkaloids exhibited strong
Table 1. Bioactive Compounds from Broussonetia kazinoki Compound type/name
Activity
Reference
ALKALOIDS Pyrrolidines Broussonetine C (1)
Inhibition of glycosidases*
[16]
Broussonetine D (2)
Inhibition of glycosidases*
[16]
Broussonetine E (3)
Inhibition of glycosidases"
[17]
Broussonetine F (4)
Inhibition of glycosidases*
[17]
Broussonetine G (5)
Inhibition of glycosidases*
[18]
Broussonetine H (6)
Inhibition of glycosidases*
[18]
Broussonetine K (7)
Inhibition of glycosidases*
[20]
Broussonetine L (8)
Inhibition of glycosidases*
[20]
Broussonetine M (9)
Inhibition of glycosidases*
[21]
Broussonetine 0 (10)
Inhibition of glycosidases*
[21]
Broussonetine P (11)
Inhibition of glycosidases*
[21]
Broussonetine Q (12)
Inhibition of glycosidases*
[21]
Broussonetinine A (13)
Inhibition of glycosidases*
Broussonetinine B (14)
Inhibition of glycosidases*
[17]
Inhibition of glycosidases*
[22]
KazinolD(16)
Cytotoxicity against human tumor cell lines'*
[25]
Ka2inolK(17)
Cytotoxicity against human tumor cell lines**
[25]
I
[17]
Pyrrolizidine Broussonetine N (15) FLAVONOIDS Diphenylpropanes
Table 1. Bioactive Compounds from Broussonetia kazinoki (continued) Compound type/name
Activity
Reference
Flavans 7,4'-Dihydroxyflavan (18)
Cytotoxicity against human tumor cell lines'*
[25]
KazinolA(19)
Antioxidant activity*" Inhibition of tyrosinase**
[26] [26]
KazinolE(20)
Antioxidant activity*^ Inhibition of tyrosinase'
[26] [26]
KazinolQ(21)
Cytotoxicity against human tumor cell lines'*
[25]
KazinolR(22)
Cytotoxicity against human tumor cell lines'*
[25]
Broussonol A (23)
Cytotoxicity against human tumor cell lines'*
[27]
Broussonol B (24)
Cytotoxicity against human tumor cell lines'*
[27]
Broussonol C (25)
Cytotoxicity against human tumor cell lines'*
[27]
Broussonol D (26)
Cytotoxicity against human tumor cell lines'*
[27]
Flavonols
1
"Glycosidase inhibitory activity expressed as ICso value (jiM); 1: p-Gal = 0.036, p-Man = 0.32; 2: P-Gal = 0.029, P-Man = 0.34; 3: a-Glc = 3.3, P-Glc == 0.055, P-Gal = 0.002, p-Man = 0.023; 4: a-Glc = 1.5, p-Glc = 0.01, P-Gal = 0.004, P-Man = 0.028; 5: P-Glc = 0.024, P-Gal = 0.003, P-Man = 0.76; 6: P-Glc = 0.036, p-Gal = 0.002, P-Man = 0.32; 7: P-Glc = 0.026, P-Gal = 0.005, P-Man = 0.3; 8: P-Glc = 0.017, P-Gal = 0.004, p-Man = 0.2; 9: P-Gal = 8.1; 10: P-Glc = 1.4, P-Gal = 0.17, P-Man = 8.2; 11: P-Glc = 2.4, P-Gal = 0.2, P-Man = 7.6; 12: P-Glc = 1.4, P-Gal = 0.6, P-Man = 20.0; 13: P-Gal = 0.016, a-Man = 0.3; 14: P-Gal = 0.01, a-Man = 0.29; 15: P-Glc = 6.7, p-Gal = 2.9, P-Man = 3.3 (P-Gal = p-Galactosidase; a-Glc = a-Glucosidase; P-Glc = pGlucosidase; a-Man = a-Mannosidase; P-Man = P-Mannosidase). '*Cytotoxicity expressed as ED50 value (^ig/mL); 16: PLC/PRF/5 = 3.3, 212 = 7.0, HT3 = 3.6; 17: HT3 = 8.6: 18: HT3 = 11.6, SiHa = 8.9, CaSki = 17.4; 21: PLC/PRF/5 = 3.5, T24 = 2.3, 212 = 3.8, HT3 = 4.3, SiHa - 4.7; 22: HT3 = 9.3, SiHa = 9.3, CaSki = 8.2; 23: A546 = 8.7, HCT-8 = 9.1; 24: A546 = 5.52, HCT-8 = 8.8; 25: A546 = 7.8, HCT-8 = 9.6; 26: KB = 4.5 (key to cell lines; 212 = inducible Ha-ras oncogene transformed NIH/3T3; A549 = human lung carcinoma; CaSki = human cervical carcinoma; HCT-8 = human ileocecal carcinoma; HT3 = human cervical carcinoma; KB = human epidermoid carcinoma; PLC/PRF/5 = human hepatoma; SiHa = human cervical carcinoma; T24 = human hepatoma). '^Antioxidant activity shown by l,l-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity (IC50 pM); 19:41.4,20:33.4. ''Activity not specified. nCso 241.3 nM.
H CH2OR2
H(?
OH 1 R,=H,R2 = H 3 R, = OH, R2 = H 7 R, = 0H,R2 = Glc 10 R, = H,R2 = H,A-3',4'
H HOHjC^i^^V-*'^
H(f
CH2OR2
OH
2 R, = H,R2 = H 4 R, = 0H,R2 = H 8 R, = 0H,R2 = Glc 11 Ri=H,R2 = H,A-3',4'
HOH2C*^^\.-*'^
Ha
OH
H
OH ^O
H0H2CM^^'\.»^'^
H(f
O
OH
CH2OH
Fig. (1). Continued H CH2OR
HO
OR
12 R = Glc 13 R==H
CH2OH
HOF^C^^'^V.^^^'
HC)
OH
14
HOH2C
I?
CH2OH
OH
16 17 A^3,4
HO.
TCO' 19
18
Fig. (1). Continued
OH
OH
O 24
Fig. (1). Structures of bioactive constituents of Broussonetia kazinoki.
glycosidase inhibitory activity with IC50 values ranging from 0.002 to 8.2 [xM. Selective inhibition of glycosidase enzymes has a number of potential therapeutic uses, including the treatment of cancer, diabetes, and HIV-AIDS [28-32]. Also, the prenylated flavonoid derivatives, kazinols D (16) and K (17) (diphenylpropanes), 7,4'-dihydroxyflavan (18),
10
kazinols Q (21), and R (22) (flavans), and broussonols A-D (23-26) (flavonols), were isolated as moderate to weak cytotoxic principles against several human cancer cell lines with ED50 values ranging from 2.3 to 17.4 ^ig/mL [25,27]. Two flavans, kazinols A (19) and E (20), were reported as antioxidative principles using the l,l-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay (IC50 41.4 and 33.4 |iM, respectively) [26]. These compounds (19 and 20) also exhibited inhibitory activity against tyrosinase, which is a key enzyme in melanin biosynthesis and plays a role in the conversion of tyrosine to DOPA and DOFA to dopaquinone [26,33]. An antioxidative effect and the suppression of melanin biosynthesis are useful for cosmetic products in relation to hyperpigmentation [34]. Broussonetine C (1), a monocyclic polyhydroxy pyrrolidine alkaloid, showed a yellow spot on TLC when sprayed with ninhydrin reagent and heated (ninhydrin reaction), and its molecular formula was determined by a positive high-resolution mass spectrometry (C18H36NO5, [M + H]^, m/z 346.2579). The IR spectrum displayed a hydroxy band at 3370 cm'^ and a carbonyl band at 1706 c m \ The ^H- and '^^C-NMR signals were assigned using the ^H-^H correlated spectroscopy (^H-^H COSY), heteronuclear single quantum coherence (HSQC), and distortionless enhancement by polarization transfer (DEFT) pulse sequences. The position of the carbonyl carbon and the linkage of the pyrrolidine ring and the aliphatic side chain were determined using the heteronuclear multiple bond coherence (HMBC) NMR technique [HMBC correlations were observed for the carbonyl carbon signal (5c 210.8) with the proton signals at 6H 2.71 (H-11') and 6H 2.12 (H-120, and for the C-5 carbon signal (5c 62.9) of the pyrrolidine ring with the proton signals at 5H 4.44 (H-4) and 5H 2.04 (H-l'), respectively] [16]. The relative stereochemistry of the pyrrolidine ring of broussonetine C (1) was determined from its coupling constants (vicinal coupling, ^2,3 = •/3,4 = •/4,5 = 6.4 Hz) and nuclear Overhauser enhancement effects (H-2/H-4 and H-3/H-5). The absolute stereostructure was disclosed as (2i?,3/?,4i?,5/?) using the benzoate chirality method [35]. A diacetylacetoamide was prepared from broussonetine C (1) by treatment with acetic anhydride in pyridine at room temperature, and then a dibenzoate (la) was obtained by benzoylation of the diacetylacetoamide. The circular dichroism (CD) curve of l a displayed a negative Cotton
11
effect (A8237 -15.9) and a positive effect (AS223 +16.4), which indicated a negative chirality as shown in Fig. (2) [16]. C0CH3
BzO
OBz la
Ae(nin): +16.4(223) -15.9(237)
Fig. (2). Determination of the absolute stereostnicture of broussonetine C (1) by the benzoate chirality method.
Broussonetine L (8) showed similar physical and spectroscopic properties to those of broussonetine C (1) [16], except for proton signals of a p-glucose (anomeric proton, 8H4.78, 1H, doublet, J- 7.8 Hz) moiety in the H-NMR spectrum. Hydrolysis of broussonetine L (8) with 1 N HCl provided broussonetine F (4) [17] and D-glucose ([a]D +40.6°). Therefore, the structure of broussonetine L (8) was determined to be 13'O-p-D-glucopyranosylbroussonetine F due to the glycosylation shift of C13' (6c 69.3) of broussonetine L (8) (broussonetine F, 4, 6c-i3' 61.6) and HMBC long-range correlations observed between H-13' (6H 3.69 and 4.09) and an anomeric carbon (5c 104.4), and between an anomeric proton and C-13'[20]. The absolute stereochemistry of broussonetine L (8) was determined by the combination of the benzoate chirality method and the Mosher's method [35-37]. A carbamate (8a) was prepared from broussonetine F (4) by reaction with phenyl chloroformate in tetrahydrofuran-H20 (7:3), and a diacetate (8b) was prepared from 8a with acetic anhydride in pyridine. Finally, a dibenzoate (8c) was obtained by benzoylation of 8b. The CD curve of 8c showed a negative Cotton effect (AE237 -30.9) and a positive effect (Ae223 +15.9) to confirm a counter-clockwise chirality between two benzoyl groups. Fig. (3) [20].
12
OH
IN.
nd
CH2OGIC
OH
H
OH CH2OH
HOHjC*^'^.*^'^
Hcf
O—CO
OH
PhOCOCl NaHCOj
QH CH2OH
CH2OAC
HO
OH PhCOCl pyridine
O—CO
OAc CH2OAC
Bz(f
8c
OBz OBz
BzQ
A8(nm): +15.9(223) -30.9 (237)
Fig. (3). Determination of the absolute stereostructure of the pyrrolidine ring of broussonetine L (8) by the benzoate chirality method.
13
The absolute configuration of C-l' of 8 was then investigated by the Mosher's method. The di- (R)- and (S)-2-methoxy-2-phenyl-2(trifluoromethyl)-acetic acid (MTPA) esters (8dR and 8d5) and tri- (R)and (iS)-MTPA esters (SeR and SeS) prepared from 8a, were analyzed by ' H - ' H C O S Y N M R (500 MHz) and A6 values (SS-SR) were measured. These values established the R configuration of C-l' of 8 by comparison of the di-MTPA esters (8d/? and 8d5) and the tri-MTPA esters (SeR and 8fty),Fig.(4)[20]. O—CO
OR4 CH2OR1
R-,(f
OR, 8dif, 8d5: Ri = R2 = MTPA, R3 - R4 = H 8ei?, ScJ: R, = R2 = R4 = MTPA. R3 = H
A^(^S-^R)
3
4
5
r
+0.100
-0.039
-0.019
0.000
+0.020
-0.332
-0.161
-0.037
-0.060
+0.013
1"
2
8d
+0.030
1 8e
-0.154
r 0.000 1 +0.050
Fig. (4). Determination of the absolute configuration of C-l' of broussonetine L (8) by the Mosher's method.
Also, the absolute stereochemistry of the pyrrolizidine ring and Cr of broussonetine N (15) was established by the Mosher's method. The tri- (Ry and (5)-MTPA esters (15a/? and 15a5) and penta- (Ry and (5)MTPA esters (15bif and IShS) were prepared from 15 and A6 values (658R) were measured. Accordingly, the R configuration of C-l of the pyrrolizidine ring from 15a and the R configuration of C-l' from 15b were determined, respectively, Fig. (5) [22]. A biosynthetic study of the 18-carbon chain skeleton of broussonetines was reported [38]. To verify the biosynthetic route of these alkaloids, the plant was grown on an aseptic medium and the enriched ^^C of the isolated alkaloids was analyzed by NMR after feeding with [l-^^C]glucose. The labeling pattem of broussonetine J (27) obtained
14
MTPAO -Z. -0.006 "L
!?-^^^_,,, H r
C-0.029
MTPAOH2C
+0.013 +0.001
c V l
CHoOMTPA
H
+0.061
OH
MTPAO -0.075'^
''-^\,ri^ -
-^-^'^
CH2OMTPA OMTPA
15b
Fig. (5). Determination of the absolute configuration of broussonetine N (15) by the Mosher's method.
from the feeding experiment indicated that C-4 through C-18 were formed via palmitoyl CoA through the acetate-malonate pathway, whereas C-1 through C-3 were derived via serine from 3-phosphoglyceric acid. Therefore, the 18-carbon chain of broussonetine J (27) was assumed to be formed initially by condensation of serine and palmitoyl CoA [38], As shown in Fig. (6), the absolute stereochemistry of the pyrrolidine rings of the broussonetines is related to o-serine and that of broussonetine U (28) is related to L-serine. Out of a series of over 30 alkaloids obtained from B, kazinoki, some of them showed potent glycosidase inhibitory activity as shovm in Table 1. Interestingly, only broussonetines E and F (3 and 4), which have a hydroxyl group on C-T, demonstrated potent inhibitory activity against a-glucosidase [17]. However, broussonetines G and H (5 and 6), which also have a hydroxyl group on C-l', did not inhibit a-glucosidase [18]. These results suggested that the inhibition of a-glucosidase might be attributed to the hydroxyl groups on both C-T and C-13' and the keto groups of C-9' or C-10' [17,18]. However, additional studies seem to be required to verify this suggestion [24].
15
CHjOH -O.
COOH
OH
OH
OH
OH OH D-[l-^^C]glucose
O
II
^
.COOH
HO SCoA
NH2 D-serine
NH2 L-serine
CoAS
OH
HO HO
OH
Fig. (6). Biosynthesis of broussonetines J and U (27 and 28).
COOH H O - ^ '
16
BIOACTIVE PAPYRIFERA
COMPOUNDS
FROM
BROUSSONETIA
The major types of bioactive constituents reported from Broussonetia papyrifera are the prenylated flavonoids, which include compoxmds of the diphenylpropane, chalcone, flavan, flavanone, flavone, flavonol, and aurone classes (Table 2), Fig. (7). An early study on B, papyrifera resulted in the isolation of two diphenylpropanes, broussonins A (29) and B (30), and a coumarin, marmesin (52), with antifungal activity [39]. Also, a diprenylated diphenylpropane derivative, kazinol F (31) [40], was reported as an antioxidant and tyrosinase inhibitory constituent [34]. Table 2. Bioactive Compounds from Broussonetia papyrifera Activity
Compound type/name
Reference(s)
FLAVONOroS Diphenylpropanes Broussonin A (29)
Antifungal activity* Inhibition of aromatase**
[39]
Broussonin B (30)
Antifungal activity*
[39]
Kazinol F (31)
Antioxidant activity (scavenging free radicals)^ Inhibition of tyrosinase**
[34]
[34]
1
Antioxidant activity (inhibition of lipid peroxidation)^ Inhibition of cyclooxygenase* Inhibition of nitric oxide production'^ Inhibition of respiratory burst in neutrophils* Platelet aggregation inhibitory activity**
[42]
1
Inhibition of aromatase**
[41]
Isogemichalcone C (34)
Inhibition of aromatase*'
[41]
1 2,4,2',4'-Tetrahydroxy-3'1 prenylchalcone (35)
Inhibition of aromatase**
[41]
1
[41]
1
Chalcones
Broussochalcone A (32)
1 3'-[y-Hydroxymethyl-(£)-ymethylallyl]-2,4,2',4'. tetrahydroxychalcone 1 r - 0 coumarate (33)
[43] [42] [44] [431
Flavans Broussoflavan A (36)
Antioxidant activity (inhibition of lipid peroxidation)*^ 1 Platelet aggregation inhibitory activity**
1 1
[43] [45]
1
17
Table 2. Bioactive Compounds from Broussonetia papyrifera Compound type/name KazinolA(19) KazinolB(37)
(continued)
Activity
RefereDce(s)
Antioxidant activity' Inhibition of tyrosinase' Platelet aggregation inhibitory activity** Inhibition of cyclooxygenase* Platelet aggregation inhibitory activity**
1
[26] [26] [43]
[43] [43]
1 1
Flavanones (25)-Abyssinone II (38)
Inhibition of aromatase**
[41]
(2.S)-2',4'-Dihydroxy-2"-(lhydroxy-1-methylethyl)dihydrofuro[2,3-Alflavanone (39)
Inhibition of aromatase*'
[41]
(2iS)-Euchrenone a? (40)
Inhibition of aromatase*'
[41]
(2.S)-Naringenin (41)
Inhibition of aromatase**
[41]
Inhibition of aromatase**
[41]
Inhibition of aromatase**
[41]
Platelet aggregation inhibitory activity*"
[43]
Antioxidant activity (inhibition of lipid peroxidation)' Antiproliferative activity* Inhibition of aromatase** Inhibition of cyclooxygenase* Platelet aggregation inhibitory activity*" Antioxidant activity (inhibition of lipid peroxidation)*^ Antiproliferative activity*
[45] [45] [41] [43] [43]
Inhibition of aromatase*'
[41]
(25)-5,7,2',4'1 Tetrahydroxyflavanone (42) Flavone 1 5,7,2',4'-Tetrahydroxy-31 geranylflavone (43) Flavonols Broussoflavonol £ (44)
Broussoflavonol F (45)
Broussoflavonol G (46) Isolicoflavonol (47)
[45]
'
Aurone Broussoaurone A (48)
[45]
Antioxidant activity (inhibition of lipid peroxidation)' Inhibition of cyclooxygenase* Platelet aggregation inhibitory activity*"
[45] [43]
L. _ [43I_.
MISCELLANEOUS AlbanoIA(49)
Inhibition of aromatase**
[41]
Betulinic acid (50)
Selective cytotoxic activity against melanoma cell lines'"
[46]
1
18
Table 2. Bioactive Compounds from Broussonetia papyrifera (continued) Compound type/name
Activity
Reference(s)
Demethylmoracin I (51)
Inhibition of aromatase**
[41]
Marmesin(52)
Antifungal activity*
[39]
MoracinN(53)
Inhibition of aromatase**
[41]
Ursolic acid (54)
Inhibition of HIV-1 protease dimerization''
[47]
"Antifungal activity (presented as a range) expressed as the minimum concentration (mM) required for complete inhibition of fungal growth including Fusarium roseum, F. lateritium, F. solani, Diaporthe nomuraU Stigmina mori, Sclerotinia sclerotiorum, Bipolaris leersiae, and Rosellinia necatrix\ 29: 0.2-0.9, 30: 0.05-0.9, 52: 0.9-4.0. ''Aromatase inhibitory activity determined as IC50 value (^iM); 29: 30.0, 33: 0.5, 34: 7.1, 35: 4.6, 38: 0.4, 39: 0.1,40: 3.4,41: 17.0,42: 2.2,43: 24.0,45: 9.7,47: 0.1,49: 7.5,51: 31.1,53: 31.1. ^Antioxidant activity expressed as IC50 value (pM); 31: 6.7 (jig/mL); 32:0.63,36: 2.1,45:2.7,46:1.0,48: 1.2. ''The tyrosinase inhibitory activity of 31 was IC50 0.39 |ig/mL. *Cyclooxygenase inhibitory effect determined as IC50 value (ng/mL); 32: 19.4,37: 155.3,45: 17.5,48: 22.7. ^Inhibitory effect (IC50) of 32 on nitric oxide production was 11.3 ^iM. ^Compound 32 inhibited O2 consumption in formylmethionyl-leucyl-phenylalanine- and phoibol 12-myristate 13-acetate-stimulated rat neutrophils with IC50 values of 70.3 and 63.9 ^M, respectively. •^Antiplatelet activity induced by arachidonic acid was expressed by IC50 value (^M); 19: 11.4, 32: 6.8, 36: 86.7,37: 32.6,44: 39.9,45:16.9,48: 15.4. 'Activity found as a constituent of Broussonetia kazinofd. ^Antiproliferation activity shown by the inhibition of ['H]thymidine incorporation into DNA in the proliferation of rat vascular smooth muscle cells. The effect was expressed as % of control; 45: 0-7.8,46: 0-0.4. ''Activity found as a constituent of a plant other than a Broussonetia species.
Broussochalcone A (32) [48], a prenylated chalcone, is one of the most completely studied constituents of B. papyrifera biologically. Broussochalcone A (32) inhibited platelet aggregation induced by arachidonic acid with an IC50 value of 6.8 |aM as well as induction by adrenaline in human platelet-rich plasma. The antiplatelet effect of 32 was partially due to an inhibitory effect on cyclooxygenase activity and by reducing thromboxane fomiation [43]. Also, broussochalcone A (32) inhibited O2 consiraiption in fomiylmethionyl-leucyl-phenylalanine- and phorbol 12-myristate 13-acetate-stimulated rat neutrophils with IC50 values of 70.3 and 63.9 jiM, respectively. This inhibitory effect of 32 on respiratory burst in neutrophils was not mediated by the reduction of phospholipase C activity, but was mediated by the suppression of protein kinase C activity through interference with the catalytic region and by the
19
RiO
29Ri = CH3,R2 = H 30Rj = H,R2 = CH3
31
33R = H 34R = OCH3
OH
36
20
Fig. (7). Continued
OH
OH
37
OH
OH
41R = H 42R = OH
21
Fig. (7). Continued
HO,
48
49
22
Fig. (7). Continued
COOH
50
HO-
51
o ^ ^ ^ -o- - o 52
CCX)H
54
Fig. (7). Structures of bioactive constituents of Broussonetia papyrifera.
attenuation of O2*" generation from the NADPH oxidase complex, which might inhibit the generation of toxic oxygen radicals and terminate the tissue damage [43]. Furthermore, broussochalcone A (32) showed antioxidant activity in iron-induced lipid peroxidation in a rat brain
23
homogenate model with an IC50 value of 0.63 |iM as well as in the DPPH system, and exhibited an inhibitory effect on nitric oxide (NO) production with an IC50 value of 11.3 jaM. This potent inhibitory effect on NO production was mediated by suppression of nuclear factor (NF)-KB activation, phosphorylation and degradation of iKBa (an inhibitory protein of NF-KB), and inducible NO synthesis expression, which have been associated with autoimmune or inflammatory diseases [42]. In an effort to investigate antioxidant constituents with antiproliferative effects in rat vascular smooth muscle cells (VSMC), broussoflavan A (36) [49], broussoflavonols F (45) [50] and G (46) [51], and broussoaurone A (48) [49] were found to inhibit the Fe^^-induced thiobarbituric acid-reactive substance formation in rat brain homogenate. Furthermore, broussoflavonols F (45) and G (46) inhibited fetal calf serum-, 5-hydroxytryptamine-, or ADP-induced [^H]thymidine incorporation into rat VSMC [45]. Antioxidant activities and inliibitory effects on proliferation of rat VSMC with potent antiplatelet activities of 45 and 46 may be useful for vascular diseases and atherosclerosis [43,45]. The concept of cancer chemoprevention is becoming wellestablished and refers to the pharmacological intervention to arrest or reverse the process of carcinogenesis, and thus prevent cancer [52,53]. It has become evident that various phytochemical components of the diet are able to prevent cancer formation in full-term carcinogenesis inhibition studies in animal models [54]. As part of a U.S. National Cancer Institutefunded program project conducted at the University of Illinois at Chicago [55-57], an ethyl acetate extract of the whole plants ofB, papyrifera was found to significantly inhibit aromatase activity in an in vitro assay [58,59] (74% inhibition at 80 |ig/mL) [41]. This was only one of a handful of extracts found to significantly inhibit aromatase activity with the bioassay protocol used, out of over 1,000 extracts screened [60]. This target was chosen for investigation, because aromatase catalyzes the final, rate-limiting step in estrogen biosynthesis [61], and is regarded as a target relevant to the treatment or prevention of breast and prostate cancers [62]. Several synthetic aromatase-inhibitory drugs have been developed, including aminoglutethimide, substrate androstenedione derivatives, imidazoles, and triazoles [63-65]. From the active extract of B, papyrifera were isolated several aromatase inhibitors with IC50 values in the range 0.1-31.1 ^M, inclusive of broussonin A (29) [66], 3'-[Y-hydroxymethyl-(£)-y-methylallyl]-
24
2,4,2',4'-tetrahydroxychalcone 11 '-O-coumarate (33) [41], isogemichalcone C (34) [41], 2,4,2',4'-tetrahydroxy-3'-prenylchalcone (35) [67], (25)-abyssinone II (38) [68], (25)-2',4'.dihycIroxy-2"-(lhydroxy-1 -methylethyl)-dihydrofuro[2,3-A]flavanone (39) [41], (25)euchrenone a7 (40) [69], (25)-naringenin (41) [70], (25)-5,7,2',4'tetrahydroxyflavanone (42) [71], 5,7,2',4'-tetrahydroxy-3-geranylflavone (43) [41], broussoflavonol F (45) [50], isolicoflavonol (47) [72], albanol A (49) [73], demethylmoracin I (51) [41], moracin N (53) [74]. Of these aromatase inhibitors, five of the compounds were new (33, 34, 39, 43, 51), and details of structure elucidation of 33, 34, and 43 are presented as examples in the following two paragraphs. The isolates 3'-[y-hydroxymethyl-(£^-Y-methylallyl]-2,4,2',4'. tetrahydroxychalcone 11'-O-coumarate (33) and 3'-[Y-hydroxymethyl(£)-y-methylallyl]-2,4,2',4'-tetrahydroxychalcone 11 '-O-ferulate (isogemichalcone C, 34) were obtained as orange powders and were shown by positive HRFABMS to possess molecular formulas of C29H26O8 (m/z [M + Na]^ 525.1884) and C30H28O9 (m/z [M + N a ] \ 555.1577), respectively. The ^H- and ^^C-NMR spectra of 33 and 34 exhibited characteristic chalcone signals, and signals for a coumarate group for 33 at 6H 7.54 (2H, 7 = 8.6 Hz, H-2" and H-6"), 6H 6.87 (2H, J = 8.5 Hz, H3" and H.5''), 5H 7.59 (IH, 7 = 16.0 Hz, H-T'), and 5H 6.35 (IH, J = 16.0 Hz, H-8") and signals for a ferulate group for 34 at 6H 7.34 (IH, 7 = 1.6 Hz, H-2"), 6H 6.85 (IH, / = 8.1 Hz, H.5"), 6H 7.12 (IH, 7 = 1.7 and 8.2 Hz, H-6"), 5H 7.57 (IH, J = 16.0 Hz, H.7"), 5H 6.40 (IH, J= 15.9 Hz, H8"), and 6H 3.91 (3H, singlet, OCH3). Based on these observations, the structures of 33 and 34 were concluded to be prenylated chalcones with a coumarate and a ferulate unit attached, respectively, which were confirmed by 2D-NMR techniques. Fig. (8). In case of isogemichalcone C (34), it was concluded to be a regioisomer of gemichalcone C by comparing its spectra with those of the latter compound [75]. This was confirmed using a NOESY NMR experiment. Thus, the NOE correlations between H-7' and H-10', and H-8' and H-IT clearly indicated E stereochemistry of the prenyl group. Moreover, the chemical shift differences at positions C-10' and C-1T of the E and Z isomers supported the stereochemistry proposed. Fig. (8) [41,75,76]. 5,7,2',4'-Tetrahydroxy-3-geranylflavone (43) exhibited a molecular ion [M]"^ at m/z 422.1719 by HREIMS, consistent with an
25
Carbon
6c 33
34
Gemichalcone C [75]
10'
14.2
14.2
64.2
ir
70.2
70.2
22.8
'
Fig. (8). Selected HMBC (->) and NOE (<^) correlations of isogemichalcone C (34), and comparison of *^C NMR data of 3'-[y-hydroxymethyl-(£)-y-methylallyl]-2,4,2',4'tetrahydroxychalcone 1 T-O-coumarate (33), 34, and gemichalcone C.
elemental formula of C25H26O6. In its ^H-NMR spectrum, characteristic proton signals for a geranyl unit [5H 3.12 (2H, •/= 6.9 Hz, H-l"), 5H 5.14 (IH, multiplet, H-2"), 5H 1.89 (2H, multiplet, H-4"), 6H 1.43 (3H, singlet, H-5"), 5H 2.00 (2H, multiplet, H>6"), 6H 5.04 (IH, multiplet, H-?''), 5H 1.61 (3H, singlet, H.9"), and 5H 1.55 (3H, singlet, H-IO")], a set of metacoupled proton signals [5H 6.25 (IH, broad singlet, H-6) and 6H 6.33 (IH, broad singlet, H-8)], and proton signals of an ABX system [6H 6.57 (IH, broad singlet, H-3'), 6H 6.51 (IH, 7 = 8.3 Hz, H-5'), and 5H 7.19 (IH, J = 8.3 Hz, H-6')] were observed. These data suggested that 43 has a flavone skeleton with four hydroxyl groups and one geranyl substituent, and these inferences were confirmed using the APT, COSY, and HMQC NMR techniques. The positions of the substituents were deduced as occurring at C-5, C-7, C-2', and C-4' (four hydroxyls) and C-3 (geranyl) using the HMBC NMR technique, Fig. (9). Additionally, NOE correlations
26
between H-6' and H-l", and H-2" and H-4" confirmed the position of attachment and the E stereochemistry of the geranyl group [41].
Fig. (9). Selected HMBC (-^) and NOE (<^) correlations of 5,7,2',4'-tetrahydroxy-3geranylflavone (43).
Out of a series of 42 compounds isolated and characterized in our investigation on B. papyrifera [41], comprising benzofurans, coumarins, and various types of flavonoids (biphenylpropanes, chalcones, flavans, flavanones, and flavones), only certain representatives of the latter class of compounds showed potent aromatase inhibition activity (Table 2). Flavanone 39 {(25)-2',4'-dihydroxy-2"-( 1 -hydroxy-1 -methylethyl)dihydrofuro[2,3-/z]flavanone, IC50 0.1 \xM) [41] and flavone 47 (isolicoflavonol, IC50 0.1 \\M) [72] were the most potent flavonoids isolated, exhibiting potencies that were approximately 60-fold greater than aminoglutethimide, the positive control used for this assay. The functionalized chalcone 33 {3'-[Y-hydroxymethyl-(jE)-y-methylallyl]2,4,2',4'-tetrahydroxychalcone 1 T-O-coumarate, IC50 0.5 |aM} [41] and the flavanone 38 [(25)-abyssinone II, IC50 0.4 |iM] [68] were both approximately 10 times more active than aminoglutethimide. Interestingly, the various benzofurans {demethylmoracin I (51) [41], moracins D [41], I [77], M [77], and N (53) [74]}, biphenylpropanes {broussonins A (29) [39], B (30) [39], E [66], and F [66], l-(2,4dihydroxyphenyl)-3-(4-hydroxyphenyl)propane [41], l-(2,4-dihydroxy-3prenylphenyl)-3-(4-hydroxyphenyl)propane [41], and 1 -(4-hydroxy-2[41]}, methoxyphenyl)-3-(4-hydroxy-3-prenylphenyl)propane
27
flavanonols {(2i?,3i?)-lespedezaflavanone C [78], (2i?,3i2)-katiiranin [79], and (2/?,3/?)-5,7,2',4'-tetrahydroxyflavanonol [80]}, and flavans {(25)7,4'-dihydroxy-3'-prenylflavan [41], (25)-2',4'-dihydroxy-7-methoxy-8prenylflavan [81], and (25)-7,4'-dihydroxyflavan [66]} tested, which are quite closely related structurally to the active compounds, did not show potent anti-aromatase activity. It was noted that a carbonyl group in compounds of the chalcone, flavone, andflavanoneclasses is required for the exhibition of potent aromatase inhibition activity. However, the presence of a C-5 hydroxyl group among the flavanones decreased activity significantly {(25)-naringenin (41) [70] and (25)-5,7,2',4'tetrahydroxyflavanone (42) [71]} and flavones or flavanones with a prenyl or geranyl unit at C-6 {bavachin [82], gancaonin P [83], 5,7,3',4'tetrahydroxy-6-geranylflavonol [41], and 5,7,3',4'-tetrahydroxy-3methoxy-6-geranylflavone [41]} were not active. Presumably, such a bulky substituent at C-6 prevents these compounds from interacting with the enzyme [41]. In our study, the inhibition of aromatase was achieved at physiologically relevant concentrations (100-1000 nM) of dietary flavonoids. Initially, some of the compounds isolated from B, papyrifera were tested for binding to the estrogen receptor - a or -p [84]. Interestingly, none of the aromatase-active compounds showed significant binding to the either of these receptors. Also, the effectiveness of some of the flavonoids against the inhibition of quinone reductase, a phase II enzyme involved in detoxification mechanisms, was evaluated [85]. No significant inhibition of the enzyme was observed by any of the agents tested. (25)-2',4'-Dihydroxy.2"-( 1 -hydroxy-1 -methylethyl)dihydroftiro[2,3-/i]flavanone (39) also was effective in inhibiting (50%) the formation of alveolar lesions in a mouse mammary organ culture model when tested at 100 ng/ml [86]. Moreover, the fiiiits of B, papyrifera have been consumed by individuals in the People's Republic of China, albeit for the treatment of various medical disorders, rather than as an edible plant [14,87], Accordingly, these potent aromatase inhibitor compounds show significant potential to be developed as cancer chemopreventive agents [41,88].
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COMPOUNDS FROM BROUSSONETIA ZEYLANICA Only three constituents have been reported from Broussonetia zeylanica, all by a group at the University of Peradeniya in Sri Lanka [89-91], A major alkaloid, 8-hydroxyquinoline-4-carbaldehyde (55), was identified as an antimicrobial agent active against Staphyllococus aureus and Candida albicans (the levels of activity were not specified) [89] and then two minor compounds, 3,4'-dihydroxy-2,3'-bipyridine (56) and 3,4-bis(8hydroxyquinolin-4-yl)-y-butyrolactone (broussonetine, 57), were reported. Fig. (10) [90,91], However, the structure of 3,4'-dihydroxy-2,3'bipyridine (56) was revised to 8-hydroxyquinoline-4-carbaldehyde oxime (58) by synthesis [92,93]. Also, it was noted that an artefactual origin of this oxime (58) could not be ruled out due to the presence of the corresponding aldehyde (55) [93]. CHO OH
N
56
58
Fig. (10). Structures of constituents of Broussonetia zeylanica.
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CONCLUSIONS Two species of the genus Broussonetia, B. kazinoki and B. papyrifera, have been of considerable interest for their ethnomedical uses for the treatment of various human diseases. There is a substantial literature on the utilization of 5. papyrifera, in particular, in the paper industry in East Asia. Extracts of Broussonetia species have exhibited various biological activities including antifungal, antiinflammatory, antioxidant, and antispasmodic activities. Broussonetines, a class of pyrrolidine alkaloids from B. kazinoki, have shown potent but generally non-specific glycosidase inhibitory activity. The structures of these alkaloids were elucidated by analyzing NMR data including ^H-^H COSY, ^H-^^C HSQC (HMQC), and ^H-^^C HMBC 2D-NMR techniques and the absolute stereochemistry was determined by a combination of the benzoate chirality and Mosher's methods. Also, prenylated flavonoids were reported with antioxidant, cytotoxic, and tyrosinase inhibitory activities from this species. From B. papyrifera, variousflavonoidswere isolated as active principles. Of these, broussochalcone A (32) and broussoflavonol F (45) showed broad activities in several types of assay system, and (25)-2',4'-dihydroxy-2"(1 -hydroxy-1 -methylethyl)-dihydrofuro[2,3-/z]flavanone (39) and isolicoflavonol (47) displayed the most potent aromatase inhibitory activity (IC50 100 nM) of any natural products reported to date. Furthermore, compound 39 was effective in inhibiting the formation of alveolar lesions in a mouse mammary organ culture model. Therefore, the secondary metabolites from Broussonetia seem worthy of more extensive biological and chemical investigation, particularly from the point of view of their role as potential cancer chemopreventive agents. ACKNOWLEDGEMENTS The laboratory work on potential cancer chemopreventive agents carried out at the University of Illinois at Chicago was supported by program project POl CA48112 (Principal Investigator, John M. Pezzuto, Ph.D.), funded by the National Cancer Institute, NIH, Bethesda, Maryland, U.S.A.
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CHEMICAL AND BIOLOGICAL STUDIES ONLICANIA GENUS ALESSANDRA B R A C A \ ANNA RITA BILIA^ JEANNETTE MENDEZ^ COSIMO PIZZA^ IVANO MORELLl\ NUNZIATINA DE TOMMASf ^ Dipartimento di Chimica Bioorganica e Biofarmacia, Universitd di Pisa, Via Bonanno 33, 56126, Pisa, Italy ^ Dipartimento di Scienze Farmaceutiche, Universitd di Firenze, Via Gino Capponi 9, 55100 Firenze, Italy ^ Grupo de Productos Naturales, Centra de Quimica Organica, Escuela de Quimica, Facultad de Ciencias, Universidad Central de Venezuela, Apartado de Correos 47102, Caracas 1020'A, Venezuela "^ Dipartimento di Scienze Farmaceutiche, Universitd di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy ABSTRACT: This paper reports the ph)^ochemical and biological studies carried out on several species of the Licania genus (Chrysobalanaceae). This genus includes many species mainly distributed in neotropical South American countries such as Venezuela, Brasil, and Mexico, most of them not yet investigated from the chemical and biological point of view. The name Licania derives from the anagram of the indigenous Venezuelan name "Calignia". Phytochemical studies of these species led to the isolation and structural characterization, by NMR and MS analysis, of many secondary metabolites, mainly flavonoids, and expecially flavonol glycosides, clerodane diterpenes, and triterpenes having the lupane, ursane, and oleanane skeletons. Particularly flavonoids and their glycosides have a chemotaxonomic interest in the genus and in general in the Chrysobalanaceae family. Furthermore, several biological studies were performed on some crude extracts of these plants. They were found to be cytotoxic to cultured human hepatoma (HepG2), colon carcinoma (Caco-2), melanoma B16, and CA-9KB cells. In addition a moUuscicidal activity against Biomphalaria glabrata was also demonstrated. Pure triterpenes showed antibacterial activity on Gram positive and yeast and a moderate cytotoxic action against KB assay, while diterpenes showed antifungal properties. Flavonoids were also tested for their antioxidative action as radical scavengers.
INTRODUCTION The Licania genus, until the last decade, v^as almost completely unexplored and little, if anything, was knov^n about the chemistry of its family: the Chrysobalanaceae. This family includes 17 genus and 420
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species of which Chrysobalanus, Acioa, Licania, Parinari, Hexellodendrum, Hirtella, and Couepia are the neotropical ones growing in Venezuela. In the past the Chrysobalanaceae family was separated from the Rosaceae due to a difference in the sexual organ morphology: the flowers in the Chrysobalanaceae have linked and partially sterile stamen, while the gynaecium has lateral styles and two ovules [1]. However, a chemotaxonomic study on 31 species of Parinari genus showed a similar flavonoid pattern between the two families [2]. This study evidenced also that the Neotropical and Asiatic taxa of Parinari genus, a complex of closely related species, were chemically very similar to each other and lack myricetin; African species were split in two groups based on the presence or the absence of myricetin glycosides. Since myricetin was considered a primitive flavonoid character, it suggested that the African species producing this metabolite represented a primitive nucleus from which a non-myricetin group may have evolved giving rise, by subsequent eastward and westward expansion, to two myricetinlacking phytogeographic lines. These chemical/phytogeographic correlations could be extended also to Licania genus which is mainly neotropical. The Licania genus (the name Licania derives from the anagram of the indigenous Venezuelan name "Calignia") comprises 150 species, mainly trees or shrubs, distributed in neotropical South American countries such as Venezuela, Brazil, Guyana, and Mexico. In Venezuela these plants grow in the "bosque nublado", a rainy zone between 1500 and 3000 m in altitude [3]. The aims of our work were to investigate the selected plants: (1) to isolate as many secondary metabolites as possible for better phytochemical knowledge of the genus with particular interest to their flavonoidic fractions, whose occurrence in Licania could be useftil for the taxonomy of the Chrysobalanaceae; (2) to evaluate biological effects of both extracts and isolated compounds on the basis of their structural relationship with other active molecules. GENERAL STRATEGIES The phytochemical study consisted of fractionating plant extracts leading to the isolation of various "novel" and known derivatives. The choice of appropriate separation methods was crucial for this type of research: the extracts may be very complex, thus separation of one single component
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can be difficult. Multistep chromatographic separations were the norm for the isolation of pure compounds. In our chemical and biological investigations the isolation and purification steps were mainly performed by extraction of the plant material with solvents of varied polarity (i.e. with petroleum ether, chloroform, chloroform-methanol, methanol, water) followed by crude separation by silica gel column for hydrophobic extracts and by gel filtration on dextran gel (Sephadex LH-20 and/or LH60) columns and droplet counter current chromatography (DCCC) for polar extracts. Less polar glycosides were usually isolated from the chloroform-methanol 9:1 and methanol extracts. Highly polar watersoluble glycosides present in the aqueous extracts were separated by purification through an Amberlite XAD-2 column of the lyophilized aqueous extract followed by gel filtration on Sephadex LH-20. Bioassayguided fractionation was employed in both cases. Prior to final separation by HPLC on RP-18 columns, an optimization of separation parameters was performed whereby mobile phase, stationary phase, and flow rate were adjusted by using TLC and other suitable techniques. The structures of the compounds were elucidated by a combination of NMR techniques (^H-, ^^C-, and ^^C-DEPT NMR) and chemical transformation, enzymatic degradation, and as well as mass spectrometry, which gives information on the saccharide sequence. A more recent approach consists of an extensive use of high-resolution 2D NMR techniques, such as homonuclear and heteronuclear correlated spectroscopy (DQF-COSY, HOHAHA, HSQC, HMBC) and NOE spectroscopy (NOESY, ROESY), which now play the most important role in the structural elucidation of intact glycosides. These techniques are very sensitive and non destructive and allow easy recovery of the intact compounds for subsequent biological testing. Detailed biological or pharmacological screenings were performed also in cooperation with scientists in different laboratories.
PHYTOCHEMICAL STUDIES OF PLANTS BELONGING TO LICANIA GENUS Our standard procedure consisted of first defatting the air-dried plant material with «-hexane followed by extraction with solvents of increasing polarity starting from chloroform, chloroform-methanol 9:1, methanol, and water in a Soxhlet apparatus or at room temperature. Only in a few
38
cases, after defatting with «-hexane, the plant material was extracted directly with methanol, and the crude methanol residue was then partitioned between solvents with different polarity. Thus, non-polar compounds, such as triterpenes were found in the less polar extract, while flavonoids in the more polar fractions. The chloroform extracts were usually subjected to fractionation over silica gel columns with CHCI3MeOH gradients, followed by reversed phase HPLC, Lobar on C-18 and RP-18 columns with MeOH-H20 mixtures as the eluents. Fractionation of the CHCb-MeOH 9:1 and MeOH extracts was conducted by gel filtration over Sephadex LH-20 using MeOH as eluent sometimes preceded by partition of the extracts between A2-butanol and H2O. The Sephadex LH20 fractions were finally purified by RP-HPLC, Lobar RP-8 and/or RP18, and flash column chromatography. The structures of the isolated compounds were elucidated by a combination of NMR techniques, chemical transformation, and EI-MS or FAB-MS spectroscopy in positive or negative ion mode. In addition, high resolution NMR techniques, which are very sensitive and non-destructive were used. In the course of our phytochemical work we studied seven Licania species all belonging to Venezuelan flora, collected in Puerto Ayacucho, Estado Amazonas and in the Parque Nacional Henry Pittieri, Maracay, Estado Aragua. A number of new and known secondary metabolites, mainly flavonoids, especially flavonol glycosides, sterols, and triterpenes having the lupane, ursane, and oleanane skeleton were isolated and characterized. The last part of this chapter deals also with the isolation of clerodane diterpenes from the methanol extract of L, intrapetiolaris by Oberlies et al, 2001 [4]. All the Licania species investigated up to now are reported in Table 1. Table 1. Licania species investigated Species Licania pittieri Prance Licania carii Cardozo Licania pyrifolia Grisebach Licania densiflora Kleinhoonte Licania heteromorpha var. heteromorpha Bentham Licania licaniaeflora (Sagot) Blake Licania apetaia var. apetala (E. Mey) Fritsch Licania intrapetiolaris Spruce (ex. Hook)
Source Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Ecuador
References
rsi [6,7] [8,91
no. Ill ri5,161 ri7,181 [181 [4]
Table 2 and Table 3 list all triterpenes and flavonoids obtained from the genus, respectively, to date. Flavonoids and their glycosides were
39
used as chemotaxonomic markers in the Chrysobalanaceae family and it was noted that in the genus Licania glycosylation at C-3 of kaempferol, quercetin, and myricetin and at C-4' only of the myricetin nucleus was found to be the most common trend. Glucose, galactose, arabinose, rhamnose, and rutinose are the most common sugars found as glycones of the flavonoids glycosides. The presence of the aglycon myricetin in all our investigated species except L. pittieri suggests that chemical/phytogeographic correlations between and within genera of Chrysobalanaceae family are probably more complex than those observed by Coradin et al, 1985 [2]. On the other hand, our results support a strong relationship between the families of Chrysobalanaceae and Rosaceae; thus the presence of the flavonoids and triterpenoids in the species investigated could justify the previous classification that included the Chrysobalanaceae family into the Rosaceae, and the uselessness of their separation, at least from a phytochemical point of view.
40
Table 2. Distribution of trierpenes in the genus Licania
A 1 B 1
Species Lupanes
Betulinic acid Alphitolic acid
+ 1 1
C
I
E 1
F
+ +
+
1 1
+ 1 alphitolic acid + 3p-0-c/5-/7-coumaroyl 1 alphitolic acid 1 la-hydroxybetulinic acid | + 1 6p-hydroxybetulinic acid | + 1 + 2a-hydroxybetulinic acid (3',4'-dihydroxy)-benzoyl ester + 2a, 27-dihydroxybetulinic acid (3',4'-dihydroxy)-benzoyl ester + Oleananes Oleanolic acid , + + Oleanolic acid 3-0-a-L-arabinoside + + 4Maslinic acid + 3 p-0-rran5-/7-coumaroyl maslinic acid + 3P-0-cz.s-/7-coumaroyl maslinic acid + Arjunic acid 28-p-D-glucosyl ester + Olean-12-ene-2a,3p-diol + + + Ursanes Ursolic acid + + 2a-hydroxy ursolic acid + Ursolic acid 3 -0-a-L-arabinoside + 2a,3a-dihydroxyurs -12-ene-28-oic + 3 p-0-/ran5-/7-coumaroyl -2a-hydroxy ursolic acid + 3 P-0-cw-/7-coumaroyl -2a-hydroxy ursolic acid + Euscaphic acid + Euscaphic acid 28-P-D-glucosyl ester + Tormentic acid + + Tormentic acid 28-P-D-glucosyl ester + Pomolic acid Legend: A=L. pittieri; B=L. carii; C=L pyrifolia; E=L. heteromorpha var. heteromorpha; ¥=L. licaniaeflora Z^-O'trans-p-coMvasnoyX
41
Table 3. Distribution of flavonoids in the genus Licania Species Flavonols
Dihydro flavonol
A Kaempferol (Kae) Kae 3-rha Kae 3-ara Kae 3-rut Kae 3-(2"-xyl)rha Kae 3-(6"-/?-coum)glc Quercetin (Que) Que 3-gIc Que 3-gal Que 3-ara Que 3-rha Que 3'0Me-3-glc Que 3-rut Que 3-(2"-xyl)rha Myricetin (Myr) Myr 3-glc Myr 3-gal Myr 3-ara Myr 3-rha Myr 3-xyl Myr 3'0Me-3-glc Myr 3'0Me-3-gal Myr 4'0Me-3-glc Myr 4'0Me-3-gal Myr4'OMe-3-rha Myr 3'0Me-3-rut Myr 3',4'-cliOMe -3-glc Myr 3',5'-diOMe -3-glc Myr3',5'-diOMe -3-rha Myr 3-rut Myr 3-(2"-xyI)rha Myr3-(2"-rha)rha Myr 4'-rha Myr 3,4'-dirha Myr 7-OMe-3,4'-dirha Taxifolin 3-rha
B
C + + +
D
E
F
G
+ +
+ + + + + +
+ + + + +
+ + + + +
+ + +
+
+ + +
-)+ +
+ + +
+ + +
+ + + + + + +
+
+
+
+ +
+
+ + +
+ + + + + +
+ + + + + +
+
+ Dihydromyr 3-rha + + (+)-Catechin + (-)-£/7/catechin + Flavones 8-OMe apigenin + 8-OH luteolin + Flavanones 8-OH eriodictyol + + 8-OH naringenin 1 + 4'-OMe-8-OH naringenin Legend: A=I. pittieri; B=L. carii; C=I. pyrifolia; D=Ldensiflora; E=I. heteromorpha var. heteromorpha; F=I. licaniaeflora\ G=JL. apetala var. apetala; rha=rhamnopyranoside; ara=arabinopyranoside; rut=rutinopyranoside; xyl=xylopyranosyl or xylopyranoside; coum=coumaroyl; glc=glucopyranoside; gal= galactopyranoside Catechins
42
Licania pittieri Prance Licania pittieri Prance is a tree up to 15 meters high growing in the cloud forests at 1800 m in altitude. From the methanol extract of the leaves, collected in the Parque Nacional Henry Pittieri, oleanolic acid (1), ursolic acid (2), catechin (3), epicatechin (4), quercetin (5), and four glycosidic derivatives, quercetin 3-0-(3-D-galactopyranoside (6), quercetin 3-0-(3-Dglucopyranoside (7), quercetin 3-0-a-L-rhamnopyranoside (8), and quercetin 3-0-a-L-arabinopyranoside (9) were identified directly by comparing their spectroscopic data with those reported in the literature and/or authentic samples [see Fig. (1)] [5].
Licania carii Cardozo Licania carii Cardozo is a tree up to 18-20 meters high, growing in the cloud forests at 1800 m in altitude and is a new neotropical species discovered and collected in the Parque Nacional Henry Pittieri, Venezuela [6]. The chloroform-methanol residue afforded a series of triterpenes: ursolic acid (2), 2a-hydroxyursolic acid (10), betuHnic acid (11), maslinic acid (12), together with p-sitosterol 3-(9-|3-D-glucopyranoside (13) and several flavonol glycosides: myricetin 3-0-(2"-0-P-D-xylopyranosyl)-a" L-rhamnopyranoside (14), quercetin 3-0-(2"-0-p-D-xylopyranosyl)-a-Lrhamnopyranoside (15), quercetin 3-O-P-D-galactopyranoside (6), quercetin 3-0-p-D-glucopyranoside (7), myricetin 3 - O - p - D glucopyranoside (16), myricetin 3-(9-P-D-galactopyranoside (17), quercetin 3-0-rutinoside (18), myricetin 3'-methyl-3-(9-rutinoside (19), and myricetin 3-0-rutinoside (20) that were identified by comparison of their spectroscopic data with those reported in the literature and/or authentic samples [see Fig. (1) and (2)] [7].
Licania pyrifolia Grisebach Licania pyrifolia Grisebach is a small tree widely cultivated in the Amazonian regions of Venezuela for their edible fruits called "merecure". The chloroform extract yielded a-amyrin, p-sitosterol, lupeol, betulin.
43
Fig. (1). Compounds 1-9 and 12 from L pittierU L carii, L, pyrifolia, L. licaniaeflora^ and L apetala
COOH
R-R"-H 2 R-CHj 12 R«H
3 OHcq 4 OH ax
R'«CHj R'«R''-H R'«CHj R"«OH
5 6 7 8 9
Fig. (2). Triterpenes and flavonoids from L. cariUL, heteromorpha, L licaniajlora, and L apetala
R=H R"gal R-gIc R»rha R = ara
pyrifolia.L,
COOPT
R'O
10 25 27 30 59
R=OH R'^R^-R-'-H R-R''»R"'»H R'=ara R«R-=0H R'=H R-'-gIc R«R-=OH R'R'^^H R«R'=R"^'=H R - - 0 H COOR
26 R-glc R'«OH 2S R-R'-H 29 R=ll R'=OH
44
14 R«H R*-Tha(l-2)xyl 16 R-H R'-glc 17 R«H R'«g«l 19 R=CHj R'«glc(1-6)rha 20 R-H R'-g'c(l-6)rha
15 R=rha(l-2)xyl 18 R=glc(I-*6)rha
uvaol, oleanolic acid (1), ursolic acid (2), and betulinic acid (11). The CHCl3-MeOH extract was fractionated by column chromatography on Sephadex LH-20 with MeOH. The portion containing the bulk triterpenes was chromatographed over Si gel with CHCla/MeOH mixtures followed by low-pressure CC on Lichroprep RP-18 with MeOH/HiO mixtures to yield four new compounds: lla-hydroxybetulinic acid (21), 6(3hydroxybetulinic acid (22), 2,3-dihydroxylup-12-en-28-oic acid 3-(3\4*-
45
dihydroxybenzoyl ester) (23), and 2,3,27-trihydroxylup-12-en-28-oic acid 3-(3\4*-dihydroxybenzoyl ester) (24) [see Fig. (3)]. The following constituents of known structures were also obtained: p-sitosterol 3-(9-(3D-glucopyranoside (13), ursolic acid 3-(9-a-L-arabinopyranoside (25), euscaphic acid 28-p-D-glucopyranosyl ester (26), tormentic acid 28-|3-Dglucopyranosyl ester (27), 2a-hydroxyursolic acid (10), 2a,3adihydroxyurs-12-ene-28-oic acid (28), euscaphic acid (29), tormentic acid (30), and maslinic acid (12). All compounds were identified by comparison of their spectroscopic data with those reported in the literature and/or authentic samples [8]. The new derivatives 21-22 were identified by means of their spectroscopic data. The EIMS spectra of both compounds 21 and 2 2 showed a molecular peak [M]^ at m/z 472 corresponding to the formula C30H48O4 (confirmed by ^^C-NMR and DEPT analysis). These data indicated a triterpenoid skeleton with one carboxyl and two hydroxyl moieties and one double bond. The ^^C-NMR spectra of both compounds 21 and 22 revealed 30 carbon signals which were sorted by ^^C-DEPT NMR into six methyls, nine methylenes, five methines, five quartemary carbons, two alcoholic methines, one carboxylic acid, and two olefinic carbons (one =CH2 and one quartemary). The 20,29-functionality of a lupene skeleton was inferred for both compounds from the resonances of the sp^ carbons at C-29 at 109 ppm and C-20 at 150 ppm. A detailed analysis of the ^H-NMR of 21 confirmed the characteristic features for a betulinic acid parent structure bearing one -OH group at C-11 (one carbinolic proton at 8 3.85, ddd, J = 10.5, 10.5, and 5.0 Hz). The 7 values (two diaxial couplings and one axial/equatorial spin-spin coupling) were in accordance with an a-hydroxyl moiety. The substitution at C-11 was revealed by a shift of the carbon signal at 20.9 (C-11) of betulinic acid to 69.8 (d) and by the downfield shifts of signals of C-1 (2.9 ppm), C-9 (2.0 ppm), and C-12 (1.6 ppm) due to 8 and y steric effects of the a configuration of this hydroxyl moiety. This conclusion was confirmed by the downfield shift in the ^H-NMR spectrum of the methyl protons linked at C-10 (Me-25) due to the same steric effects. The 6|3-OH substitution of compound 22 was deduced in an analogous manner.
46
Fig. (3). Lupane derivatives 21-24fromL pyrifolia
COOH
21 R=OH R'=H 22 R»H R'«OH
COOH
23 R-H 24 R«OH
The EIMS of compounds 23 and 24 showed molecular peaks [M]^ at m/z 608 and 624 corresponding to the formulas C37H52O7 and C37H52O8, respectively. The ^^C-NMR spectrum of both compounds revealed 37 carbon signals. Those of 23 were sorted by ^^C-DEPT NMR into seven methyls, eight methylenes, five methines, five quatemaries, two alcoholic methines, one carboxylic acid, one -COOR, and two olefinic carbons (one =CH and one quatemary). Furthermore, a nonsymmetrically trisubstituted aromatic ring was also present. The signals of 24 were similar to those of 23 except that one -CH3 was replaced by a -CH2OH. These data indicated the presence in both 23 and 24 of a triterpenoid skeleton with two secondary hydroxyls (plus one primary OH in 24) and one carboxyl moiety, one double bond, and a 3,4-hydroxybenzoic unit. A 12,13-double bond and the fact that both derivatives had a lup-12-ene skeleton was indicated by resonances of the sp^ carbons C-12 (methine) at 126.3 ppm
47
and C-13 (quaternary carbon) at 138.0 ppm and by the analysis of the methine and methylene resonances. A detailed analysis of the ^H-NMR spectrum of 23 confirmed the characteristic features for a lup-12-en-28oic acid derivative bearing an a-OH at C-2 and a |3-0H at C-3. The carbinohc region revealed a doublet of doublets at 6 3.82 (IH, J= 4.5, 9.6, and 10.8 Hz) and a doublet at 8 4.63 (IH, / = 9.6 Hz), whose chemical shifts and J couplings were typical for a 2a,3(3-dihydroxyl substitution pattern. Furthermore, the ^H-NMR spectrum revealed the presence of a 3,4-dihydroxybenzoic unit by the presence of signals at 8 6.90 (2H, m, H-5' and H-6') and 8 7.02 (IH, br s, H-20. Moreover, the linkage of this moiety to C-3 of the triterpene was derived from the downfield shift (1.6 ppm) of C-3 when compared with other derivatives with the same substitution pattem. Compound 24 had resonances similar to those apparent in the ^H-NMR spectrum of 23 for a 2a,3^dihydroxylup-12-en-28-oic acid derivative. In addition, two signals for an oxymethylene group replaced the signal due to a rer/-methyl of the aliphatic region (Me-27). Further support for this additional substitution was obtained from NOESY experiments (Table 4). Thus, NOEs were observed between H-5 and H-9, H-23 and H-6a; between H-9 and H-23, H-3, H-27a, and H-27b; between H-27b and H-20; as well as between H18 and H-19. These results showed that the primary hydroxyl and isopropyl groups both had a-dispositions, whereas the OHfimctionat C-3 was p-oriented. Table 4. Interactions observed in the NOESY NMR spectrum of compound 24
proton 5 5 5 9 9 9 9 18 27b
^6H
0.72 0.72 0.72 1.56 1.56 1.56 1.56 1.38 3.52
Correlated signal Proton 5H 1.56 9 1.04 23 1.52 6a 1.52 6a 1.04 23 27a 3.18 27P 3.52 1.40 19 0.88 20
Successively from the chloroform-methanol and methanol extracts also some flavonoids were obtained: kaempferol 3-0-a-L-rhamnopyranoside (31), quercetin 3-0-a-L-rhamnopyranoside (8), myricetin 3-0-a-Lrhamnopyranoside (32), myricetin 3-0-(2"-0-p-D-xylopyranosyl)-a-L-
48
rhamnopyranoside (14), quercetin 3-0-(2"-(9-p-D-xylopyranosyl)-a-Lrhamnopyranoside (15), kaempferol 3-0-(2"-(9-p-D-xylopyranosyl)-a-Lrhamnopyranoside (33), kaempferol 3-0-a-L-arabinopyranoside (34), quercetin 3-(9-a-L-arabinopyranoside (9), myricetin (35), quercetin (5), kaempferol (36), 8-hydroxy-naringenin (37), 8-hydroxy-eriodictyol (38), and 8-hydroxy luteolin (39) [see Fig. (1), (2), and (4)]. Compound 38 was a new natural product: the unusual 5,7,8-trihydroxy substitution of ring A was determined by the presence of the singlet due to H-6 in the ^H NMR spectrum and by the evaluation of the resonances of C-2—C-10 of ypyronic ring A. The upfield shift of C-8 and the downfield shifts of C-7, C-5, and C-9 with respect to the same signals of eriodictyol led to the complete structural elucidation of compound 38 [9]. ^^^» (4). Flavonoids from L pyrifolia and L licaniaeflora
31 R-rha 33 R = rha(l-2)xyl 32 R - r h a 35 R » H 63 R-ara
34 R = ara 36 R » H
OH
0 37 R - H 38 R - O H
49
Licania densiflora Kleinhoonte Licania densiflora Kleinhoonte, synonym Licania kanukuensis Standley, is a tree 30 meters high, growing in forests and hills of Venezuela, Brasil, and Guyanes. Especially in Venezuela it grows in the Bolivar and Delta Amacuro regions; its common names are "Guanay", "Hierrito", and "Merecurillo" [3]. The chloroform-methanol and methanol extracts of the plant's leaves afforded eight flavonoids [compounds 40-47; see Fig. (5)] from which three were new natural products: naringenin 8-hydroxy-4'methyl ether (40), myricetin 3-0-(2"-0-a-L-rhamnopyranosyl)-a-Lrhamnopyranoside (41), 3',4'-dimethylmyricetin-3-0-P-Dglucopyranoside (42), myricetin 3'-methylether-3-0-(3-D-glucopyranoside (43), myricetin 3'-methylether-3-0-P-D-galactopyranoside (44), myricetin 4'-methylether-3-0-a-L-rhamnopyranoside (4 5 ), myricetin 3\5'dimethylether-3-0-(3-D-glucopyranoside (46), and myricetin 3',5'dimethylether-3-O-a-L-rhamnoside (47) [10, 11]. All compounds were characterised by spectroscopic methods including mono- and bidimensional NMR techniques. Fig. (5). Compounds 40-47fromL densiflora .OCH,
41
50
42 43 44 45 46 47
R = glc R«gal R-rha R«glc R-rhi
R'^CHj R-'R^'H R'«CHj R-'-R'-^H R'-R^^H R"«CHj R'=R'"=CH3 R*»H R'-R^-CHj R--H
In particular compound 40 was purified by Sephadex LH-20 column from the chloroform-methanol extract, while compounds 41-42 were obtained similarly by using Sephadex LH-20 column and Lobar Lichroprep RP 8 chromatography. The structures and molecular formulas of these new derivatives were determined bv positive ion FAB MS and UV spectra, ID and 2 D - ' H , ' ^ C , and C DEPT NMR data. Furthermore, the absolute configurations of the sugar moieties were confirmed referring to the optical rotation after hydrolysis of the glycosides. Compound 40 had molecular formula C16H14O6. Mass spectrometry, ^H, and ^^C NMR analysis indicated its flavanoidic structures and particularly it was evidenced from the ^H-NMR spectrum for the presence of the ABX spin system: H-2 (X part of the system) appeared as a dd at 8 5.49 (/BX= 3.4 Hz and /AX= 13.0 Hz), while H2-3, (AB part), were represented by two dd, one at 8 2.89 and one at 6 2.72 (7AB= 17.2 Hz). The presence of one singlet at 6 5.83, attributed to H-6, determined the unusual 5,7,8-trihydroxy substitution of ring A. Furthermore, the ^H NMR data also revealed one signal at 8 3.75 (3H, s) ascribable to one methoxyl group that resonated at 56.1 ppm in the ^^C NMR spectrum. The exact position of the methoxyl group was established by typical methoxylation shift observed in the C NMR spectrum with respect to 8-hydroxy naringenin: downfield shift of C-4' (ca. 5 ppm) and upfield shifts of C-3* (ca. 1.3 ppm), C-5' (ca. 1.3 ppm), and C-T (ca. 0.5 ppm) suggested the presence of the methoxyl group at C-4'. This was confirmed by bidimensional NOESY experiment which showed a correlation between the signal at 8 3.75 with the one at 8 6.95 (H-3* and H-5').
51
Mass spectrometry, ^^C, and ^^C DEPT NMR analysis indicated the flavonoidic nature also for compound 41, and in particular 15 carbon atoms ascribable to the aglycon and 12 to the sugar moieties. In the H NMR spectrum the chemical shifts and the proton coupling constants indicated a 5,7-dihydroxylated pattern for ring A (two meta-couplQd doublets at 6 6.21 and 6.42, J= 2.0 Hz) and a 3',4\5*-trihydroxylation for ring B (a two-proton singlet at 86.95), permitting recognition of the aglycon as myricetin. Furthermore two anomeric protons were easily identified in the ^H NMR spectrum. Mass spectra evidenced the presence of two flanked deoxyhexose moieties due to the presence of a molecular peak at 611 m/z and two prominent peaks were evidenced at 465 m/z [(M-f-H)-146]"^ due to the loss of a deoxyhexose unit and at 319 m/z [(M+H)-(146+146)]^ due to the loss of another deoxyhexose unit; actually they were identified by ^^C NMR data as two rhamnopyranosyl moieties with a-configuration at the anomeric carbon [12]. The position of the disaccaridic moiety at C-3 was determined by typical glycosylation shift observed in the ^^C NMR spectrum with respect to the aglycon myricetin: upfield shifts of C-2 and C-4 (about 5.0 and 3.7 ppm, respectively), and a downfield shift of C-3 (about 3.5 ppm). Actually myricetin 3-dirhamnoside was previously isolated from Azara microphylla leaves [13], but the authors did not specify sugar interlinkage. Therefore, from high digital resolution ^H NMR spectral data and from ^^C NMR, we determined without a doubt this linkage which was confirmed by bidimensional COSY and HETCOR experiments. After hydrolysis L-rhamnose was also identified by TLC and optical rotation value by comparing their data with those of an authentic sample. ^^C NMR of compounds 40 and 41 are reported on Table 5.
52
Table 5."CNMR< lata (200 MHz) for compounds 4( and 41. DEPT DEPT 40 (DMSO-c/6) Carbon 41 (CD3OD) 157.4 78.0 C 2 CH 42.0 C 136.0 3 CH2 196.4 C 178.2 C J 41 C 156.8 163.9 5 1 CH 95.3 99.8 CH 6 C 7 160.8 165.9 C 128.2 i CH 94.7 C 8 C 154.3 158.5 9 C 103.4 C 10 104.5 C 130.4 C 121.8 C 128.4 CH 2' 109.5 CH C 146.9 3' 114.6 CH 162.4 C 4' 137.3 C 114.6 C 146.9 5' CH 128.4 CH 6' CH 109.5 56.1 OCH3 CH3 rha 1" CH 103.8 CH 2" 79.1 CH 3" 71.8 4" CH 72.0 CH 5" 70.3 6" 17.8 CH3 CH 102.4 rha 1'" -^111 72.2 1 CH CH 3'" 73.6 4"» 73.9 1 CH 5"' 70.8 CH 17.8 6'" CH3 -
c
r
Compound 42 had molecular foraiula C23H24O13. In the positive ion FAB MS spectrum, a molecular peak was observed at 509 m/z and two prominent peaks were evidenced at 479 m/z [(M+H)-30]^ due to the loss of two methyl groups and at 347 miz [(M+H)-162]'^ due to the loss of a hexose unit. Comparing spectral data of compound 42 with those of 41, close similarities were observed between the signal values of the aglycon of both compounds, while sugar moiety provided the point of difference. Except for myricetin signals, ^H NMR spectrum revealed the presence of a one-proton doublet at 6 5.27, J= 7.5 Hz, representative of one anomeric proton, and two singlets at 6 3.93 (3H), and 3.95 (3H), ascribable to two methoxyl groups. The sugar was identified as P-D-glucopyranoside from absolute values of the coupling constants in the ^H-NMR spectrum and the evaluation of TLC and optical rotation of the sugar moiety after hydrolysis of the glycoside by comparison with an authentic sample. Relative positions of p-D-glucopyranoside and of the methoxyl groups were determined by UV spectra registered with AICI3, AICI3 + HCl, and
53
NaOMe establishing respectively the absence of two ortho OH residues and substituted 3, 3', and 4' positions. L. densiflora produced methoxylated flavonoid glycosides that are quite unusual in the plant kingdom and often play an important role of protection from attacks by herbivores or pathogens, so that their biosynthesis could be related to local environmental factors [14].
Licania heteromorpha van heteromorpha Bentham Licania heteromorpha var. heteromorpha Bentham is a tree up to 30 m high native to the Amazonian forest. Phytochemical study of its aerial parts yielded both triterpenes and flavonoids; triterpenes were obtained from the chloroform extract by silica gel column followed by RP-HPLC and were characterised as: betulinic acid (11), alphitolic acid (48), 3|3-6>trans'P'COumaiVoyX alphitolic acid (49), 3p-0-c/5-/7-coumaroyl alphitolic acid (50), 3p-C>-/ra«5-/7-coumaroyl maslinic acid (51), 3P-0-c/5-/7coumaroyl maslinic acid (52), 3(3-0-fraw5-/?-coumaroyl-2a-hydroxy-urs12-en-28-oic acid (53), 3(3-0-d5-p-coumaroyl-2a-hydroxy-urs-12-en-28oic acid (54) [see Fig. (2) and (6)] [15]. Compounds 11 and 48-54 were identified comparing their ^H and ^^C NMR data with those previously described. Triterpenoids 48-54 were found for the first time in Licania, while betulinic acid had been isolated previously from L carii [9]. On the other hand, flavonoids were isolated from the chloroform-methanol and methanol residues by Sephadex LH-20 and HPLC; they were identified as myricetin 3-(9-p-D-galactopyranoside (17), myricetin 3-0a-L-rhamnopyranoside (32), myricetin 4'-methylether-3-(9-(3-Dglucopyranoside (55), myricetin 4'-methylether-3-0-a-Lrhamnopyranoside (45), myricetin 3,4*-di-(9-a-L-rhamnopyranoside (56), myricetin 7-methylether 3,4*-di-(9-a-L-rhamnopyranoside (57), and myricetin 4'-methylether-3-0-p-D-galactopyranoside (58) [see Fig. (2), (4), and (6)]. The last three myricetin derivatives were new natural compounds [16]. Known compounds were identified by comparison of their ^H and ^^C NMR spectra with those reported in the literature [15]. Compounds 56-58 were purified by Sephadex LH-20 column and RPHPLC from the methanol extract. After spraying the TLC with Naturstoff reagent, they gave red spots typical of 3*, A\ 5'-trihydroxyflavonols showing that they all have the same aglycon. The structure and molecular
54
Fig. (6). Compounds 48-58 from L heteromorpha
COOH
COOH
R'O'
51 52 53 54
48 R«H 49 R«franj-p-coumaroyl 50 R«c»-p-coumaroyt
R«H R»H R*CHj R-CHj
m^trans-p-cQWMtxoyX R^-CHj R'«cfe-/'-coumaroyl R^^CHj R'e(7-an5-p-couinaroyi R''"H R'"ar-p-coumaroyi R-^H
.OR-
R'O,
55 56 57 58
R-gIc R'-H R--CH, R=R"«rha R'-H R=R"=Tha R'^CHj R=gal R'=H R"=CHj
formulae of new compounds 56-58 were determined by negative ion ESIMS spectra, ID and 2D-^H, ^^C and ^^C DEPT NMR data. Compound 56 showed in its ^^C, and ^^C DEPT NMR analysis 15 carbon atoms ascribable to the aglycon and 12 to the sugar moieties. In the ^H NMR spectrum the chemical shifts and the coupling constants of protons indicated a 5,7-dihydroxylated pattern for ring A (two metacoupled doublet at 8 6.22 and 6.36, 7= 1.8 Hz) and a 3\4\5*trihydroxylation for ring B (two-proton singlet at 6 6.94), permitting recognition of the aglycon as myricetin. Two anomeric protons were easily identified in the ^H NMR spectrum. They resonated at 8 5.31 {J= 1.5 Hz) and 5.57 (y= 1.5 Hz), and correlated respectively with 103.9 and 103.1 ppm in HSQC spectra. Chemical shifts, multipHcity of the signal.
55
absolute values of the coupling constant and the magnitude in the ^H NMR spectrum as well as ^C NMR data indicated the presence of two rhamnopyranosyl moieties with a-configuration at the anomeric carbon. The exact position of rhamnose units was determined by typical glycosilation shifts observed in the ^^C NMR spectrum with respect to the aglycon myricetin: upfield shifts of C- 2 (ca. 5.0 ppm ), and C-4 (ca. 3.7 ppm), and a downfield shift of C-3 (ca. 3.5 ppm) suggested the presence of a rhamnose unit at C-3; in the same way an upfield shift of C-4' (ca. 2.0 ppm) and downfield shifts of C-3' and C-5' (ca. 0.4 ppm) implied the position of the other a-L-rhamnose at C-4' [6]. This was confirmed by HMBC experiment correlations [8 5.31 with 136.8 ppm (C3) and 8 5.57 with 151.9 ppm (C-4')]. Compound 57 had molecular formula C28H32O16. Its ^H NMR and ^^C spectra compared with those of 56 each revealed one more signal respectively at 6 3.76, (3H, s) and 51.9 ppm assigned to one methoxyl group. Its position is established from HMBC correlation data. The methoxylation shift observed at C-6 and C-8 supported the position of the methoxyl group at C-7. When 56 is used as reference compound in the spectral analysis of compound 58, close similarities are observed between spectral data of the aglycon of both compounds, while sugar moiety provided the point of difference. Except for aglycon signals, ^H NMR spectrum revealed the presence of a one-proton doublet at 8 5.24, / = 7.5 Hz representative of one anomeric proton of a hexose unit, and one singlet at 8 3.92 (3H) again ascribable to one methoxyl group. Selected ID-TOCSY obtained irradiating anomeric proton signal (8 5.24) yielded the subspectrum of sugar residue with high digital resolution. The result of ID-TOCSY compared with those of ^^C NMR experiment allowed the identification of sugar as galactopyranoside; its (J-configuration at anomeric position is derived combining ^H NMR and ^^C NMR data. The relative position of p-D-galactopyranose and of the methoxyl group was again established from HMBC experiment correlations. ^^C NMR data of compounds 5658 are reported in Table 6.
56
Table 6/^CNMR data (600 MHz, CD3OD) for compounds 56-58. 56 I 57 I 58 Carbon I DEPT 1583 158^6 157.8 2 C 136.8 136.7 136.5 3 c 179.3 179.1 4 179.5 c 162.8 162.8 162.8 5 c 100.8 CH 100.5 99.5 6 165.6 167.7 165.6 7 C 94.4 95.5 95.1 s 1 CH 158.2 158.0 9 158.6 C 104.4 105.6 105.56 10 C 127.3 126.9 126.8 1' C 110.1 109.6 CH T 109.9 138.0 y 136.3 136.0 C 151.4 152.3 151.9 4' C 138.0 136.0 136.3 5' C 110.1 6' 109.9 109.6 CH 105.5 RhaatC-3 1" CH 103.9 103.9 73.2 73.4 2" 73.8 CH 3" 72.2 71.8 75.1 CH 4" 70.1 71.7 72.1 CH 77.3 71.0 CH 71.9 5" 18.0 6" 17.6 CH3 62.0 CH2 Rha at C-4' 103.1 102.7 1'" CH •^iii 73.3 CH 72.9 3.,, 71.7 72.1 CH 4'" 72.0 71.5 CH 71.3 70.6 CH 5'" 17.4 17.8 6"' CH3 -_ OCH3 51.9 60.8 CH3
1 1
Similarly to L. densiflora, L, heteromorpha is unusual for its biosynthesis of methoxylated flavonoids glycosides (with the methoxyl groups at C-4' of ring B and C-7 of ring A). The production of such flavonoids could be a response to local environmental factors. In comparison to the other Licania species, L. heteromorpha predominantly yields flavonol glycosides instead of flavones or flavanones.
Licania licaniaeflora (Sagot) Blake Licania licaniaeflora (Sagot) Blake is a tree native to the Amazonian forest where the leaves were collected. The plant's leaves were defatted with «-hexane and then extracted at room temperature with methanol to yield a residue which was subsequently partitioned between AcOEt, nBuOH, and H2O. From the AcOEt fraction, nine triterpenes were isolated
57
and identified as: oleanolic acid (1), ursolic acid (2), betulinic acid (11), maslinic acid (12), tormentic acid 28-P-D-glucosyl ester (27), pomolic acid (59), oleanolic acid 3-O-a-L-arabinopyranoside (60), arjunic acid 28-(3-D-glucosyl ester commonly known as arjunetin (61), and olean-12ene-2a,3|3-diol (62) [see Fig. (1), (2), and (7)] [17]. Their structural identification was achieved by ^H and ^^C NMR experiments. The nbutanol fraction submitted to Sephadex LH-20 and RP-HPLC afforded 10 flavonoids, both flavonol and dihydroflavonol glycosides that were characterised on the basis of spectral data (UV, MS, ^H and ^^C NMR) and by comparison with literature data or authentic samples: quercetin 30-p-D-galactopyranoside (6), quercetin 3-0-a-L-rhamnopyranoside (8), myricetin 3-0-(3-D-galactopyranoside (17), myricetin 3-0-a-Larabinopyranoside (63), kaempferol 3-0-a-L-rhamnopyranoside (31), kaempferol 3-(9-(2"-p-D-xylopyranosyl)-a-L-rhamnopyranoside (33), quercetin 3-(9-a-L-arabinopyranoside (9), 8-hydroxy naringenin (37), taxifolin 3-0-a-L-rhamnopyranoside (64), and dihydromyricetin 3-0-aL-rhamnopyranoside (65) [(see Fig. (1), (2), (4), and (7)] [18]. The presence of dihydroflavonol glycosides such as taxifolin 3-0-a-Lrhamnoside and dihydromyricetin 3-O-a-L-rhamnoside in the genus is reported now for the first time and thus may have some chemotaxonomic implications. Fig. (7). Compounds 60-62 and 64-65 from L licaniaeflora and L. apetala
60 R«ara R'-R-'-H R-«C00H 61 R=H R'^R-'OH R"«COO-glc 62 R-R-'H R'=OH R-=CH3
64 R»H
65 R«OH
58
Licania apetala var. apetala (E. May) Fritsch Licania apetala var. apetala (E. May) Fritsch is a tree native to the Amazonian forest. Its leaves were extracted with the same method used for L. licaniaeflora. From the w-butanol extract of the leaves seven flavonoids were obtained: quercetin 3-0-(3-D-galactopyranoside (6), quercetin 3-O-a-L-rhamnopyranoside (8) quercetin 3-0-rutinoside (18), quercetin 3-0-a-L-arabinopyranoside (9), taxifolin 3-O-a-Lrhamnopyranoside (64), myricetin 4*-0-a-L-rhamnopyranoside (66), and kaempferol 3-0-rutinoside (67), [see Fig. (1), (2), (7), and (8)] [18]. Fig. (8). Compounds 66-67 from L apetala
Orha
'Oglc(1-^)rh«
66
Licania intrapetiolaris Spruce ex Hook Licania intrapetiolaris Spruce ex Hook, a large tree that may grow up to 30 m in height and is widely distributed in tropical South America, including Columbia and Peru, was recently studied by Oberlies et al., 2001 [4]. The plant root, collected in a tropical rain forest of Ecuador, was subjected to a bioactivity-directed fractionation procedure through KB cytotoxicity assay. Using a series of chromatographic techniques, including flash silica gel and RP-HPLC, three bioactive compounds intrapetacin A, intrapetacin B, and cucurbitacin B were isolated. Intrapetacin A and B are new structures in a class of similar compounds that have been termed variously as either the clerodane diterpenoids, the kolovane diterpenoids, or the kovalene diterpenoids, while cucurbitacin B is a triterpenoid [see Fig. (9)] [4]. The structures of the new compounds were deduced from ID- and 2D-NMR experiments.
59
including relative stereochemical assignments based on NOESY correlations and COSY coupling constants. All these compounds have been unreported in the genus Licania until now. Fig. (9). Intrapetacin A and B and cucurbitacin B from L intrapetiolaris
I \'*°"J intrapetacin A R^CHj intrapetacin B R"Ac
OAc
cucurbitacin B
BIOLOGICAL STUDIES OF PLANTS BELONGING TO LICANIA GENUS
MoUuscicidal activity Schistosomiasis is a parasitic disease on the increase, affecting millions of people in Africa, South America, and the Far East. A general strategy for schistosomiasis control is to remove a link in the vital cycle, i.e. the
60
snail. The use of molluscicides provides a way to control the multiplication and spreading of snails. In this context, we performed in our laboratories a permanent programme for random screening of plants of South American flora, especially from Venezuela, with attributed moUuscicidal properties. Leaves of six species of Licania genus, L. pittieri^ L. carii^ L, pyrifolia, L, densiflora, L. heteromorpha var. heteromorpha, andZ. licaniaeflora, were investigated for a total of 15 extracts. Previously only L, tomentosa had been investigated for this activity [21]. A preliminary screening, by testing a standard concentration in water (1.000 ppm/24 hours), was carried out to differentiate extracts without any moUuscicidal activity from those with strong, some or weak toxicity. As a first step in our studies we distinguished the extracts with high or weak toxicity from those without any moUuscicidal response. Then the LCioo values (the concentration in water that kills 100% of a target snail population) of the moUuscicide were evaluated, and finally, for the active extracts, the toxicity species was tested on two fishes to consider the risks to nontarget organisms. MoUuscicidal tests were carried out with snails of Biomphalaria glabrata Say reared in aquaria with a continuous circulation of water through a filter system, with a temperature of about 24°C. Aquatic snails employed for the tests were young-mature and relatively uniform in age and size (average diameter of shell 8 mm). The snails were placed in dechlorinated tap water at 26-28° C under laboratory lighting conditions with normal diurnal light changes. Exposure of 24 hours to potential molluscicides was performed and death was established by examination (discoloration, heart rate, activity of muscle) and/or crushing. Acute toxicity on B, glabrata was evaluated by rapid screening procedure: the extracts were dissolved or suspended in water at a concentration of 1.000 ppm. Two containers at this concentration with two snails each were used, with the volume per snail not less than 40 ml. As control, one container with two snails each was included. In the event of both snails' death, a series of dilutions (e.g. 400, 200, 100, 50 ppm), were tested. For these assays, two containers at each concentration with 10 snails each were used, with the volume per snail not less than 40 ml. As control, one or two containers with 10 snails each were included as well. In order, to confirm the mortality of the snails, they were placed in a beaker containing distilled water alone; after 24 hours their condition was rechecked. The minimal lethal concentration required to kiU both snails after 24 hours was thus established and tested for the potential toxicity
61
on two fish species with a great adaptabihty: Poecilia reticolata XIPHO Viemef and Carassius auratus L. Fishes were reared in aquaria under the same conditions previously described for snails. The piscicidal test of the active extracts was carried out in duplicate also, with a reference, using an analogue method described for the rapid screening procedure of the moUuscicidal activity; in this case the volume per fish was not less than 1000 ml. The tested extract was considered piscicidal if the mortality of both fishes was recorded after 24 hours. All methanol extract and one-less polar extracts of the species examined killed snails at 1.000 ppm within 24 h (Table 7). Table 7. MoUuscicidal activity of Licania genus plant extracts.* Activity (ppm)
Species Extract
400 200 100 -H++ ++ MeOH ++ ++ ++ MeOH ++ ++ + CHCl3/Me0H ++ ++ ++ MeOH ++ L, densiflora MeOH ++ ++ MeOH L. heteromorpha L. licaniaeflora MeOH * Activity at a maximun concentration of 1000 ppm after a time interval of 24 hours. Legend: ++ means LCioo; + for LC50; and - for LCo
L. carii L, pittieri L, pyrifolia
50 ++ ++
25
++
+
+
1
+
On the contrary watery residues were completely inactive at the same concentration. The active fractions were susequently diluted to the minimum toxic concentration to determine the lethal concentration for 100% of exposed snails. As suggested from the literature, the data were judged positively if a plant extract killed snails at concentrations of 100 ppm or less [21]. Among the extracts investigated, four extracts showed LCioo at 100 ppm and three of them had the same toxicity in snails at 50 ppm. Therefore, these active samples were tested on two species of fishes, Poecilia reticularis and Carassius auratus, starting from the minimum effective toxic dose on snails and continuing with increasing concentrations, to find the LCioo/24 h concentration (Table 8). All extracts showed a lack of toxicity in fishes (toxicities were generally comparable in the two fish species examined) even when tested at 10.000 ppm, a value 2.000 times greater than the LCioo/24 h for snails.
62
Table 8. Toxicity (LCioo/24 h) on fishes of Licania leaf extracts Species (MeOH Extract)
Toxic dose (mg/1) Poecilia reticolata XIPHO Carassius auratus L. ViERNEF
L, carii L. pittieri L, pyrifolia
> 10,000 > 10,000 > 10,000
>10,000 > 10,000 >10,000
Three methanol extracts from the six Licania plants showed activity at 50 ppm within 24 hours on snails, a potency comparable with those reported in the literature for other tested plants of this family; one killed snails at 200 ppm, another one at 400 ppm, and the last one is active only at 1.000 ppm. Furthermore, extracts from L, carii, L. pittieri, and L. pyrifolia had an high selectivity for snails without toxicity in fishes. All the tested extracts contained mostly compounds belonging to the class of flavonoids and triterpenes, derivatives. Studies on the moUuscicidal activity of triterpenes with oleanane, ursane, and lupane nuclei isolated from Z. pyrifolia and I . licaniaeflora showed no mortality on snails and catechins did not exhibit any moUuscicidal activity up to 400 ppm. On the contrary flavonols glycosides of myricetin were lethal at 100 ppm; their high content in L carii, L. pittieri, and L. pyrifolia is assumed to be responsible for the strong moUuscicidal activity of these plants. These constituents, although characteristic of Z. densiflora andZ. heteromorpha were not in such high concentration to exhibit moUuscicidal activity at 100 ppm [22]. Antimicrobial activity Pure compounds 11 and 48-52 isolated from the aerial parts of Z. heteromorpha var. heteromorpha showed antimicrobial activity, with a different spectrum of action, on Gram-positive bacteria and yeasts (Table 9). Table 9. Antimicrobial activity of compounds 11 and 49-53: Minimum Inhibitory Concentration, Minimum Bactericidal Concentration is reported in parentheses (M-g/ml) C krusei Triterpenoids S. aureus S. capitis S. agalacticae C albicans 11 ~ >48 50(50) 100(100) ~ ~ 100(>100) 49 12.5(12.5) 12.5 (12.5) 25 (50) — 50 100(100) ~ ~ ~ ~ 51 25 (25) 25 (>100) 100(>100) 52 25 (100) 25 (25) 100(>100) 25 (50) — no effect 11 betulinic acid, 48 alphitolic acid, 49 3^-0-trans-p-couTmiroy\ alphitolic acid, 50 3P-0-cw-/7-coumaroyl alphitolic acid, 51 3P-0-fra/is-p-coumaroyl maslinic acid, 52 3p-0-cw-p-coumaroyl maslinic acid.
63
The minimal inhibitory concentration (MIC) was determined on 96well culture plates by a microdilution method using MuUer-Hinton Broth. Eleven two-fold dilutions of test substances were carried out starting from the concentration of 100 jxg/ml (2.5 % of ethanol). The wells were inoculated with a microorganism suspensions at a density of 10^ cells/ml. The plates were incubated for 24 hr (or 48 hr for the yeast) at 37 °C. The cultures that did not present growth were used to inoculate plates of solid medium in order to determine the minimal bactericidal concentration (MBC). Proper blanks were assayed simultaneously. The broadest activity was observed for compounds 49 and 52 which inhibited the growth of S. capitis, S. agalacticae, C. albicans, and C krusei; in particular 49 showed a MIC of 12.5 jxg/ml on S. capitis and C albicans, AlphitoUc acid (48) inhibited S. aureus (100 jxg/ml) and C. albicans (50 (xg/ml); 50 was active at the maximum concentration tested (100 |xg/ml) only against S, agalacticae. Compound 51 inhibited S. aureus, S. agalacticae, and C albicans with different activity. The antimicrobial activity of the triterpenoids tested was generally bactericidal. Betulinic acid (11) was inactive against all microorganisms tested. None of these triterpenoids inhibited the Gram-negative bacteria Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa. The stereoisomery of the compounds appeared to affect their activity particularly in the case of Sp-O-^ran^-p-coumaroyl alphitolic acid (49) and 3(3-0-c/5-/?-coumaroyl alphitolic acid (50); the latter inhibits only one bacterium at the maximal concentration tested, while the former inhibited almost all tested microrganisms at low concentration.
Antioxidant activity Pure flavonoids 6, 8, 9, 31, 33, 37, and 64, both glycosides and aglycons, isolated from the leaves of Z. licaniaeflora, were tested for their antioxidant activity by DPPH assay [23]. Oxidation is well known to be a major cause of material degradation. More recently, oxygen-reactive species, in particular free radicals, have been recognized as involved in several diseases, including cancer and atherosclerosis. Ageing may also be the result of the deleterious free-radical reactions which occur throughout cells and tissues. In this context, natural antioxidants are receiving increasing attention; particularly, flavonoids have been
64
reported to be efficient antioxidants by scavenging oxygen radicals, having interesting anti-cancer, hypolidaemic, anti-ageing, and antiinflammatory activities. The use of the DPPH radical as TLC spray reagent proposed for the first time in 1994 [24] for screening antioxidants in marine bacteria, appears to be also well suited for the detection of antioxidants in crude plant extracts or pure compounds isolated from plant material. The DPPH assay measured hydrogen atom (or one electron) donating activity and hence provided an evaluation of antioxidant activity due to free radical scavenging. DPPH (2,2-diphenyl-l-picrylhydrazyl radical), a purplcr coloured stable free radical, is reduced into the yellow coloured diphenylpicryl hydrazine. The test was first performed with a rapid TLC screening method using a 0.2% DPPH in MeOH. 30 min after spraying active compounds appear as yellow spots against purple background. Subsequently, spectrophotometric assay was carried out by the following method: 30 \xl of a methanolic solution containing the pure compound were added to 3 ml of a 0.004% MeOH solution of DPPH. Absorbance at 517 nm was determined after 30 min, and the percentage of activity was calculated. Quercetin was used as reference compound. The results of our experiments demonstrated that flavonoids possess a potent antioxidant activity which is consistent with other reports [25]. DPPH on TLC revealed all tested compounds as yellow spots against purple background, but 8-hydroxy-naringenin (37) and kaempferol 3-(9a-L-rhamnopyranoside (31) gave pale yellow spots when compared with the others. All compounds were also tested against DPPH in a spectrophotometric assay. As shown in Fig. (10), the activity of quercetin 3-0-(3-D-galactopyranoside (6), quercetin 3-O-a-L-arabinopyranoside (9), and quercetin-3-O-a-L-rhamnopyranoside (8) remained slightly lower than that of the reference aglycon, indicating that glycosylation at 3-0 position with different sugar moieties reduced the activity of the quercetin. Taxifolin 3-0-a-L-rhamnopyranoside (64), the dihydroflavonol derivative of quercetin 3-(9-a-L-rhamnopyranoside (8), showed stronger radical-scavenger activity compared with this one. Interestingly, kaempferol 3-0-(2''-13-D-xylopyranosyl)-a-Lrhamnopyranoside (33) had stronger activity than its biosynthetic precursor kaempferol 3-(9-a-L-rhamnopyranoside (31), but both were less efficient compared with quercetin derivatives: this is due to the presence in kaempferol derivatives of only one hydroxy group in the ring B of the flavonol. Finally 8-hydroxy-naringenin (37) exhibited similar
65
activity to kaempferol 3-(9-a-L-rhamnopyranoside (31) but with a different behaviour according to the concentration: stronger activity was shown up to 50 |uiM, while lower activity between 50 and 80 ^iM. Fig (10). Scavenging activity of flavonoidsfromL licaniaeflora leaves on DPPH radical. 100
^
1
90 1 80
»
»
»• » 1
: r ^ ^ ^
—
70 60
1
50 ^
III '"^"'^
#
40
Qiiefcetin
—A—33
30 B •
20 10 VM[
^^X^^""^
——37
0 10
64 9
20
30
40
50
Concentration
i&M
60
70
J 80
Cytotoxic activity The first screening on cytotoxic activity regarding plants of Licania genus was accomplished by Suffness et al. in 1988 on the ethanolic extract of Z. heteromorpha [19]. The plant showed cytotoxic activity in vitro in colon carcinoma 38 and B16 melanoma models. Subsequently, also the L. michauxii root methanol extract was found to be cytotoxic to cultured human hepatoma (HepG2) and colon carcinoma (Caco-2) cells, inducing necrotic death. There was no report on the isolation and characterisation of the active compounds present in this extract [20]. Finally, the triterpenoid cucurbitacin B, isolated from L intrapetiolaris, showed activity in the KB (human oral epidermoid carcinoma) assay with EC50 value of 0.008 tig/ml. The clerodane diterpenes intrapetacin A and intrapetacin B displayed moderate activity vs KB as well [4].
66
Antifungal activity Intrapetacin A and intrapetacin B isolated from L. intrapetiolaris, when examined in antifungal assay with Aspergillus niger spores, induced a good zone of inhibition of fungal growth if compared with amphotericin B activity [4]. REFERENCES [I] [2] [3] [4]
[5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]
Chaffaud, M; Emberger L. Traite' de Botanique Systematique, Vol.11, pp. 1338-40, Masson et C. Editeurs, Paris, 1960. Coradin, L.; Giaimasi, D.E.; Prance, A.T.; Brittonia, 1985, 37, 169-178. Toledo, C.L.; Kubitzki, K.; Prance G.H. Flora de Venezuela, Vol.1 V, pp. 326350, Ediciones Fundacion Educacion Ambiental, Caracas, 1982. Oberlies, N.H.; Burgess, J.P.; Navarro, H.A.; Pinos, R.E.; Soejarto, D.D.; Famsworth, N.R.; Kinghom, A.D.; Wani, M.C.; Wall, M.E.; J. Nat. Prod., 2001,(5^,497-501. Mendez, J.; Bilia, A.R.; Morelli, I.; Pharm. Acta Helv., 1995, 70,223-226. Cardozo, A.; Ernstia, 1992,1, 143-146. Bilia, A.R.; Mendez, J.; Morelli, I.; Pharm. Acta Helv., 1996, 71, 191-197. Bilia, A.R.; Morelli, I.; Mendez, J.; J. Nat. Prod., 1996, 59, 297-300. Bilia, A.R.; Ciampi, L.; Mendez, J.; Morelli, I.; Pharm. Acta Helv., 1996, 71, 199-204. Braca, A.; Bilia, A.R.; Mendez, J.; Morelli, I.; Phytochemistry, 1999, 51, 11251128. Braca, A.; Bilia, A.R.; Mendez, J.; Morelli, I.; Fitoterapia, 2001, 72, 182-185. Agrawal, P.K.; Bansal, M.C. Carbon-13 NMR of flavonoids, Elsevier, Amsterdam, 1989. Sagareishvili, T.G.; Alaniya, M.D.; Kemertelidze, E.P.; Khim. Prir. Soedin, 1983, 3, 289-293. Wollenweber, E. In: J.B. Harbome, The Flavonoids: Advances in research since 1986. Chapman & Hall, London, 1990. Braca, A.; De Tommasi, N.; Mendez, J.; Morelli, I.; Biochem. Syst. EcoL, 1999,27,527-530. Braca, A.; De Tommasi, N.; Mendez, J.; Morelli, I.; Pizza, C ; Phytochemistry, 1999,57,1121-1124. Braca, A.; Sortino, C ; Mendez, J.; Morelli, I.; Fitoterapia, 2001, 72, 585-587. Braca, A.; Luna, D.; Mendez, J.; Morelli, L; Biochem. Syst. EcoL, 2002, 30, 271-273. Suffness, M.; Abbott, B.; Statz, D.W.; Wonilowicz, E.; Spjut, R.; Phytotherapy Res., 1998,2, 89-97. Badisa, R.B.; Chaudhuri, S.K.; Pilarinou, E.; Rutkoski, N.J.; Hare, J.; Levenson, C.W.; Cancer Lett., 2000,149, 61-68. Mott, K.E. Plant Molluscicides, John Wiley & Sons LTD., Chichester, Great Britain, 1987.
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[22] [23] [24] [25]
Bilia, A.R.; Braca, A.; Mendez, J.; Morelli, I.; Life Sciences, 2000, 66, PL 5359. Braca, A.; Sortino, C ; Politi, M.; Morelli, I.; Mendez, J.; J. EthnopharmacoL, 2002, 7P, 379-381. Takao, T.; Kitatani, F.; Wataanabe, N.; Yagi, A.; Sakata, K.; Biosci. Biotechnol. Biochem., 1994, 51, 1780-1783. Hanasaki, Y.; Ogawar, S.; Fukui, S.; Free Rad. Biol Med., 1994,16, 845-850.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry^ Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
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RECENT PROGRESS IN RETINOID CHEMISTRY ALAIN R. VALLA VA R&D Pepiniere d'entrephses, 140 Bd de Creac 'h Gween 29561 Quimper France DOMINIQUE L. CARTIER and ROGER LABIA FRE 2125 CNRS, 6 rue de IVniversite 29000 Quimper France ABSTRACT: Natural retinoids, especially retinol, are present in all living organisms. They are known as fundamental mediators for many biological processes, e.g. vision, cellular growth, differentiation of epithelial tissue, reproduction, etc. Although retinol is an omnipotent compound, natural retinoids like all E, 9Z and dehydroretinoic acids are clearly more potent outside the retina and trigger gene expression via binding to nuclear retinoid receptors. Retinaldehyde occupies an intermediate position in this respect, with the ability to convert to both retinol and retinoic acid (RA). Retinol has an exceptional position among the retinoids, both in terms of production and its field of applications. In 1995, the sales of retinol reached US $ 500 million. Considering the interplay between the stereochemistry of retinoids and their biological activities, any synthetic approach to these compounds must meet the requirement of stereochemical control, in order to obtain the desired isomer in a highly stereoselective manner. Despite the recent achievements in the preparation of conjugated E/Z-dienes and trienes using a variety of synthetic procedures, there is still a need for versatile and efficient approaches to higher unsaturated E/Z-polyolefins with the appropriated configuration. This review covers the major synthetic literature on natural retinoids from 1990 to the present.
INTRODUCTION Retinol (vitamin A) is found in foods of mammalian origin in the form of retinyl ester, or in fruits and vegetables as carotenoids w^ith provitamin A activity, especially P-carotene (provitamin A). In enterocytes, retinol binds to cellular retinol-binding protein type II (CRBPII), w^hich directs the esterification by the enzyme lecithin: retinol acyltransferase (LRAT). The p-carotene may undergo central or eccentric cleavage with the formation of retinoids, or may be released unchanged into the bloodflow^.
70
Retinyl esters and the P-carotene are incorporated into chylomicrons and taken up mainly by hepatocytes. In the liver retinol may be stored in stellate cells as retinyl esters, oxidized to retinoic acid or liberated into cells bound to retinol-binding proteins (RBP). All E retinoic acid and its 9Z isomer have an affinity for nuclear receptors. They activate the transcription and bind as dimers to specific nucleotide sequences, present in promoters of target genes. Retinoids also modulate gene expression acting and the posttranscriptional level. The P-carotene has also important implications from the clinical point of view. All E retinoic acid is used for patients with acute promyelocytic leukemia [1]. They have been found to be active in the regulation and differentiation of many cell types, including epithelial. Therefore, many natural retinoids have been tested therapeutically against skin diseases, including some forms of malignancies. The best effects were achieved with retinol, retinaldehyde, 13Z-RA and all E-RA [2]. The latter has selective biological activity for normal growth and epithelial differentiation. It has been shown that retinoic acid receptors (RARs) have a high affinity for both all E retinoic acid and its 9Z isomer; which is the natural ligand of the retinoid X receptors (RXRs) [3]. The six isoforms of these proteins (RARa, RARp, RARy and RXRa, RXRp, RXRy) are transcription factors interacting with their target genes through specific DNA-binding domains. European researchers found recently that retinoic acid-induced apoptosis in leukemia cells was mediated by paracrine action of tumorselective death ligand trail (tumor necrosis factor-related apoptosisinducing ligand) [4].
P-carotene
71
CHEMISTRY Since its discovery in 1909, the elucidation of its structure by Karrer in 1931 [5] and its first total synthesis [6], vitamin A has represented a challenging target molecule for chemists [7]. The first industrial synthesis of retinol was performed at Hoffmann-La Roche (H-L R) [8], followed by other approaches of the Baadische Anilin-& Soda Fabrik (BASF AG) [9], and Rhone-Poulenc (R-P) (today: Aventis) [10] Both companies possess today the economically most important processes, and use p-ionone as starting material. Fig. (1).
6c^'
vitamine A OR
H-LR
PhjP
OH
•---
P'^Phs, CI"
vitamine A
NaOMe
HCl ' BASF AG "OAc
PhS02Na
OH
^Y^^^^
vitamine A
/BuOK
R-P OAc
Fig. (1). Industrial syntheses of vitamin A. The emphasis for the past twenty years has been the stereoselective syntheses of vitamin A, and its derivatives, retinal and retinoic acid [11]. A range of recent conceptions, using tricarbonyliron complex [12,13], Palladium-catalyzed cross-coupling reactions such as: Negishi reaction [14] (organozinc reagents and organotriflates or halides), Suzuki coupling [15] (orgaboron reagents and organotriflates or halides), Stille reaction [16] (organostannate and electrophiles), Heck reaction [17] (organotriflates or halides with olefins),
72
Sonogashira coupling [18] (copper (I) acetylenes with iodoalkenes)... were described. Each strategy could be achieved by two methods: e.g. for the construction of the C(6)-C(7) bond or C(7)-C(8), either the coupling of C9 with Cii or of Cio with Cio synthons had been done, Fig. (2).
Fig. (2). Strategies C(6)-C(7) strategies: Using a Stille's reaction, De Lera et al connected a tetraenic stannate with a triflate derived from the trimethylcyclohexanone [19,20]. First, the tetraenylstannate was prepared by Horner-Wadsworth-Emmons condensation of the known stannate [21] and the carbanion of the phosphonate [22, 23], Fig (3).
CHO
(EtO)2(0)P>
COOEt Bu.Sn^
^COOEt
Fig (3). Treatment of 2,2,6-trimethylcyclohexanone with lithium diisopropylamide (LDA) followed by phenyltriflimide (7V-phenylbis (trifluoromethanesulphonimide) gave the corresponding triflate [24]. The
73
best coupling reaction could be achieved with Farina's 'soft' palladium (Pd2(dba)3) with AsPhs as ligand and DMPU, Fig (4) [25]. OTf
a)LDA
^COOEt
b) Bu3Sn"
phenyltriflimide
Pd2(dba)3, NMP, DMPU, AsPh3
^^COOU
COOEt c) K:OH, EtOH, H2O
Fig. (4). Dominguez, Iglesias, and De Lera (1998, 2001). As an extension of this procedure, they synthesized the side chain of 9Z-retinoate stereoselectively and attached it to the hydrophobic ring by a high yielding thallium accelerated Suzuki cross-coupling reaction, Fig. (5) [26]. The tetraenylstannate used for the coupling reaction was obtained by Mn02 oxidation of the known stannyldienol [27], to the corresponding aldehyde (86%), followed by condensation with the phosphonate (52%) and reaction of the tetraenylstannate with a solution of iodine. The product was immediately added to the organoborane, in the presence of Pd(PPh3)4 then TIOH was added, to provide the 9Zretinoate in 84% yield. The organoborane was freshly prepared from the cyclohexanone, via its hydrazone, which was transformed into the iodide. COOEt
a) BuLi,
1 BuSn
BuSnH, CuCN OH4
BuSn
^
^
b) Mn02 K2C03^
[ ""OH
-^:^ ^
BuSn''
OEt ^j^Q
c) BuLi, DMPU
•
COOEt
COOEt
I J) /BuLi, B(0Me)3
e) H2N>fH2 I2, Et3N, DBN
"
Pd(PPh3)4
*"
Fig. (5). Pazos, and De Lera (1999).
B(0H)2 J ^ ^ ^
kjl\
COOEt
74
In this exhaustive work De Lera et al described the syntheses of the retinoid skeleton via the Stille coupUng for the formation of side-chain single bonds [28]. C(7)-C(8) strategies: A stereoselective synthesis of all E retinal, via a condensation of a Cio chloroacetal with p-cyclogeranylsulfone was described by Julia et al. [29]. The chloroacetal was reacted with the silylenol ether, using TiCl4/Ti(OMe)4, to give in 63% yield, the chloromethoxyacetal derivative as a mixture of ElZ isomers (80/20). The aldehyde was converted in 97% yield into the corresponding acetal with HC(0Me)3 and camphorsulfonic acid in methanol, Fig. (6).
OMe OMe
OMe
OMe
a) Ti(0Me)4 TiCl4
OMe
b) HC(0Me)3 CHO
OMe CI
01
CI
Fig. (6). This building block was condensed with the anion of (icyclogeranylsulfone. During flash-chromatography the intermediate was hydrolyzed to the sulfone-aldehyde, as a mixture of three isomers in 95% yield. Retinal was obtained from this sulfone by treatment with MeONa, for 10 days, in the dark (90%), Fig. (7).
OMe I
OMe OMe
SO2
I
OMe I
CHO
Fig. (7). Chemla, Julia, and Ugen (1993).
75
Chabardes developed a process for the preparation of vitamin A and its intermediates, from cyclogeranylsulfone and Cio aldehyde-acetals [30], For example, chlorocitral reacted with ethylene glycol, HC(0Me)3 and pyridinium tosylate to provide the chloroacetal (40%), as a mixture of two isomers. Reaction of this allylchloride with A^-methylmorpholine oxide (NMO) and Nal furnished the aldehyde, as a mixture of four isomers. These compounds underwent condensation with pcyclogeranylsulfone. Further chlorination of the sulfone-alkoxide salts, led to a mixture of sulfone-chloride acetals and their products of hydrolysis in 45-50% yield. Double elimination of the chloride and the sulfone, followed by hydrolysis with pyridinium tosylate (PPTS) gave retinal, as a mixture of all E and 13Z isomers (78/22). The overall yield from the chloroacetal was 18%. In another 'one-pot' example, retinal was obtained in 52% yield from the aldehyde, and was then isomerised and reduced to retinol (all E: 95.5, 13Z: 4, 9Z: 0.5) Fig. (8).
s
a) NMO CI
I
Nal, DMF
I
O-^ ^) L A
^2
/PrMgCl c) SOCI2, pyridine
d) MeOK
j^^^^'-'^^::^.-'^^
e) PPTS
Fig. (8). Chabardes (1994). Honda et al described a highly Z stereoselective [2,3]-sigmatropic rearrangement that provided trisubstituted E,Z synthons, starting from A^tiglyl-p-methallyldimethylammonium salts [31]. The application of this key triene synthon to the stereoselective synthesis of 13Z-retinol was reported from a trieneester. Thus, prenylbenzyl ether was converted via ene-type chlorination followed by amination into internal allylamine. This was reacted with ethyl 3-bromotyglate in acetonitrile to give the
76
quaternary salt. Treatment of the latter with EtOK in ethanol resulted in the formation of an ylide. This latter underwent [2,3]-sigmatropic rearrangement to furnish the diene that possessed a newly formed Z and tiglyl-origin E stereochemistry, Fig. (9). a) Br
COOEt
OSi/BuMeo
OSi/BuMeo
h) EtOK
NMeo
"^ EtOOC 0SirBuMe2 COOEt
Fig. (9). Treatment of this synthon with peracetic acid resulted in the formation of a A^-oxide intermediate. A Cope elimination gave the triene, Fig. (10).
c) AcOOH
EtOOC
EtOOC 0Si/BuMe2
0Si/BuMe2 ^0°C
EtOOC 0Si/BuMe2
Fig. (10). ?BuMe2Si was then replaced by rBuPh2Si and the transformation of the ester group to formyl group was carried out by treatment with aluminium hydride (AIH3), followed by manganese dioxide oxidation. This triene aldehyde was reacted with the anion of p-cyclogeranylsulfone and quenched with AC2O. Desilylation to the acetoxysulfone (80%), and
77
reductive cleavage with sodium amalgam gave the desired 13Z-retinol (63%), Fig. (11). e)Bu4NF,y)TBDPSCl
»• OHC
EtOOC
^
g) AlCl3/LiAlH4, h) Mn02 OSi/BuMeo
OSi/BuPh,
OSi/BuPho
Fig. (11). Honda, Yoshii, and Inoue (1996). A one-pot procedure was developed by Otera et al from pcyclogeranylsulfone [32]. Its lithium salt reacted with 3,7-dimethyl-8oxo-2,6-octadienyl acetate to the sulfone-alcohol. The hydroxyl group was protected to the MOM ether with MeOCH2Cl. Double elimination could be achieved with potassium MeOK to provide vitamin A in 50% yield. Fig. (12).
.o 9 o.
OHC
^ ^ so. OAc
OAc OH
a) Nal, BuLi
b)MOM-ci
y^^^Yi^^^^ ^
\ X \
c) MeOK
OMOM
Fig. (12). Orita, Yamashita, Toh, and Otera (1997).
^^^^^^^^^^^V^^OAc
78
A similar synthesis was patented by Odera [33]. Two patents by Takahashi et al reported the synthesis of vitamin A via a Cio dihalogeno derivative [34,35]. In one procedure the halogenodiene was prepared by bromination of 3,7-dimethyl-2,5,7octatrien-1-yl acetate. Addition of the latter and /BuOK in DMF to the Cio sulfone provided the retinol sulfone (34%). Again, double elimination (MeOK), gave vitamin A acetate, Fig. (13).
a) Br2
Brv
OAc b) /BuOK, DMF
PY
SOo
I
Br
c) MeOK
-^::s^^^^
OAc
Fig. (13). Takahashi, Furutani, and Seko (2000). They also developed a second process via other dihalo-compounds [36]. Treatment of the 1,2-bromo-hydroxy chain with TiCU in DME, gave mainly the l-bromo-4-chloro unit. Condensation with the Cio sulfone in DMF, in the presence of /BuOK gave the retinylsulfoneacetate. Elimination of the tolylsulfmate with KOH in DMF produced vitamin A acetate in 87% yield. Fig. (14).
Fig. (14). Takahashi, and Seko (2001).
79
In a similar route, Takahashi et al made use of non-halogenated sulfones [37]. Similar processes were related. TiCU was added to a solution of the diol to give a crude mixture of isomers in which the 5-chlorosulfone was the main compound in 95% yield. The mixture was treated with MeOK to produce crude retinol. Acetylation with acetic anhydride (AC2O) in pyridine, in the presence of DMAP, provided the retinyl acetate in 70% from the diol [38,39], Fig. (15).
OH
c) AC2O, DMAP
Fig. (15). Takahashi, Furutani, and Seko (2000). C(io)-C(ii) strategies: Mestres et al [40] published a regioselective addition of a lithium trienediolate (generated from hexa-2,4-dienoic acid or dihydropyran-2one) to p-ionone. Dehydration of the hydroxyacid, afforded a mixture of 9EIZ, 13£'/Zretinoic acids which, isomerised in the presence of I2, led to all E retinoic acid in 35% and 30% yield, starting from dienic acid and pyranone, respectively, Fig. (16).
COOH
COOH
Fig. (16). Aurell, Parra, Tortajada, Gil, and Mestres (1990); Aurell, Came, Clar, Gil, Mestres, Parra, and Tortajada (1993); Aurell, Ceita, Mestres, Parra, and Tortajada (1995).
80
A concise preparation of retinoids via new enaminodiesters synthons was described by Valla et al [41]. For example, all £-retinoic acid was synthesized within one day by a 'one-pot' process. The enaminodiester synthon was prepared from methyl isopropylidenemalonate and dimethylformamide dimethylacetal (DMF-DMA) and then condensed with the lithium enolate of p-ionone. A Grignard reaction with the obtained ketodiester led to the retro carbomethoxyretinoate. Saponification and concomitant decarboxylation, provided mainly all E retinoic acid {all E/UZ: 90/10, 72% from (J-ionone), Fig. (17).
^v
COOMe
a)LDA
l^^^J^
b) MeMgBr
COOMe
COOMe COOMe COOMe
COOH
c)K0H,Me0H,H20
^ COOMe
^HCllM
Fig. (17). Valla, Cartier, Labia, and Potier (2001). A short synthesis of retinal was described by Taylor et al. [42] based on the addition of a Cn vinylalane to a methylpyrylium salt. The 13Zretinal (48%) was isomerised to all E retinal by a previous procedure [43]. p-Ionone was first converted into the alkyne and then into the vinylalane, using the Negishi methodology [44]. Addition of an excess of this alane to 4-methylpyrilium tetrafluoroborate [45] gave 13Z-retinal, being isomerized to the all E isomer (I2 in benzene/ether). Fig. (18). AlMejBuLi
a) MejAl, ZrClz
^ (TI-C5H5)2, BuLi
c)l2 CHO
Fig. (18). Hemming, De Meideros, and Taylor, (1994); Taylor, Hemming, and De Meideros (1995).
CHO
81
Through two successive Stille reactions, Parrain et al [44] realized a stereo selective synthesis of all E, 13Zand 9-A2or-retinoic acids. First, the coupling of £'-l,2-bis(tributylstannyl)ethene and Z- or E-tributylstannyl3-iodoalk-2-enoates was performed, followed by iododestannylation. The second step involved another vinyltin which was synthesized by stannylation of the Negishi dienyne, derived from p-ionone [47]. To obtain the substituted vinylstannate, the dienyne was treated with lithium butyltributylstannylcyanocuprate (Lipshutz reagent) [48] to yield the intermediate vinylcuprate, which was trapped with an excess of Mel in the presence of hexamethylphosphoramide (HMPA). The reaction occurred to the advantage of the terminal vinylstannate (up to 92%). The coupling partner was obtained from tetrolic acid, which was converted into E vinyliodide by stannylcupration of the generated stannate [49]. The Z vinyliodide was more classically obtained by hydroiodination [50]. Stille coupling of the P-iodovinylic acids (protected as the corresponding tributyltin esters) with £'-l,2-bis(tributylstannyl)ethene, catalyzed by dichlorobis(acetonitrile)palladium provided dienyltins with retention of the configuration of the two double bonds in fair yields. Iododestannylation yielded quantitatively the dienic acids, Fig. (19). a) Bu3SnBuCuLi, yr-——
^^
j T-
C00HBu3Sn^
^COOH
^
d) BuSnOMe, PdClzCMeCN); e)l2./)KF,HC\
V c)HI
j
I
SnBu3 g)PdCl2(MeCN)2,DMF
COOH
I
I
^ O ^ : ^ ^ ^ ^
^ \^-!v
COOH
Fig. (19). Thibonnet, Abarbri, Duchene, and Parrain (1999). The first palladium-catalyzed cross-coupling reaction used in the synthesis of retinoids was described by Negishi and Owczarczyk from a Ci4 alkenylzinc [51]. The synthesis was carried out via a Pd(PPh3)4
82
catalyzed coupling of the C14 alkenylzinc (obtained from the iodide) with the Ce iodide (derived from 3-methyl-2£'-penten-4-yn-l-ol), followed by further deprotection with BU4NF. Vitamin A was obtained in 38% yield based on p-ionone, with complete control of stereo- and regiochemistry, Fig. (20).
Q
a) LDA, Cl-P(0)(0Et)2, LDA b) MesAl, Cl2ZrCp2,12,
c) DIBAL-H, I2 ClSiPh2/Bu, EtsN, DMAP
d) /BuLi, ZnBr2 P(i(PPh3)4 e) BU4NF
Fig. (20). Negishi, and Owczarczyk (1991). A highly stereoselective synthesis of retinol vz^ a CM + C6 route was depicted by De Lera et al [52]. A Suzuki reaction of a C14 alkenyliodide with a C6 alkenylboronic acid afforded retinol in 83% yield, with retention of the geometries of the coupling partners. The alkenyliodide was obtained by a zirconium-mediated methylalumination and a subsequent Al/I exchange by slow addition of ICN. Coupling with the C6 boronic acid (12 hrs to reach completion), afforded retinol in 83% yield [53], Fig. (21).
Fig. (21). Torrado, Iglesias, Lopez, and De Lera (1995).
83
C(ii)-C(i2) strategies: Stereoselective syntheses of all E, 9Z-retinoic-acids and llZ-retinal were developed from p-ionone-tricarbonyliron complex [12]. Treatment of the complex (prepared from p-ionone and dodecacarbonyliron, (Fe3(CO)i2)), with the lithium salt of acetonitrile, Wada et al obtained the nitrile, in 88% yield, Fig. (22). CHO
O
a) LDA, MeCN ^ Fe(C0)3 Q (EtO)2P''''~V^COOEt
^ c) BuLi
COOEt
COOH
,)j^30H MeOH, HjG
Fig. (22). Contrarily, the reaction of the lithium enolate of ethyl acetate with subsequent dehydration gave predominantly the ethyl 9Zionylideneacetate in 89% yield, Fig. (23).
O
a) LDA, MeCOOEt ^
Fe(C0)3
/'-p^
CHO ^)BuUTHF
(C0)3 g) NaOH, ^
^ COOEt
MeOH, H2O COOH
COOEt Fig. (23). Wada, Hiraishi, Takamura, Date, Aoe, and Ito (1997); Wada, (2000).
84
These compounds were converted to the corresponding all E and 9Zretinoic acids via P-ionylideneacetaldehydes. Thus, the reaction with the Uthium sah of (EtO)2P(0)CH2C(Me)=CHCOOEt in THF made possible the C20 ester-complex. The complex was removed by CUCI2 in EtOH (98%) and saponification of the ethyl retinoate, the retinoic acids could be obtained {all E: 89%, 13Z: 8% and 9Z: 59%, 9Z,13Z: 12%, respectively). The Peterson reaction of the chlorovinyl-complex with ethyl trimethylsilylacetate provided the HZ isomer preferentially (77%), and the 1 IJE" isomer as a secondary product (15%). The ester was transformed into the Cig ketone (Ph3SnCH2l, BuLi, Et20, 79%). Reaction with (/PrO)2P(0)CH2CN afforded the llZ-retinonitrile in 73% yield. The complex was removed by CuCb (72%) and DIBAL-H reduction led quantitatively to llZ-retinal, Fig. (24). EtOOC
Fig. (24). Wada, Hiraishi, Takamura, Date, Aoe, and Ito (1997); Wada (2000). Wada et al. [13] have previously reported similar syntheses of all E, 9Z-retinoic acids and 1 IZ-retinal. A short access to retinal was reported by Duhamel et al. [54,55] via the enolate of prenal, prepared from the corresponding silyl enol ether or enol acetate. The diene reacted with p-ionylideneacetaldehyde to give the dihydropyranol as the single reaction product. The dihydropyranol was
85
easily converted into retinal (43% yield) by dehydration, ring opening and further dehydration in the presence of a catalytic amount of pyridinium chloride or boric acid, Fig. (25).
OAc
a) MeLi
J^„." t ^
OSiMe,
pyridinium chloride, DMF
Fig. (25). Duhamel, Guillemont, and Poirier (1991); Cahard, Duhamel, Lecomte, and Poirier (1998). These authors also described a three-step synthesis of 13Z-retinoic acid [56]. The obtained hydroxydihydropyrane (66%) was oxidized either by Jones's reagent (CrOs, water, H2SO4, 90%) or Corey's reagent (pyridinium chlorochromate (PCC), 65%). Finally, the dihydropyranone was transformed into retinoic acid (as a mixture of9E, 13Z, and 9Z,13Z), by /BuOK, according to a known procedure [57], Fig. (26).
or PCC
GOGH
Fig. (26). Cahard, Mammeri, Poirier, and Duhamel (2000); Cahard, Duhamel, Lecomte, and Poirier (1998). This French group patented a process for the preparation of vitamin A from vinyl-P-ionol, by BF3-Et20 catalyzed condensation with a C5 sulphide (50% yield) [58]. The phenylthioretinal was reduced with NaBH4 to give the corresponding alcohol (99.5%), which was acetylated (AC2O, -100%).
86
The resulting sulphide-acetate was oxidized with w-chloroperbenzoic acid (MCPBA) and the sulfoxide was eliminated by heating in CCI4 to supply vitamin A acetate in 76% yield. Fig. (27). SPh OH
OMe
^^O 6)NaBH4
a) BF3-Et20
c) Bi^N, AC2O
•Qll
^Y^vA.^^
SPh
d) MCPBA
^Y^^^A-^^^^
ecu ^
kA.
reflux
SOPh
OAc
Fig. (27). Ancel, Bienayme, Duhamel, and Duhamel (1992). Another work of Duhamel and Ancel [59] related this synthesis of retinal via p-ionylideneacetaldehyde. Condensation of methallylmagnesium chloride with diethyl phenyl orthoformate (Et02CH0Ph) led after bromination of the ene-acetal, deshydrohalogenation (NaOH 50%), ethanol elimination with hexamethyldisilazane (HMDS) and ISiMes, to the bromo-dienol ether. This latter was submitted to bromine lithium exchange and the lithio enol ether was then condensed with pionylideneacetaldehyde to give retinal. Fig. (28). GEt MgCl (Et0)2CH0Ph
GEt Br,
GEt
GEt
GEt NaGH
GEt Br
/BuLi
HMDS <^^GEt
». ISiMe3
Br
CHO
>Ov-^CHG
Fig. (28). Duhamel, and Ancel (1992). In a similar approach, Duhamel et al [60] studied the catalyzed condensation (BF3-Et20 or ZnCb) of vinyl-P-ionol with a chloroenolether. The intermediary aldehyde {all EI9Z\ 65/35) had been
87
dehydrohalogenated (l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 86% or LiCl, 75%), to a mixture of retinals. This mixture had been isomerized to all E retinal, according to literature procedures, [61] Fig. (29).
a) BF3-Et20 or ZnCl2^
CHO orLiCl, DMF
Fig. (29). Duhamel, Duhamel, and Ancel (1994). In connection with a work related to the syntheses of C5 building blocks, Quintard et al [62] described a synthesis of retinal from Pcyclocitral. This aldehyde was condensed with the vinyl lithium salt of the C5 acetal. The lithiated compound was obtained via the vinyltin derivative which was first converted into the vinyl iodide before doing the halogen-metal exchange. Fig. (30 and 31). OEt J^^^^
OEt
.;Bu3SnMgMe,CuCN
i ^ J . ^ ^ ^ ^
c)BuUort^.U
u^J.^^^ OEt
b)\2
Fig. (30). In an iterative fashion, the hydroxyacetal (intermediately formed by condensation of vinyllithium salt with p-cyclocitral) was dehydrated with aqueous HBr. This allowed the simultaneous hydrolysis into pionylideneacetaldehyde, as a mixture of7E,9E (80%) and 7£,9Z (20%). The reaction had been repeated with the same C5 unit and finally, retinal could be obtained as a mixture of isomers, containing 68% of all E isomer (47%) yield from P-cyclocitral), Fig. (31).
>CcCHO
Fig. (31). Beaudet, Launay, Parrain, and Quintard, (1995); Launay, Beaudet, and Quintard (1997). Bienayme and Yezeguelian [63] described a new synthesis of retinal via a Heck vinylation of a C15 tertiary allylic alcohol with a C5 iodoacetal. Thus, the bromo acetal was prepared by a known procedure [64], by a bromination-dehydrobromination reaction sequence (E and Z isomers: 40/60). The iodo acetal could be easily obtained (as a mixture of E and Z isomers, 40/60), by a nickel catalyzed iodine-bromine exchange. This synthon reacted smoothly with the C15 tertiary allylic alcohol in the presence of a catalytic amount of palladium acetate and a stoechiometric amount of either a silver or a thallium salt. The C20 hydroxy-acetal was obtained in 38% yield, as a mixture of E and Z isomers (48/52). Finally retinal was obtained by treatment with dilute HBr in refluxing acetone, as a mixture of £" and Z isomers (C(9)=C(io) and C(i3)=C(i4)), Fig. (32).
I
GEt ""^^ 1 ^ 3
I
OEt
I ' c)lK,NiBrJn
^
OEt ^^^
I
OH
^Pd(OAc),
Fig. (32). Bienayme, and Yezeguelian (1994) In another study, Bienayme [65] obtained retinal in three steps from pionone, involving a Pd-catalyzed rearrangement of a mixed carbonate, derived from ethynyl-retro-ionol.
89
Thus, the P-ionone was smoothly deconjugated and ethynylated to give ethynyl-retro-ionol as a mixture of ElZ stereoisomers. Formation of the carbonate and its Pd-catalyzed rearrangement produced straightforward a mixture of aldehydes and a allene compound. After silica-gel chromatography, the allenic-aldehyde was conjugated with a catalytic amount of HBr in acetone. Retinal was obtained as a mixture of E and Z isomers (75/25), which could be converted into the all E isomer by simple equilibration. Fig. (33).
uC^^ /^^^N.x^^
T^
...^^
c) Pd(0Ac)2
^.
fl)MeONa,NMP
?$^ ^ " ^
^ QC
r^y^^^^'''''^^
—"
^^ ^E~MgCl
o 6)Pd(dba)3,P(napht)3
Y^
CHO
^HBr
CHO
Pd(dba)3, P(napht)3
Fig. (33). Bienayme (1994, 1995). A similar route was patented by Ancel and Meilland [66]. The ethynyl-retro-ionol was acetylated (Ac20-DMAP-Et3N) and this propargylic acetate was reacted with methyl butadiene acetate in the presence of BF3-Et20. The allenic-retinal, obtained in 61% yield was isomerised in retinal by HBr in acetone (yield: 50%), Fig. (34).
CHO
Fig. (34). Ancel, and Meilland (2000).
90
Salman et al [67] described a process for the preparation of 13Zretinoic acid (isotretinoin) in a single step from piony lideneacetaldehy de. Thus, isotretinoin was obtained by treating methyl-3,3dimethylacrylate with LDA, followed by addition of pionylideneacetaldehyde and further hydrolysis with 10% sulphuric acid. The pH had to be adjusted to 2.8 ±0.5, Fig. (35).
'
b)
MeO a) LDA
c)H2S04,pH=2.8
\ X \
COOH
Fig. (35). Salman, Kaul, Babu, and Kumar (2001). Recently Valla et al showed that new 'P-methylenealdehydes' synthons could be substituted to 7jE',9£'-ionylideneacetaldehydes (derived from a and P-ionones) in a Stobbe reaction [68,69]. Regioselective isomerization of these P-methylenaldehydes in Et2NH produce the compound {EIZ\ 97/3). These synthons were synthesized by formylation of ionones and concomitant acetalysation of the sodium salts of the hydroxymethylenic compounds. Wittig reaction and acidic hydrolysis of the p-methyleneacetals produced the pmethy lenealdehydes. Hence, Stobbe-like condensation with dimethyl-isopropylidene malonate and saponification of malonic acid, half-esters afforded the corresponding 14-carboxyretinoic acids, as a mixture of all E and 9Z isomers (80/20). The all E diacid was easily removed by crystallization from MeCN or ether, Fig. (36). A stereospecific decarboxylation in 2,6dimethylpyridine led to isotretinoin.
91
O >Q
a) MeONa, HCOOMe
OMe "OMe
c) ?h^?CH2
b) H2SO4, MeOH COOMe
OMe OMe
d) HCOOH
COOMe y)NaOH COOH
'
COOH
g) ether
COOH
h) 2,6.dimethyl pyridine
COOH
Fig. (36). Valla, Andriamialisoa, Prat, Giraud, Laurent, and Potier (1999); Giraud, Potier, Andriamialisoa, and Valla (1999). A related stereoselective synthesis of all E retinoic acid was also performed by Valla et al [70] from the 14-carboxyretinoic acid, derived from p-ionone, using pyridine (2 eq.) at room temperature for 20 hrs. The crude retinoic acid mixture {all E/13Z: 97/3) was crystallized in MeCN or AcOEt to provide pure all E retinoic acid. Fig. (37). COOH
COOH
a) pyridine ^
COOH
Fig. (37). Valla, Andriamialisoa, Prat, Laurent, Giraud, and Potier (2000). A new preparation of the Cig ketone, an important synthon for the synthesis of vitamin A had also been published by Valla et al [71]. Hence P-ionone and acetonitrile were condensed in the presence of KOH, to afford the nitrile (80%, ElZ isomers: 80/20). A Reformatsky reaction of ethyl bromoacetate with the nitrile provided the ethyl Pionylideneacetoacetate in 70% yield. Subsequent reduction with NaBH4, followed by esterification (MeS02Cl) and desulfonation of the unstable
92
ester, led to the acid {ElZ isomers, 80/20) in 80% yield. Reaction of the latter with MeLi afforded the Cig ketone in 70% yield, as a mixture of 9£/Z isomers (80/20), Fig. (38).
^V-'^^O
CN
ci) MeCN
b) Zn, BrCHsCOOEt
KOH
V^^^^^^/J^^^^
c) NaBH4
COOH
d) MeS02Cl-Me3N
Fig. (38). Andriamialisoa, Valla, Zenache, Giraud, and Potier (1993). In addition, these researchers described a series of 9- and 13methylene analogues. The synthesis of 9 and 13-methylene isomers of retinal has also been reported [72]. Hence, the above described Pmethylenealdehyde was condensed with the carbanion of diethyl 2oxopropylphosphonate, to give the methylene ketone in 51% yield. Condensation of the ketone with A^-ethylidenecyclohexylamine afforded the 9-methylene isomer of retinal, as a 13£/13Z mixture (80/20), Fig. (39).
CHO
a) (EtO)2POCH2COMe
^)MeCH=NC6Hii c) (C00H)2
Fig. (39). Laurent, Prat, Valla, Andriamialisoa, Giraud, Labia, and Potier (2000).
93
The synthesis of the 13-methylene isomer was performed from Pionylideneacetaldehyde {ElZ: 80/20). Condensation with acetone provided the conjugated ketone which, after formylation (MeONa/HCOOEt) and ketalisation (H2SO4/CH3OH), produced the pketoacetal {9EIZ\ 80/20). A Wittig reaction with methyltriphenyl phosphorane (/BuOK/PhaP^CHs, Br") followed by hydrolysis of the pmethyleneketal, produced the 13-methylene isomer of retinal, as a 9E and 9Z mixture (80/20), Fig. (40). O P>
a) MeCOMe
Ijl
MeONa O
^^^^V^^V
c) H2S04/MeOH
OMe OMe ^Ph3PCH2
e)HCOOH
b) HCOOMe/MeONa
I II OMe X^^^:^A,.^^%AAoMe
/K.^--:^:^^^^
pentane
Fig. (40). Laurent, Prat, Valla, Andriamialisoa, Giraud, Labia, and Potier (2000). In a comparable approach, Valla et al [73] described the synthesis of 9-methylene analogues of retinol, retinal, retinonitrile and retinoic acid, using the p-methylenealdehyde derived from p-ionone. Homer-Emmons condensation with ethyl 4-(diethoxyphosphoryl)-3-methylbut-2-enoate carbanion afforded the ester in 55% yield, as a mixture of 13£'/13Z isomers (50/50). This ethyl 9-methylene-retinoate was saponified with ethanolic NaOH to give the corresponding 9-methylene-retinoic acid in 55% yield (13£/13Z: 50/50). The retinol analogue was obtained by DIBAL-H reduction of the ethyl ester (75%, 13£/13Z isomers: 65/35). In the same way, the anion of ethyl 3-cyano-2-methylprop-2-enylphosphonate was reacted with the P-methylenealdehyde to give the 9methylene-retinonitrile in 50% yield, as a mixture of 13£'/13Z isomers (65/35). DIBAL-H reduction of the latter compound provided the related retinal (70%, 13£/13Z: 65/35). Alternatively, Mn02 oxidation of the
94
alcohol gave retinal in 75% yield, as a mixture of isomers (13E/13Z: 65/35), Fig. (41). b) NaOH
- " \ ^ - ^ ^ \ ^ ^ .--^-.^ COOH
jCOOEt ^ - " ^
^
O a) (EtO)2P>.
i.
|c)DIBAL-H
r^^^'^Y^''^^:-^^
y)Mn02
.COOEt
CHO
v^^^^^CHO O
^
(EtO)2P'xA^CN x ^ e) DIBAL-H CN
Fig. (41). Valla, Prat, Laurent, Andriamialisoa, Giraud, Labia, and Potier (2001). These French chemists described a synthesis of ethyl 9-methylene13£ and 13Z-retinoates via the Julia strategy [74]. The required new C15 sulfone was prepared by O-silylation of p-ionone, followed by catalytic condensation (ZnBr2) of the enol with PhSCH2Cl. A Peterson olefmation of the ketosulphide led to the methylenic sulphide. Oxidation (using bis(trimethylsilyl) peroxide [75]), gave the Ci5 9-methylenesulphone, without any detectable oxidation of the double bonds. Thus, condensation with ethyl 4-bromo-3-methyl-2-butenoate (2£/2Z: 50/50) provided the sulphone-ester, as a mixture of isomers (13£'/13Z: 50/50). Elimination to the ethyl 9-methylene-retinoate (2£/2Z: 50/50) was done by treating the crude mixture with EtONa in cyclohexane. Fig. (42).
95
OSiMe Q
b) PhSCHsCl, ZnBr2
a) LDA, Me3SiCl
O l^'^^^Y'^^
^) Me3SiCH2MgCl peroxyde
X^^^^^^A^ I
H
Br-
COOEt
SOoPh
r^^^Y^^^/^^^
COOEt
e) BuLi
Fig. (42). Valla, Laurent, Prat, Andriamialisoa, Cartier, Giraud, Labia, and Potier (2001). These researchers also described further syntheses of modified retinoids such as: 9-demethyl-14-carboxyretinoic acid [76], 9-methylene13-demethyl analogues of natural retinoids [77], aromatic 9-methylene and 13-demethyl-retinol, retinal, and ethyl 13-demethyl-9-methylene retinoate [78], Fig. (43). COOH COOH
R = CH20H;CH0; COOEt
Fig (43). Giraud, Andriamialisoa, Valla, Zennache, and Potier (1994); Valla, Prat, Laurent, Andriamialisoa, Cartier, Labia, and Potier (2001).
96
The Wittig reaction of lithium a-(dimethylamino)-alkoxydes and a Ci5 alkyltriphenylphosphonium salt was used by Wang et al to elaborate the ethylenic linkage of retinol [79]. This in situ method offers the unique advantage in its application to labile aldehydes, which otherwise would become isomerised or self-condensed, Fig. (44).
P"Ph3,Br- ^> /Bir^^'^0 /BuLi, /BuOK
Fig. (44). Wang, Wei, and Schlosser, (1999). Three analogous processes involved the reaction of the C15 phosphonium salt with the 5-hydroxy-4-methyl-2(5/i/)-furanone, in the presence of a base, as described below. To generate the phosphorane, Magnone [80,81], Wang et al [82] and John and Paust [83] used respectively sodium methoxide, triethylamine/MgCb in A^,A^-dimethylacetamide and LiOH in A^,A^dimethylformamide. For the isomerization step, the two first authors emploied rose Bengal as photosensitizer and the latter Erythrosine B, to give isotretinoin. Fig. (45). a)
BrMg"^
b) PhsP, HCl, EtOH OH
*^
c) NaOMe or Et3N, MgClj, AcNMe2 or LiOH, DMF
e) KOH, rose Bengal or Erythrosine B
Fig. (45). Magnone (1996,1999); Wang, Bhatia, Hossain, and Towne (1999); John, and Paust (1994).
97
White et al. developed a stereospecific synthesis of Z-olefins, including isotretinoin [84]. Thus, isotretinoin was obtained by a Reformatsky reaction of p-cyclocitral with the C5 bromoester, followed by DIBAL-H lactone reduction, lactol ring opening, selective olefin bond formation with ethyl 4-diethoxyphosphoryl-3-methyl-2-butenoate and further saponification, Fig. (46). OH
0
\
/
II
t^
\ /
\ >
>C^"« .)EtO^^^^
Uk
'A
1
zii
>
^
b) DIBAL-H
^
" L0
°
d) KOH, EtOH, H2O
JC"
I
•rS r^
r^^
COOH
Fig. (46). White, Hwang, and Winn (1996). Tanaka et al reported a synthesis of vitamin A derivatives from C15 phosphonates [85]. Vitamin A acetate was prepared in 92% yield by reaction of the C15 phosphonate with 2-methyl-4-acetoxy-2-butenal, Fig. (47).
a) /BuONa, DMF, PhMe
Fig. (47). Tanaka, Hanakoa, and Takanohashi (1994). Babler and Schlidt [86] described a route to a versatile C15 phosphonate, used for a stereoselective synthesis of all E retinoic acid and p-carotene. Base-catalyzed isomerization of the vinyl-phosphonate afforded the corresponding allyl-phosphonate as the sole product. Horner-Emmons olefination with ethyl 3-methyl-4-oxo-2-butenoate concluded the facile synthesis of all E ethyl retinoate. The C15 phosphonate was synthesized starting from the epoxide of p-ionone. Subsequent isomerization with MgBr2, afforded the C14 aldehyde in 93%
98
from p-ionone. A modified Homer-Emmons olefmation with tetraethyl methylenediphosphonate led to the vinyl phosphonate in 93% yield. Isomerization to the allylic phosphonate was perfomied with /BuOK. The synthesis of ethyl retinoate was carried out via Homer-Emmons olefination with ethyl 3-methyl-4-oxo-2£-butenoate (61%), Fig. (48).
^v
>Q fl)Me2S=CH2
c)CH2(P(OEt)2)2
Z>)MgBr2
/ ^ ^ ^ ^ C H O
r^V^V-^^^-<^P(0Et)2 ^
Q) tBuOK
Fig. (48). Babler, and Schlidt (1992). Ancel et al patented a procedure for the preparation of vitamin A from p-ionol and C5 synthons [87]. For example, condensation of 4chloro-1,1 -dimethoxy-3 -methy l-2£'-butene with benzenethiol and distillation of the crude acetal gave 80% of the sulphide enolether which, condensed with vinyl-p-ionol to phenylthioretinal (50%). Retinal was obtained in 92% yield from the corresponding sulfoxide (obtained by MCPBA oxidation) and heating in CCI4. In a similar fashion, the retinyl acetate (76%) could be synthezised: quantitative borohydride reduction of phenylthioretinal to the alcohol, followed by its acetylation gave the acetate, which was oxidized to the sulfoxide by MCPBA and then eliminated by heating in CCI4, Fig. (49).
99
.OH
^^
^OMe
SPh^ a) BF3-Et20
d) NaBH4. e)Ac20, Et^N
^^Y^""^^
CHO
J) MCPBA CHO L^^^A^
SOPh
g)CCl4 A
OAc
Fig. (49). Ancel, Bienayme, Duhamel, and Duhamel (1993).
C(i2)-C(i3) strategies: Wada et al described a new strategy for the stereoselective syntheses of llZ-retinal by palladium-catalyzed cross coupling of a Ci6 tetraenyl stannate with a C4 £" or Z-vinyl triflate [88]. P-Ionylideneacetaldehyde was dibromomethylenylated at 0°C with CBr4 and triphenylphosphine. Stereoselective hydrogenolysis of the dibromo compound with tributyltin hydride in the presence of a catalytic amount of Pd(PPh3)4 proceeded cleanly to the C16 bromotetraene in 86% yield. The synthesis of llZ-retinal required the boronic-partner, which was prepared from 2-butyn-l-ol by addition of the tributylstannyl cuprate (83%), followed by protection of the alcohol with /BuMeiSiCl (TBDMSCl) (93%). The tributylstannyl group was substituted with boronic acid in three steps: lithiation, quenching alkenyllithium with triisopropyl boronate and hydrolysis to the boronic acid. The Suzuki coupling of the C16 tetraene with the boronic compound was carried out in THF at room temperature, in the presence of a catalytic amount of
100
Pd(PPh3)4, Ag2C03, and an aqueous solution of KOH (77%). Removal of the silyl ether with tetrabutylammonium fluoride (TBAF) and successive oxidation with BaMn04 provided the 1 IZ-retinal in 85% yield, Fig. (50). ^ ^ ^ a)CBr4,PPh3
/ K ^ ^ ^
L^Jls,
c)Pd(PPh3)4,KOH,Ag2C03
Br
B(0H)2 OTBDMS OTBDMS
f) BU4NF, BaMn04
e) TBDMSCl, KH BuLi, B(0/Pr)3, aq. HCl
d) (Bu3Sn)2CuCNLi2
SnBu CHO OH
Fig. (50). Uenishi, Kawahama, Yonemitsu, Wada, and Ito, (1998). A similar strategy had been developed for the preparation of all E, 9Z and 13Z-retinoic acids [89]. P-Ionylideneacetaldehydes {E or Z) were converted to the corresponding alkynes (ICHiF'^Phs, Y and NaN(TMS)2) in 70% yield. Stannylcupration of the alkynes with butyltributylstannylcyanocuprate (BusSnCuBuCNLii) afforded the tetraenylstannates, which were coupled with the vinyl triflates, using tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) and triphenylarsine (AsPPhs) as a ligand. Thus, 9£-stannate and ^-triflate furnished ethyl all E'-retinoate in 60% yield. Under the same conditions, the coupling reactions of 9£'-stannate and 9Z-triflate and £'-triflate led to ethyl 13Zretinoate and 9Z-retinoates in 43 and 39% yields, respectively. The coupling of E tetraenyl-stannate with Z-triflate and Z-tetraenyl-stannate with £-triflate afforded ethyl 13Z- and 9Z-retinoates in 43 and 39% yields, respectively. Fig. (51).
101
^ " O a) ICHzPhj?""!- | - ' ' ^ [ t ^ ' " ^ = > ' - " " W ^
b) BujSnCuBuCNLij
NaN(TMS)2
COOEt
j^^K-^^V-^^
nBu, c)Pd2(dba)3,AsPh3 COOEt
EoY Z
Zstannate
COOEt
Fig. (51). Wada, Fukunaga, and Ito, (2001). Conclusions The retinoid activities are influenced by the complex interactions of their nuclear receptors that mediate their effects. Free vitamin A, its esters and the retinoic acids, ATRA, 9Z-RA and 13Z-RA, currently are the most widely tested in clinical studies. These naturally occurring retinoids tend to be in vivo pan-activators of receptors (non-receptor specific). Since these retinoic acids are readily interconverted in vivo, each can activate a wide spectrum of retinoid receptors, signalling pathways, and biological effects. The receptors are involved in regulating transcription of specific genes. They regulate cell differentiation, proliferation, loss... The relationship between variations in receptor expression by organ site and responsiveness to chemo preventive agents is currently under study. These established facts made that retinoid chemistry of today, more than ever, a challenge for chemists. This account, relative to retinoid chemistry, was in ad equation and corroborated the older methodologies of retinoid syntheses, which favored the C(ii)-C(i2) way. Significantly, since 1990, more than forty references have linked P-ionylideneacetaldehyde as an intermediate in the syntheses of retinoids (ref: CAS, Sci. Finder). This choice could be
102
attributed in parts to the fact, that this C15 aldehyde could be synthesized in many ways from p-ionone, a relatively Cn inexpensive product.
ADCDP BASF AG CRBPII dba DBN DBU DIBAL-H DMAP DMF DMF-DMA DMPU HMDS HMPA HMPT H-LR LDA LDE LRAT MCPBA MOM NMO NMP PCC PhH phenyltriflimide PhSH PTSA PPTS RA RARs R-P RBP
= 1, r (azodicarbony l)-dipiperidine = Badische Anilin-& Soda Fabrik AG = cellular retinol-binding protein type II = dibenzylideneacetone = l,5-diazabicyclo[4.3.0]non-5-ene = 1,8-diazabicyclo[5.4.0]undec-7-ene = diisobutylaluminium hydride = 4-dimethylaminopyridine = A^,A^-dimethylformamide = A^//-dimethylformamide, dimethylacetal = 1,3-dimethyl-3,4,5,6-tetrahydro-2(l^-pyrimidone = hexamethyldisilazane = hexamethylphosphoramide = hexamethylphosphorous triamide = Hoffmann-La Roche = lithium diisopropylamide = lithium diethylamide = lecithin:retinol acyltransferase = /w-chloroperbenzoic acid == methoxymethyl = A^-methylmorpholine oxide = 1 -methyl-2-pyrrolidinone = pyridinium chlorochromate = benzene = A^-phenylbis(trifluoromethanesulphonimide) = benzenethiol = para toluene sulfonic acid = pyridinium paratoluenesulfonate = retinoic acid = retinoic acid receptors = Rhone-Poulenc = retinol-binding protein
103
RXRs TBAF THF TMS Tf TBDPSCl
= = = = = =
retinoid X receptors tetrabutylammonium fluoride tetrahydrofurane trimethylsilyl trifluoromethanesulfonyl (triflyl) /butyldimethylchlorosilane
DEDICATE This review is dedicated to Professor Pierre Potier, Member of the Sciences Academy of France, for his 68th birthday. ACKNOWLEDGEMENTS The authors gratefully acknowledge Pr B. Corbel (UBO Brest) for helpful discussions. Also, we are indebted to Pr J.-J. Le Yeuc'h (UBO Quimper) and Dr N. Boeker for assistance in the revision of this manuscript.
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106
[49] [50] [51] [52] [53]
[52] [55] [56] [57]
[58] [59] [60] [61] [62] [63]
[64] [65] [66] [67] [68] [69] [70] [71]
Thibonnet, J.; Launay, V.; Abartri, M.; Duchene, A. and Parrain, J.-L. Tetrahedron Lett 1998, 39, 4277-4280. Abarbri, M.; Parrain, J.-L. and Duchene, A. Tetrahedron Lett. 1995, 36, 24692472. Negishi, E. and Owczarczyk, Z. Tetrahedron Lett. 1991, 32, 6683-6386. Torrado, A.; Iglesias, B.; Lopez, S and De Lera, A. R. Tetrahedron 1995, 51, 2435-2454. Roush, W. R.; Brown, B. B. and Drozda, S. E. Tetrahedron Lett. 1988, 29, 35413544. Roush, W. R.; Moriarty, K. J. and Brown, B. B. Tetrahedron Lett. 1990, 31, 6509-6512. Roush, W. R. and Brown, B. B. J. Am. Chem. Soa 1993, 115, 2268-2278. Aurell, M. J.; Parra, M.; Tortajada, A.; Gil, S. and Mestres, R. Tetrahedron Lett. 1990,57,5791-5794. Duhamel, L.; Guillemont, J. and Poirier, J.-M. Tetrahedron Lett. 1991, 32, 44994500. Cahard, D.; Duhamel, L.; Lecomte, S. and Poirier, J.-M. Synlett 1998, 13991401. Cahard, D.; Mammeri, M.; Poirier, J.-M. and Duhamel, L. Tetrahedron Lett. 2000, 41, 3619-3622. Cahard, D.; Duhamel, L.; Lecomte, S. and Poirier, J. M. Synlett 199S, 1399-1401. Cainelli, G., Cardillo, G.; Contendo, M.; Trapani, G. and Ronchi, U. J. Chem. Sac, Perkin Trans. 1 1973, 400-404. Ancel, J.-E.; Bienayme, H.; Duhamel, L. and Duhamel, P. Eur. Pat. EP 92403167.7 25Nov. 1992 - Rhone-Poulenc Nutrition Animale. Duhamel, L. and Ancel, J.-E. Tetrahedron 1992, 48, 9237-9250. Duhamel, L.; Duhamel, P. and Ancel, J.-E. Tetrahedron Lett. 1994, 35, 12091210. a S. Pat. Eastman Kodak 1961,3, 013, 080; Pat. AEC 1, 288, 972, C07c. Beaudet, I.; Launay, V.; Parrain, J.-L. and Quintard, J.-P. Tetrahedron Lett. 1995, 36, 389-392 - Launay, V.; Beaudet, V. and Quintard, J.-P. Bull. Soc. Chim. 1997, 134, 937-946. Bienayme H. and Yezeguelian, C. Tetrahedron 1994, 50, 3389-3396. Picotin, G. and Miginiac, P. Chem. Ber. 1986,119, 1725-1730. Bienayme, H. Tetrahedron Lett. 1994, 35, 7383-86. Bienayme, H. Bull. Soc. Chim. Fr. 1995,132, 696-708. Ancel, J.-E. and Meilland, P. PCT 00 02854 20 Jan. 2000 - Rhone-Poulenc Agro. Salman, M.; Kaul, V. K.; Babu, J. S. and Kumar, N. PCT Int. Appl 009089 08 October 2001 - Ranxaby Labs, Ltd, India. Valla, A.; Andriamialisoa, Z.; Prat, V.; Laurent, A.; Giraud, M. and Potier, P. Tetrahedron Lett. 1999, 40, 9235-9237. Giraud, M.; Potier, P.; Andriamialisoa, Z.and Valla, A. U.S Pat. 5925797 20 July 1999. Valla, A.; Andriamialisoa, Z.; Prat, V.; Laurent, A.; Giraud, M. and Potier, P. Tetrahedron 2000, 56, 7211-7215.
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[72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90]
Andriamialisoa, Z.; Valla, A.; Zenache, S.; Giraud, M. and Potier, P. Tetrahedron Lett 1993, 34, 8091-8092. Laurent, A.; Prat, V.; Valla, A.; Andriamialisoa, Z.; Giraud, M.; Labia, R. and Potier, P. Tetrahedron Lett. 2000, 41, 7221-7224. Valla, A.; Prat, V.; Laurent, A.; Andriamialisoa, Z.; Giraud, M.; Labia, R. and Potier, P. Eur. J. Org. Chem. 2001, 1731-1734. Valla, A.; Laurent, A.; Prat, V.; Andriamialisoa, Z.; Cartier, D.; Giraud, M.; Labia, R. and Potier P. Tetrahedron Lett. 2001, 42, 4795-4797. Kocienski, P. and Todd, M. J. Chem. Soc. 1982, 1078-1079. Giraud, M.; Andriamialisoa, Z.; Valla, A.; Zennache, S. and Potier P. Tetrahedron Lett. 1994, 35, 3077-3080. Valla, A.; Prat, V.; Laurent, A.; Andriamialisoa, Z.; Cartier, D.; Labia, R. and Potier P. Synth. Commun. 2001, 57, 3423-3427. Valla, A.; Prat, V.; Laurent, A.; Andriamialisoa, Z.; Cartier, D.; Giraud, M.; Labia, R. and Potier P. Helv. Chim. Acta 2001, 84, 3423-3427. Wang, Q.; Wei, H.-X. and Schlosser, M. Eur. J. Org. Chem. 1999, 3263-3268. Magnone, G. A. Eur. Pat. Appl. EP 959069 11 Nov 1999 Laboratori Mag S.P.A., Italy. Magnone, G. A. Eur. Pat. Appl. EP 742204 13 Nov. 1996 Laboratori Mag S.P.A., Italy. Wang, X. C ; Bhatia, A. V.; Hossain, A. and Towne, T. B. PCT Int. Appl 9948866 30 Sep. 1999 Abbot Laboratories, USA. John, M. and Paust, J. Ger. Offen. DE 4313089 27 Oct. 1994 BASF A.-G., Germany. White, S. K.; Hwang, C. K. and Winn, D. T. US Pat. US 5567855 22 Oct. 1996 Ligand Pharmaceuticals Inc, USA. Tanaka, M.; Hanakoa, T. and Takanohashi, K. Jpn. Kokai Tokkyo Koho JP 06329623 29 Nov. 1994 Takeda Chemical Industries, Ltd, Japan. Babler, J. H. and Schlidt, S.A. Tetrahedron Lett. 1992, 33, 7697-7700. Ancel, J. E.; Bienayme, H.; Duhamel, L. and Duhamel, P. Eur. Pat. Appl. EP 544588 02 June 1993 Rhone-Poulenc Nutrition Animale. Uenishi, J.; Kawahama, R.; Yonemitsu, O.; Wada, A. and Ito, M. Angew. Chem. Int. Ed 1998, 37, 320-323. Wada, A.; Fukunaga, K. and Ito, M. Synlett 2001, 800-802.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
109
BIOACTIVE TETRAMIC ACID METABOLITES EMILIO L. GHISALBERTI Department of Chemistry^ University of Western Australia, Nedlands 6009 WA. Australia ABSTRACT: Secondary metabolites containing the tetramic acid (2,4-pyiTolidine-2,4dione) ring system have been known for almost half a century, and well before the parent system was synthesised. Although the first naturally occurring tetramic acids were identified because of their activity as antibiotics and/or mycotoxins, more recently tetramic acid-containing compounds have been found to display a remarkable diversity of biological activities. The often unusual and intricate substituents modifying the tetramic acid structural unit make the synthesis of these metabolites a challenging target. Recent studies has confirmed that these metabolites have a wide distribution and play a significant role in ecological interactions. They have been isolated from marine mollusks, sponges and cyanobacteria, terrestrial and marine microorganisms, particularly endophytic fungi. In an attempt to bring all these strands together, this review will consider the structure, chemistry, biosynthesis, bioactivity, distribution and ecology of this diverse group of metabolites.
INTRODUCTION The term ^tetramic acid_ was coined in 1901 to refer to the heterocycUc system l,5-dihydro-4-hydroxy-2H-pyrrol-2-one (1), a tautomer of 2,4pyrrolidinedione (2) which is the predominant species in solution [1]. Although tetramic acid was apparently synthesised only in 1972 [2], a number of natural substances had been identified as derivatives of tetramic acids well before that time. Secondary metabolites containing the tetramic acid ring constitute an increasingly important class of bioactive natural products. Compounds containing this structural unit, sometimes intriguingly camouflaged, exhibit a diverse range of biological activities and have attracted the interest not only of natural products chemists, but also of chemical ecologists, medicinal and synthetic chemists. The tetramic acid moiety, in most cases, is present as a 3-acyl derivative with the acyl group varying from the simple acetyl group to structurally complex entities incorporating several stereogenic centres. The increasing activity in this area, stimulated by an increasing number of bioassays and a larger resource pool, makes periodic reviews of the field useful.
no
It is somewhat surprising to find that there have been only three published reviews on tetramic acids. The first reports on the advances in tetramic acid chemistry up to 1993 [1], another describes the synthesis of tetramic acid antibiotics [3], and a third, dealing with the structure, isolation and synthesis of naturally occurring tetramic acids, was published in 1995 [4]. The present review is an attempt to cover the field of tetramic acid metabolites with particular emphasis on the structure, biosynthesis and biological activity of these compounds.
For the purpose of this review, it has been necessary to circumscribe the type of tetramic acid metabolites to be considered. Thus, the large group of cytochalasins, chaetoglobosins and penchalasins, e.g. 3, will not be included here as they have been covered in detail elsewhere [5-7], For these metabolites, a tetramic acid species has been postulated as the putative precursor 4 that undergoes a Diels-Alder reaction [8-10].
From a biosynthetic perspective, naturally occurring tetramic acids can be regarded to arise from the assembly of an amino acid and an activated acyl entity derived from an acetyl group or a more complex activated ester. Fig. (1). Alternatively, the simple tetramic acid formed can undergo substitution at C-3 with a second acyl group. On this basis, compounds such as lactacystin (5) have not been considered [11]. The carboxylic acid
Ill
group of the a-amino acid (from L-leucine) is not involved in the heterocyclic ring in (5), Fig. (2).
Fig, (1). Biosynthetic pathways to 3-acyltetramic acids
STRUCTURE AND SPECTRAL PROPERTIES Aspects of the structure and properties of tetramic acids have been presented in detail [1,3] and only a summary will be given here. Tetramic acid is a much weaker acid (pK^ 6.4 in water) than its oxygen counterpart and exists mainly in the 2,4-diketone form. The presence of an acyl group at C3 results in an increase in acidity (pK^ 3.0-3.5) and proton NMR spectra indicate complete enolisation and the presence of tautomeric forms. As shown in Fig. (3), these can be separated into two sets of _iitemaL tautomers (A/B, C/D), which are rapidly interconverting, and two pairs of __externaL isomers that interconvert slowly on the NMR time scale and often give rise to separate NMR signals [1,3]- ^^C-NMR spectroscopy is more useful in determining the predominant tautomeric species and it indicates that the one represented by D is more important. On the other hand, if the nitrogen is acylated, the preferred form is A since the lone pair on nitrogen does not enhance the proton acceptor ability of the lactam carbonyl at C2 [3,1]. Selected examples of differently substituted tetramic acids and their spectral parameters are given in Fig. (4).
112
Of some interest is the ability of 3-acyltetramic acid to form stable complexes with ions of transition metals, e.g. Cu^^, La^"", Sm^"^, Eu?'^, Gd^^, and boron complexes [1]. Indeed, examples are available in which the tetramic acid occurs naturally as magnesium or calcium salts. Methods of isolation or purification involving acidic solvents generate conditions that
Fig, (2). Biosynthetic assembly of lactacystin (5)
SLOW
^N^^a R
FAST
C
Fig. (3). Tautomeric forms for 3-acyltetramic acids
113
are capable of converting salts to the conjugate acid [13-15]. In one case, it was found that the free acid derived from the magnesium salt was unstable and prone to oxidation [16]. For tetramic acids embedded in a macrocyclic lactam ring, it was found that all prior isolations of these type of compounds involved acidification during extraction or chromatography [16]. On the other hand, it is also possible for the tetramic acid to sequester metal cations as observed for the case of corresponding tetronic acid that formed the calcium salt on chromatography with silica gel 60 [17].
^^C-NMR (CD^CN) IR (film) 1726.1657-1617 cm"''(several bands) UV (CH3CN) 238. 286 nm
f^3 HaC^
26.3
TAUTOMER
H,C^-'' 27.5
178.0
172.8
i^C-NMR (CDCI3) IR(KBr), 1713,1620 cm-'' UV (CH3OH) 245, 285 nm
^^C-NMR (CD^CN) IR(ZnSe), 1690,1664 cm-'' UV (CH3OH) 235, 290 nm
Fig. (4). ^^C-NMR, IR and UV spectral parameters of typical tetramic acids
114
ACYLTETRAMIC ACIDS In this section, tetramic acids with an acyl group substituent at C-3 are discussed. The simplest of the naturally occurring 3-acyl tetramic acids, tenuazonic acid (6), was first isolated from the culture filtrate of Alternaria tenuis [18] and, subsequently, from other fungal species (A. alternata, A. longipes, Pyricularia oryzae) [19,20]. Species of Altemaria are known to produce more than 70 secondary metabolites, many of which, particularly those from the Altemaria altemata complex, are mycotoxins [19]. The absolute stereochemistry of 6 (55,65) was deduced from the formation of L-isoleucine on ozonolysis followed by acid hydrolysis [21]. Tenuazonic acid has also been isolated as a mixture of calcium and magnesium complexes from Phoma sorghina, the fungus implicated in the aetiology of onyalai, a haematologic disorder affecting Black African populations south of the Sahara [22]. Tenuazonic acid (TA) readily forms complexes with magnesium" to yield Mg"TA2, and with transition metals (Cu" TAj, Ni"TA2 ^^^ Fe"'TA3) [23]. An X-ray crystallographic study of the copper-fc/5-tenuazonate monohydrate, Cu(TA)2.H20, showed that the chelate is formed between the enolic oxygen of the acyl group and the amide oxygen [24]. With this in mind, and considering that the isolation of tenuazonic acid from the liquid cultures of A. tenuis required acidification, it is highly probable that these covalent complexes are the __naturaL form of the metabolite [22].
The biosynthesis of tenuazonic acid was studied using [l-^'^C]-labeled acetate. N-acetoacetyl-L-isoleucine (7) was detected by radioactive trapping, indicating that amide formation, rather than carbon-carbon bond formation is probably the first step. None of the simple tetramic acid (8)
115
was detected [16,25]. It was also found that the analogues of tenuazonic acid (9-11) were obtained when the medium was supplemented with Lvaline [27] L-leucine [27] or L-norvaline [20].
T °
9 R = i-Pr 10R = i-Bu
11 R = n-Pr
Tenuazonic acid has a broad toxicity spectrum and is regarded as a mycotoxin [28]. It was first detected as a growth inhibitor of tumour cells (human adenocarcinoma), and was later shown to have weak antibacterial and, at high dose levels (100-500 fig/ml), antiviral activity towards poliovirus MEF-1, enterovirus (ECHO-9), respiratory viruses (parainfluenza3), vaccinia and Herpes simplex (HF). It has been shown to be an inhibitor of peptide bond formation in preventing substrate binding to acceptor site of peptidyltransferase in human ribosomes [29]. Tenuazonic acid is also produced by the rice fungal pathogen Pyricularia oryzae and exhibits a conspicuous stunting effect on rice seedlings. In plant cells, it also inhibits growth by interfering with protein synthesis at the ribosome level [30]. Tenuazonic acid induces a defence reaction (browning) on the leaves of varieties of rice resistant to P. oryzae. When sprayed on rice seedlings at 1000 ppm, it inhibits infection by 98.5-99.7% after 7 days from application [31]. Alternaria alternata isolated from field-grown Beta vulgaris produced 150 mg/L of the acid in the sodium form. Tenuazonic acid was toxic to 5. vulgaris in the 10"^ M range [15]. The first recorded example of a naturally occurring tetramic acid magnesium salt was isolated from a new Pseudomonas species, P. magnesiorubra, obtained from the surface washings of the marine green alga Caulerpa peltata [33]. Magnesidin is an inseparable mixture (--1:1) of the magnesium chelates of the 3-hexanoyl (12) and 3-octanoyl (13) tetramic acid derivatives; the exact proportions of the mixture vary according to methods of cultivation and isolation. They were rediscovered 20 years later as the antibiotic principles from the gram-negative bacterium Vibrio gazogenes isolated from marine mud [34]. Magnesidin inhibits various
116
gram-positive bacteria (MIC 2-7 |Lig/ml), particularly spore bearers, and prevents the decay of foodstuff caused by spore germinating organisms. The toxicity of magnesidin in mice were LD50 50 (ip) and 1000 mg/kg (orally and s.c). Replacing magnesium by other metal cations, Li^, Na^, K"", Cu^^ and H^, resulted only in reducing the activity towards Bacillus subtilis (MIC 2.5-30 ^ig/ml) [35,36]. Mg',2+
12 n = 3 13 n = 5 J 2
Ras oncogenes play an important part in cell growth and differentiation. Agents that reverse the transformed phenotype caused by ras to normal may provide selective anti-cancer drugs. In a search for such compounds, two tetramic acids (14, 15) were isolated from the marine sponge Melophlus sarassinorum collected at Spermonde Islands in Indonesia [37]. The gross structures were deduced from spectral data, although the NMR spectra were complicated by the fact that the compounds exist as tautomers, exo and endo, in the ratio -9:1.
HdC(CH2)ir
A
^N—CH3
H3C(CH2W ^ . ^ > ^ A ^ ^ ^^^ "Y N—CH3
14
15 Hcp(CH2)l3
N—CH3
16 AcO
117
This complication could be overcome by acetylation which provided the C-4-O-acetyl derivative, e.g.l6. For melophlin B (15), the configuration at C-5 was determined by classical methods. Thus, treatment with NaI04 and KMn04, followed by hydrolysis with HCl, gave N-methyl alanine which was derivatised with N-a-(2,4-dinitro-5-fluorophenyl)-Lleucinamide. HPLC analysis of the derivative showed it to correspond to that prepared from L-alanine. The melophlins exhibited moderate cytotoxic activity against HL60 cells at 0.2 and 0.4 ^ig/ml respectively, and reversed the morphology of H-ra^ transformed NIH3T3 fibroblasts at 5 |LXg/ml. At a concentration of 1 |Lig/ml, they arrested the NIH3T3 cells in the Gl phase of the cell cycle, an indication that they act on the rasmediated signal transduction pathway. The P-triketone (17) was isolated from the fermentation broth of an isolate of the bacterium Apiosordaria ejfusa. The structure of 17 was determined from NMR spectral studies [38]. If the broth was acid treated prior to extraction, a mixture of the two isomers, a- and p-apiodionen (18, 19) were isolated and were found to be in equilibrium in solution [39]. The absolute stereochemistry of these compounds has not been assigned. Apiodionen exhibited weak activity as inhibitor of topoisomerase I and II [40].
A similar compound, bripiodionen (20), has been isolated from a Streptomyces sp [41]. NMR spectral data determined for a fresh sample of the metabolite indicated predominantly a single isomer, probably that with 5(7)£'-configuration. If a MeOH or DMSO solution was left for a few days, a 1:1 mixture of two isomers (20, 21) became evident, as found
118
for apiodionen. Bripiodionen was discovered in a screen for identifying inhibitors of human cytomegalovirus protease, a P-herpes DNA virus. This virus is an opportunistic pathogen in immunocompromised or immunosuppressed individuals. Bripiodionen displayed inhibitory activity against the protease with IC50 30 |LIM, and also showed low cytotoxicity (IC50 34 |LiM) on the murine lung carcinoma-derived cell line.
Cultures of Streptomyces rimosus var paromomycinus characteristically develop UV absorption at 240 and 278 nm due to formation of malonomicin (22), a compound that shows antiprotozoal activity towards Trypanosoma congolense, the causative agent of sleping sickness in cattle [42]. Malonomicin contains an unique aminomalonic acid unit that, on brief heating in water, undergoes decarboxylation and results in a compound devoid of biological activity [43]. Hydrolysis of the compound yielded L-serine and racemic aspartic acid. The structure was elucidated by chemical and spectroscopic methods [43,44] and was confirmed by total synthesis [45]. OH
OH
HN ^ ^ HoN
°°^" 23
Aspects of the biosynthesis have been resolved. The amino acid (see 23) involved in the heterocyclic ring arises from L-2,3-diaminopropionic
119
acid [46]. Evidence for the involvement of 3-oxoadipic acid to generate firstly the amide and then the heterocyclicringhas been obtained [47,48]. Cyclopiazonic acid (24) is pehaps the most studied of the tetramic acid group of compounds, a consequence of its intriguing structure, range of biological activity and its environmental importance. Originally isolated from Penicillium cyclopium, it was later found as a metabolite from several species of Penicillium and Aspergillus that infect a number of agricultural commodities such as groundnuts, com, cheese, meat [49]. The structure was established by chemical degradation and from NMR spectroscopy [50], and was confirmed by X-ray crystallographic studies, which also established the absolute stereochemistry [51]. The bissecodehydrocyclopiazonic acid (25) and the imino derivative (26) were also isolated [52]. The imino derivative (26) accumulates later in the fermentation and may be an artefact, whereas (25) accumulates in the early stages of fermentation, but decreases as cyclopiazonic acid is formed. The biosynthetic pathway to (24) has been shown to involve tryptophan, mevalonic acid and acetate as precursors of P-cyclopiazonic acid (25) which undergoes ring closure to (24). O
OH
24 R = OH 26 R = NH2
Cyclopiazonic acid is a potent inhibitor of calcium uptake and acts as a selective inhibitor of the sarco-endoplasmic reticulum Ca^'^ATPases (SERCAs) [53], it induces charge alteration in plasma membranes and mitochondria and it can function as an antioxidant [54]. A number of tetramic acid derivatives acylated with a liposaccharide unit have recently been discovered in marine sponges. The first example was uncovered in the sponge Ancorina sp using a bioassay to detect inhibition of blastulation in starfish embryos. The structure of the compound (27), named ancorinoside A, was determined by spectroscopic techniques
120
[55]. Interpretation of the NMR parameters was difficult since, in solution, this compound exists as a mixture of 4 tautomers (see Fig. (3)). A full analysis of the NMR parameters required spectra for pyridine and methanol solutions to be analysed. The absolute configuration of the p-glucose and |3-galactouronic acid groups was confirmed as D by GC analysis of the trimethylsilyl ethers of l(L-a-methylbenzyl-amino)-l-deoxyalditols derived from the watersoluble fraction from acid hydrolysis of (27). The stereochemistry at C-5 was secured by the usual method (hydrolysis and Lemieux degradation of
OH ^ CO2H
27 Ancorinoside A
o^^
28 Ancorinoside A Mg salt
HO\.--V^|OH
C02® Mg'*
CO2H
R
o^N.
29 R = H Ancorinoside B 30R = CH3 Ancorinoside C CO2H
31 Ancorinoside D
the chloroform-soluble fraction). This afforded N-methyl-D-aspartic acid which translates to a 5/?-configuration. Moreover, the CD spectrum of (27) showed positive Cotton effects at 285 (Ae +0.79) and 240 nm (Ae +0.76), opposite to those observed for tenuazonic acid (6) and equisetin (see below) which have the 55-configuration. Interestingly, the corresponding water-soluble magnesium salt (28) was isolated from the same sponge [56]. When fertilized starfish eggs were cultured from fertilization in the presence of 27, or the magnesium salt (0.4 |ig/ml), the development proceeded normally to 256-512 cell stage at which point, the embryos ceased to develop further without any sign of blastulation [55,56].
121
A suite of similar compounds, ancorinosides B-D (29-31), was subsequently found in another marine sponge from Japan, Penares sollasi, together with ancorinoside A [57]. These compounds, which were obtained in 0.004, 0.003, 0.001 and 0.0006% of the wet weight of the sponge, showed some activity as inhibitors of recombinant MTl-MMP. The matrix metallo-proteinases (MMPs) belong to the proteases which contain a catalytic zinc-binding domain. They are linked to a range of physiological and pathological processes including wound healing, angiogenesis, inflammation, tumor progression and metastasis. Interestingly, the ancorinosides are much less active than tenuazonic acid in which the complex architecture is reduced to the basic tetramic acid group. Ph.
"J^^^^Hs
^fr »/wvvrvAA/v»
33
HsC^
32R, =CH20H;R2 = H 3 4 R i = H ; R 2 = CH20H
Equisetin (32) was first isolated in 1974 as a metabolite of the white mould Fusarium equiseti [58,59]. It was found to exhibit a range of bioactivities, including antibiotic and HIV inhibitory activity, cytotoxicity and mammalian DNA binding [59-61]. Equisetin was reisolated in 1989 [60] following the implication of this fungus as a promoter of chronic environmental diseases, including leukemia [62]. The elucidation of the structure was complicated by the presence of an equilibrium mixture of tautomers which precluded detailed NMR analysis. This was reduced to a single tautomer by formation of the phenylboronate (33), the spectral properties of which facilitated the assignment of structure. NOE experiments and molecular mechanics calculations provided the relative sterochemistry of the decalin system. The absolute configuration of the serinederived tetramic domain was established as 5 by application of CD methods and comparison with the CD spectrum obtained for tenuazonic acid
122
(6). The assigned absolute configuration is supported by two elegant syntheses [63,64]. Fusarium equiseti and F. pallidoroseum are frequently reported as secondary colonisers of mono- and dicotyledons and cause disease on many crop plants such as snapbean, tomato, banana, cantaloup and muskmelon [65]. A numberof isolates of the two species were observed to produce equisetin (32) and epZ-equisetin (34). The two are related via epimerisation at C-5 of the tetramic unit, a reaction that in vitro can be observed under pyridine catalysis and leads to an equimolar mixture of the epimers. Both were found to suppress germination and inhibit the growth of seeds at concentrations of 2.5-10 |ig/ml. In addition, they adversely affect growth of young sedlings, cause necrotic lesions on the roots, cotyledons and coleoptiles of tested plants. Phomasetin (35), from a Phoma sp, has been shown to be the enantiomeric bishomologue of equisetin from NMR and chiroptical comparison between the two [66]. Using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer, equisetin recovered from the fungus Fusarium heterosporum and phomasetin from Phoma sp. were isolated as inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase in vitro [67]. Equisetin and related compounds inhibit 3' end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50-212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells and are mechanistically distinct from many previously described inhibitors of HIV-1 integrase. Equisetin specifically inhibits the substrate ion carriers of the mitochondrial inner membrane. It is a potent inhibitor (IC50 8 nM) of DNP-stimulated ATPase activity of liver mitochondria and mitoplast [68]. Cryptosporiopsis cf quercina is an endophytic fungus first isolated from the plant Tryptergyium wilfordii. It causes no discemable external disease symptoms on T, wilfordii or Quercus sp from which it has also been isolated. It has been suggested that it may function as a symbiot; the plant providing support and nutrition to the fungus which in turn participates in the association by producing antifungal metabolites. The fungus was grown on oat seeds in water (800 ml) for 4 weeks, and the dichloromethane soluble portion of the culture filtrate was obtained. Repeated
123
chromatography and bioassay-guided fractionation, using the phytopathogen Sclerotinia sclerotiorum, led to the isolation of cryptocin (36) (63 nig) [69]. As observed for equisetin, the NMR spectra were complicated by the existence of a mixture of tautomers. Fortunately, cryptocin crystalised from a mixture of ethyl acetate-methanol as rhomboidal crystals that were amenable to an X-ray crystallographic study. Cryptocin crystallised as a sodium salt with sodium coordinating each of the oxygen atom in the molecule. The absolute stereochemistry remains to be determined.
»AAA/\AAA/»
36
37
H3C 38
Cryptocin (36) exhibits potent activity against certain phytopathogenic fungi; the oomycetes Pythium ultimum (0.78 _g/ml), Phytophthora cinnamoni (0.78 _g/ml), P. citrophthora (1.56 _g/ml), the ascomycetes Sclerotinia sclerotiorum (0.78 _g/ml), Pyricularia oryzae (0.39 _g/ml), the basidomycete Rhizoctonia solani (6.25 _g/ml), the fungi imperfecti Geotrichum candidum (1.56 _g/ml) and Fusarium oxysporum (1.56 _g/ml). However, it showed little activity (> 80 _g/ml) against human pathogenic fungi such as Candida albicans and Aspergillus fumigatus, and in this respect does not share the activity of certain other tetramic acid metabolites such as the aurantosides that are active against C albicans. Interestingly, P. oryzae is the most sensitive pathogen to cryptocin. This fungus, which causes rice blast and is responsible for significant crop losses, is one of the five targeted diseases in the development of fungicides [70]. Cryptocin is also active against R. solani, a representative of the basidiomycetes that cause cankers, heart and stem rots, root rots, and blights of woody and viney plants. A metabolite (CJ-17,572) from a strain of the fungus Pezicula sp appears to be identical to cryptocin, although the possible identity of the two was not mooted [71]. The lack of reported details, NMR parameters for
124
cryptocin and m.p. for the Pezicula metabolite, makes comparison difficult. Of some interest is the observation that attempted acetylation of the Pezicula metabolite yielded a derivative (37) in which the secondary alcohol had been eliminated and the enol oxygen at C4 acetylated. The metabolite (CJ-17,572) inhibited the growth of multi-drug resistant strains of Staphyllococcus aureus (MIC 10 |ig/ml) and Enterococcus faecalis (MIC 20 |ig/ml) and exhibited cytotoxicity against HeLa cells (ICg^ 7.1 Jig/ml).
NH2
39
40
Yet another analogue (CJ-21,058) (38) of equisetin was isolated from an unidentified soil fungus found at Nagasaki, Japan [72]. It showed marginally greater activity than CJ-17,572 against S. aureus (MIC 5 |ig/ml) and E. faecalis (MIC 5 ^g/ml). Interestingly, CJ-21,058 was discovered using an assay for SecA inhibiting activity. Sec A is a dimer of 102 kDa subunits found in the cytoplasm and bound to the inner membrane and is the peripheral domain of a core containing an integral domain comprising SecY, SecE and SecG proteins. SecA couples the energy from ATP binding and hydrolysis to protein translocation through repeated cycles of insertion and deinsertion of SecA. Compounds that inhibit association of the enzyme complex or of ATPase activity of SecA could provide a new class of antibiotics. CJ-21,058 showed an IC50 of 15 |ig/ml. Other examples in which the decalin system has been modified have been described. The epoxide (39) (PF1052) has been reported as a metabolite from an isolate of a Phoma sp. It showed good activity against Staphylococcus aureus (MIC 3.13 |ig/ml). Streptococcus parvulus (0.78 |Lig/ml) and Clostridium perfringens (0.39 |Lig/ml) [73]. A Microtetraspora sp isolate recovered at Andhra Pradesh in India, produced a metabolite BU-4514N assigned structure (40) from NMR data
125
[74]. It has been claimed to be active against Gram-positive bacteria and to be effective as a nerve growth factor (NGF) mimic. NGF is a protein known to be essential for the development and maintenance of certain sympathetic and sensory neurons in the peripheral nervous system. NGF appears to have functions in the cholinergic neurons in the basal forebrain. BU-4514N is useful for treating neurodegenerative disorders such as Alzheimer_s disease by mimicking the effect of NGF. Cultures of PCI2 rat pheochromocytoma cells respond to NGF by differentiating into sympathetic neuron-like cells. The cells stop dividing, produce nuritelike structures and produce increased levels of neurotransmitters and neurotransmitter receptors [75].
N—r^^ CO^Hs
o»»'
42
Vermisporin (41) is produced by the fungus Ophiobolus vermisporis [76]. Its structure was determined by chemical degradation to the derivative (42) which was studied by X-ray crystallography and provided the absolute configuration [77]. Vermisporin exhibits antimicrobial activity towards Bacteroides spp (0.25-2 |ig/ml), Clostridium perfringens (0.25-2 fig/ml) and methicillin-resistant Staphylococcus aureus (0.12-0.5 |Lig/ml). A metabolite of Ophiobolus rubellus produces the tetramic acid (43) that has been claimed to be an inhibitor of proline hydroxylase (IC5019|LiM) [78]. Three related tetramic acids have been reported from Chaetomium globosum. Two (44, 45) differ in the stereochemistry of the amino acid component, and the third is the methyl ester of 44 [79]. It is claimed that these compounds are chemokine receptor antagonists and can be used to treat HIV-1 infections.
126
44Ri = C02H;R2=OH 4 5 R i = OH;
R2 = C02F
An isolate of Streptomyces lydicus gave lydicamycin (46), a metabolite that showed activity against gram-positive bacteria, Bacillus subtilis (MIC
The next tetramic acid to be considered was isolated from lactic acid bacteria and is unusual in that, while it includes an acetyl group at C-3, it contains an a,P-unsaturated fatty acid as a substituent on N-1. Lactic acid bacteria have long been used to make various dairy products, sauerkraut or also sourdough for bread. Antibiotics from Lactobacillus protect against infections of Salmonella and Helicobacter, a causative agent of stomach ulcers [83]. One of these lactobacilli, L. reuteri, is a constituent of the naturally occurring intestinal bacteria in humans and animals. In a sourdough that is used in the production of a commercial baking product, a strain of L. reuteri was found to have an inhibitory effect against gram-
127
positive bacteria [84]. The antibiotic, named reutericyclin (47), was obtained from cell extracts and culture filtrate in a yield of -Img/L [85]. The gross structural features, presence of a tetramic acid and Edecenoyl side chain, could be inferred from NMR studies. Methanolysis (HCl/MeOH) of 47 and pentane extraction of the quenched reaction mixture gave two compounds that were determined to be the methyl esters of decenoic acid and N-(2-decenoyl)leucine. The nature of the 3-acyl tetramic acid was deduced from the identification of 48 and 49 in the aqueous portion of the methanolysis reaction mixture following treatment with trifluoroacetic acid anhydride. The unusual C-C bond fragmentation under acidic conditions, and the structure of the antibiotic was confirmed by synthesis of racemic 47 [86]. The configuration at the lone chiral centre was established as R by chiral GC. The carbon NMR spectrum of 47 indicated an equilibrium between three tautomers in which the A^-pyrrolin-4one form is preferred (60%) and the two internal tautomers (50, 51) make equal contributions (20% each).
k 47
OCH3 49
50
i'^-^
ENOYLTETRAMIC ACIDS Blasticidin (52) is an antibiotic isolated from Streptomyces griseochromogenes in 1955 [87]. Detailed physicochemical properties were reported in 1968, but the structure remained undefined [88]. Interest in this metabolite was renewed recently with the isolation of the homologous compounds aflastatin A and B (53, 54), metabolites of 5. griseochro-
128
mogenes that exibit the unusual property of inhibiting aflatoxin formation by strains of Aspergillus [89]. HO, 9H0H ?H QH QH QH QH QH QH QH QH OH OH V ^ N ^ ^ ^ ^ H
OH '
' '
OH OH
OH
53 R = CH3 54R = H HO.
9HQH
?HQH OH OH OH OHOH OH OH OH OH OHVA^^^QH OHOH
OH
Aflatoxins are a group of mycotoxins produced by some strains of Aspergillus, e.g. A.flavus^A. nomius.A. parasiticus, and A. tamarii. These fungi do not always produce aflatoxins, but under certain environmental conditions, high temperature and humidity, they infect crops such as peanuts or com and produce the toxins which have been recognised as potent carcinogens towards mammals and a risk factor of liver cancer in humans [90]. Strains of Streptomycetes whose culture broths inhibited aflatoxin production in A. parasiticus, but not the growth of the fungus, were uncovered and investigated [91]. Of these, Streptomyces griseochromogenes was found to produce an inhibitor in the mycelium that was extracted into methanol. Fractionation and chromatography yielded aflastatin A (12 mg/100 ml) and B (0.14 mg/100 ml). The determination of the structure of aflastatin A (53), C62Hji5N024, relied largely on NMR analysis of the metabolite and degradation products. The ^^C-NMR of the free acid contained broad signals due to the presence of tautomers. The signals sharpened considerably in the spectrum of the diethylamine salt that was formed in the last HPLC separation in which the amine was added to the mobile phase. Fig. (5) illustrates the degradation sequences that revealed the structural components of 53. Perhaps, the most challenging task was the determination of the configuration of the 29 stereocentres and one double bond. The strategy adopted is shown in Fig. (5).
129
stereochemistry from optical rotation
1.Q3;Me2S 2. LiAIH4 3. BzCN, EtgN
OH
Nal04 1. Nal04 2. NaBH
QH
?«V^
OH OH OH OH OH OH OH OH OH OH OH 0
.OH OH
1. Nal04 2. NaBH4 3. 3N HCI 4. BzCI 5.O3
relative configuratfon by J-based method
I.O3 2. NaBH4 3. AC2O 4. NaOMe 5.H^
BzO'
OBz
comparison with I authentic sample I
I OH OH OH OH OH OH OH OH OH OH OH =
•
=
•
-"
OH OH
relative configuration by J-based method
Fig. (5). Degradation scheme to determine structure and stereochemistry of aflastatin A (53)
With the structures of aflastatin A (53) and B (N-demethyl aflastatin A, 54) secured, the structure of the related blasticidin A (52) was determined in a similar manner. Studies on the biosynthesis of aflastatin A
130
have shown that C-7, 27,29,33 and 35 arise from glycolic acid whereas all the others are as expected [6]. All the three compounds inhibit production of aflatoxins in A. parasiticus at 0.5 jxg/nil [90,92,93]. Aflastatin A inhibits the formation of norsolorinic acid, an anthraquinone precursor of aflatoxin biosynthesis [94], and melanin production in Colletotrichum lagenarium [95]. In both cases, indications are that inhibition occurs at an early step of the biosynthesis. The effect of removing the tetramic acid group has ben investigated. Thus, compound (55) and the corresponding methyl glycoside were found to significantly reduce expression of genes encoding enzymes involved in the aflatoxin pathway, and did not show antifungal activity [96]. DIENOYLTETRAMIC ACIDS A number of tetramic acids bearing a 1-oxopentadienyl substituent at C-3 have been discovered. The first of these was streptolygidin (56) isolated from the actinomycete Streptomyces lydicus in 1955 [97]. This metabolite showed strong antibiotic activity against gram-positive bacteria and is a potent inhibitor of terminal DNA transferase and bacterial RNA polymerase [98,99]. The dienoyl functionality at C-3 is crucial for its activity and the presence of 1 and 5-substituents, although not essential, does improve activity [100]. A metabolite named afragilimycin was identified as the sodium salt of streptolygidin [101].
c CONHMe H
Elucidation of the structure, first assigned a molecular formula C32H46N2O9 and later revised to C32H44N2O9 [97], was achieved by a combination of classical degradation methods, spectroscopic methods and by comparison of data from synthesised model subunits [97,102, 103]. The structure of the 2,3,6-trideoxyaldohexose forming the N-glycoside was determined by synthesis [104] thus establishing the absolute stereo-
131
chemistry of the glycoside. A complete stereochemical assignment was achieved in conjunction with work carried out in defining the structure of a related antibiotic, tirandamycin A (57) isolated from 5. tirandis [105]. The biological activity of 56 and 57 are similar [106,107], as is the mode of action [108,109], although streptolydigin is the more potent. OH o
Tirandamycin A (57) on periodate oxidation afforded tirandamycic acid (58) which was converted to the corresponding p-bromo phenacyl ester. X-ray cystallographic studies on this heavy atom derivative revealed the structure and absolute stereochemistry of the dienoyl portion of tirandamycin A [110]. Structural correlation between tirandamycic acid and the corresponding acid (59) derived from streptolydigin was achieved as shown below. The stereochemistry of the P-methylaspartic acid portion of streptolydigin had been assigned the L-threo configuration [102] and the configuration at the anomeric carbon of the Nglycoside as p- from NMR studies.
COgH
CHgOH
S.flaveolusprovided tirandamycin B (60) which contains an extra hydroxy 1 group on the 2,9-dioxabicyclononane skeleton [111]. Tirandalydigin (61) incorporates structural features of 56 and 57 and is also pro-
132
duced by a Streptomyces strain [112] and showed activity (MIC 0.5-32 mg/ml) towards a number of pathogenic anaerobes, streptococci, enterococci and legionellae [113]. OH o
Two related antibiotics have been isolated from an unidentified actinomycete strain [114,115]. The two metabolites, 62 and 63, differ only in the presence or absence of the methyl group on the nitrogen in the tetramic ring [116]. Although both are broad spectrum antibiotics with activity against anaerobic and aerobic bacteria, the N-demethyl analogue (63) is almost twice as potent [115]. The structures of these metabolites rests on NMR spectral data and on X-ray crystal structure of the pbromphenacyl ester of the acid obtained from the acyl portion of the compound [114]. The last representative of this group of antibiotics is nocamycin II which was isolated from Norcardiopsis syringae [117-119]. This is the dihydro analogue of 63 in which the ketone in the dioxabicyclononane system is replaced by an a-hydroxyl. The structure originally assigned to a congener, nocamycin I, was revised to 63 [118].
62R = CH3 63R=:H
POLYENOYLTETRAMIC ACIDS Plasmodial slime molds (Myxomycetes) are eukaryotic bacteriovores usually occurring in terrestrial ecosystems. Although a few species appear to be confined to the tropics or subtropics, the majority are cosmopolitan and approximately 1000 species have been recognised. In the assimilative
133
part of their life cycle, they form a free-living multinucleate, acellular mobile mass of protoplasm (plasmodium) which feeds on living bacteria. The Plasmodium undergoes sporulation to develop into a small funguslike fruiting bodies with unique structure and colour. Myxomycetes are particularly abundant in temperate forests where they are found on the bark of living trees,on leaf litter, soil, and dung of herbivorous animals. The chemical nature and role of the yellow plasmoidal pigments of Physarum and Fuligo species have been studied because of their involvement in phototaxis and induction of sporulation. Apart from their activity as photoreceptors, these pigments may also protect vulnerable plasmodia from microbial attack and be useful because of their metal chelating properties The Plasmodia of the wild type of Physarum polycephalum are bright yellow and produce an orange-red pigment that is also present in a white mutant [120]. This compound, named physarorubinic acid (64), binds calcium and other metals very well and this made acquisition of the ^H-NMR spectrum difficult (line broadening) unless the sample was washed with aq. EDTA solution. The structure contains a decapentaene system and signals at 5 102.4 (C-3), 172.6 (C-7), 174.5 (C-2) and 194.0 (C-4) are characteristic of C-3 acylated tetramic acid unit. OH
O N
\
CH3
The stereochemistry was determined by comparison of CD data of the decahydroderivative of compound (64) to that of the synthetic chiral analogue (65). This established the 5-configuration at C-5. Interestingly, fuligorubin (66), present as the yellow calcium salt in the aethelia of the closely related myxomycete Fuligo septica [121], incorporates /?glutamic acid whereas P. polycephalum uses ^-serine. Leocarpus fragilis, Plasmodia of which are found in autumn attached to dead conifer needles or grass, produces the group of tetramic acid pigments (67-70) that have incorporated iS-tyrosine [122].
134
'OH CH.I3
^
.CO2H
H3C
65
68 n = 3 69 n = 4
70
O
CH3
^"
P. polycephalum has an unusual life cycle. Young plasmodia live inside decaying trees and move away from light, whereas older plasmodia, at the end of their growth phase, move towards the light and sporulate. This behaviour is mediated by photoreceptors in the UV-A or blue light range which contains maxima at X^^^ 350 nm and 460 nm (blue light). It has been shown that light stimulates the formation of two orange-red metabolites (0.003 and 0.005% yield) that can be extracted into chloroform and show UV absorption at - 250 (In e 4.14) and 390 (In 8 4.74) [123]. The structures of these compounds, polycephalin C (71) and B (72), has been secured. HO
0
_
71 R = CH3 72R= H
A more elaborate C-3-polyenoyl side chain is presented by erythroskyrine (73), a pigment first isolated in 1949 from Penicillium islandicum. It was reisolated in 1954, but the gross structure was elucidated only in 1965 [124]. At that time, the only stereochemical point resolved was the assignment of the 5-configuration at C-5, deduced from the formation of L-N-methylvaline on degradation of the pigment with ozone [125,126]. Detailed NMR studies of 73, including NOE measurements.
135
and analysis by the Mosher-Trost chiral ester (esterification of the C21 hydroxyl group with /?-and 5-0-methyl mandelic acid) indicated the /?configuration [127]. This has been conclusively confirmed by synthesis [128]. Erythroslcyrine is considered to be a mycotoxin and is also active against Staphylococcus species [129]. Oleficin (74), a polyenoyltetramic acid characterised by the presence of the desoxy sugar D-digitoxose on the C-3 hexenoyl side chain, is an antibiotic isolated from a strain related to Streptomyces parvulus [130].
The pentenoyl analogue, a-lipomycin (75) and the aglycone Plipomycin (76) are metabolites of 5. aureofaciens [131] which, together with oleficin, inhibit gram-positive bacteria but are inactive to fungi [131,132]. Oleficin exhibited an LD50 40 mg/kg (i.v.) in mice and was effective against Yashida s.c. sarcoma in mice [133]. Oleficin depleted Mg^^ and Ca^"^ ions in isolated rat liver mitochondria and increases membrane permeability [134] It also induced disintegration of the mitochondrial genome in Saccharomyces cerevisiae [135]. Altamycin (77), isolated from a variety of Actinomyces pneumonicus, also belongs to this group containing a tetraene system in the side chain [136]. OH '
OH
n^\ ^
\
74n = 5 I ^"3
75n = 4 77 n = 3
I
I 76
^^^H
O
\ ^^3
Trichoderma spp are a well known fungal group that produce a wide range of metabolites with diverse activities [137]. The only tetramic acid derivative isolated so far is harzianic acid (78) which was isolated from a
136
Strain of T. harzianum collected from a water sample at Hiroshima in Japan. The structure and the configuration of the double bonds were determined from spectral parameters [138]. The tetramic acid portion of harzianic acid includes an unusual amino acid residue. Harzianic acid exhibited only weak antibiotic activity against Pasteurella piscidida (MIC 12.5 |Lig/ml) and Proteus mirabilis (MIC 25 |ig/ml). OH 0
The orange pigments aurantoside A and B have been isolated from the marine sponge Theonella swinhoei [139]. The structures contain a dichlorohexaene chain and a glutamine-derived tetramic acid that is modified by an N-trisaccharide unit. The structure and absolute sterochemistry of the trisaccharide group was established as D-xylo-D-arabino-D-arabino by GC analysis of the hydrolysis product. The configuration at C4 was S since L-aspartic acid was obtained on Lemieux oxidation (periodate/ permanganate) followed by hydrolysis. The original structure was revised to include an ^-geometry of the terminal double bond [140]. The aurantosides were first isolated as the antifungal and cytotoxic constituents and were later shown to inhibit binding of interleukin-6 to its receptors. Aurantoside C was obtained from a sponge, Homophymia conferta (Theonellidae) (Phillipines) and was mildly toxic to brine shrimp [141]. Resonances for C-1 and C-2 were not observed in ^^C-NMR spectrum but were detected in the hexylacetate derivative (5^ 174, C-1, and 5^ 100.3, C-2). Aurantoside D, E and F from the sponge Siliquariaspongia japonica (Theonellidae) were obtained by bioassay-guided fractionation [140]. Aurantoside F was four times more toxic (IC5Q 0.05 |ig/ml) than D or E (IC50 0.2 |xg/ml), whereas A and B did not show activity at 5 ^ig/ml against P388 murine leukemia cells. On the other hand, aurantoside F was inactive towards C albicans and A. fumigatus, which were sensitive to the others, in particular E (MIC 0.16 and 0.04 |ig/ml with inhibitory zones of 9.7 and 13.6 mm) [140].
137
The rubrosides are a group of eight related compounds, similar to the aurantosides, that have been obtained from Siliquariaspongia japonica [141]. The methanol extract from the sponge exhibited significant activity in the 3Y1 rat fibroblasts assay and bioassay-guided fractionation resulted in the isolation of the metabolites, e.g. 85. Compared to the aurantosides, OH HO
O'
OH
H2N79 R = CH3 Aurantoside A 80 R = H
OH O,, ^Q
Y)"CH3
Aurontoside B
or
V\
HO OH
84 Aurantoside F
•3^"
'OH
they present a slightly more elaborate polyene unit at C-3 that terminates
138
in a 4-chloro-2-methyltetrahydrofuran ring. The rubrosides induced numerous intracellular vacuoles in 3Y1 rat fibroblasts at 0.5-1.0 fxg/ml. Rubrosides A, C, D and E were cytotoxic against P388 murine lukemia cell with IC50 0.046-0.21 fig/ml and were active against Aspergillus fumigatus and Candida albicans.
H O
MACROCYCLIC LACTAMS This section covers a growing group of metabolites that contain a tetramic group, or a modified tetramic acid, embedded in a macrocyclic lactam. The first example of this class was ikarugamycin (86) which was isolated from the culture medium of Streptomyces phaechromogenes var ikaruganensin. This metabolite showed strong antiprotozoal, in vitro amoebic, and activity towards gram-positive bacteria [142]. The structure was determined using classical oxidative degradation methods and NMR spectroscopy [143]. The amino acid incorporated into the tetramic acid ring was shown to be L-ornithine which was obtained from 86 by acid hydrolysis. Ikarugamycin encapsulates sodium ions very strongly and can sequester them from solution of siUca gel [144]. Capsimycin (87), a related compound produced by a Streptomyces strain, showed antifungal activity against the phytopathogens Phytophthora capsicii and Pythium debaryanum [145]. Its structure was deduced by NMR spectral comparison with ikarugamycin and was established by X-ray diffraction studies, which also revealed the absolute stereochemistry. A collection of the deep-sea sponge Discodermia dissoluta from Grand Bahaman Island provided another example; discodermide (88) [146]. The
139
0
H^
\,.o.H
^^ Ikarugamycin 87 5,6-p-epoxy
structure and stereochemistry, with the exception of C-16 and C-17, were assigned on the basis of spectral analysis. Discodermide exhibited cytotoxic (P388; IC50 0.3 [xg/ml) and antifungal activities (C albicans; MIC 12.5 jxg/ml). A number of related metabolites have been uncovered. Alteramide A (89) was isolated from the bacterium Altermonas sp. recovered from the marine sponge Halichondria okadai [147]. An incompletely characterised congener, the 25-desoxy analogue, alteramide B, is also produced. Alteramide A shows cytotoxic activity to P388 (IC50 0.1 |Lig/ml), murine lymphoma LI210 (IC50 ^-^ ^g/i^O and human epidermoid carcinoma (IC50 5.0 ^ig/ml) cell lines. Interestingly, altemamide A on exposure to light undergoes a [4+4]cycloaddition to yield a compound devoid of theactivity associated with the parent compound. Cylindramide (90) is a cytotoxic compound isolated from Halichondria cylindrata (7.10'^% wet wt.), but very likely of bacterial origin [148]. The structure was deduced from spectroscopic analysis, the relative stereochemistry (only) of the bicyclo[3.3.0]octene unit by NOESY techniques, and the absolute stereochemistry of the amino acid of the tetramic acid ring (25,3S) from the recovery of erythtro-L-p-hydroxyornithine from oxidative degradation of 90. It showed cytotoxicity to B16 melanoma cells (IC50 0.8 |Lig/ml). Aburatubolactam A (91) and C (92) were isolated from the culture broth of Streptomyces sp recovered from a mollusk. X-ray diffraction studies established the structure of aburatubolactam A which was reported to inhibit TPA-induced superoxide anion generation by human neutrophils [149]. Aburatubolactam C appears to be cytotoxic for various proliferating tumor cells of human and murine origin (0.3-5.8 M'g/inl) via apoptotic DNA fragmentation [150,151].
140
"OH 88 Discodermide
89 Aiteramide
Stenotrophomonas maltophilia (formerly Pseudomonas maltophilia), isolated from the rhizosphere of rape plants (Brassica napus), yielded the antifungal agent maltophilin, for which only the plane structure has been described [152]. This compound inhibited the growth of various saprophytic, human-pathogenic and phytopathogenic fungi, but was inactive towards gram-positive and gram-negative bacteria. The corresponding compound in which the cyclohexanone carbonyl is reduced to the alcohol (dihydromaltophilin) cooccurs with maltophilin in an isolate of Streptomyces sp [153] A Stenotrophomonas strain SB-K88, isolated from the fibrous root surface of sugar beet cultivated in a field infested with Polymyxa betae, suppressed rhizomania and seedling damping-off of sugar beet [154]. The bacterium is therefore a member of the competitive rhizosphere microflora.. Three fungitoxic metabolites, xanthobaccin A-C (93-95), were produced by this organism grown in liquid cultures, and all inhibited the growth of several fungal phytopathogens, particularly Pythium ultimum (MIC 1 |Lig/ml) [155,156]. Although the plane structure assigned to xanthobaccin A (93) corresponds to that of maltophilin, more details have been obtained for the former, including the configuration of the amino acid group and relative configuration of the tricyclo[7.3.0.0^'^]dodecane ring system [155]. It is useful to note that dihydromaltophilin may correspond to xanthobaccin B [156]. Interestingly, the xanthobaccins and maltophilin exhibit antifungal activity in vitro but no antibacterial activity. On the other hand, ikarugamycin (86) shows antibacterial activity against gram-positive bacteria.
141
90 Cylindramlde ^ ^ OH
O
93 Xanthobaccin A 94 27-dihydro 9516-deoxy
An ethanol extract from Geodia sp., a marine sponge from the Great Australian Bight, showed activity in inhibiting larval development of the nematode Haemonchus contortus (LD99 14|Lig/ml) [16]. The active material (LD991 |Lig/ml) was soluble in BuOH. Energy dispersive spectroscopy (EDS) indicated the presence of magnesium and HR-ESIMS(-ve) and AA spectroscopy was consistent with the formula [C27H3iN205]2Mg. The structure of geodin A magnesium salt (96) was deduced from spectroscopic analyses, but the relative stereochemistry of the amino acid component of the tetramic acid and the absolute stereochemistry remain to be assigned [16]. Attempts to convert the salt to its conjugate acid with either HCl or TEA. gave an unstable compound that could not be characterised. Mg2^
96
J 2
142
N.ACYL.4.METHOXY-3.PYRROLIN.2-ONES Compounds containing the 4-0-methyl ether of N-acylated tetramic acids form a significant group of derivatives that, in the main, are found in marine organisms and, more specifically, are biosynthesised by marine cyanobacteria. The first example of this class was dysidin (97), isolated from an Australian variety of the marine sponge Dysidea herbacea [157]. The structure was determined by X-ray diffraction studies which also disclosed the 5, S absolute configuration of the two stereocentres [157]. Blue-green algae are frequently associated symbiotically with D. herbacea, and often constitute 50% of cellular material of the sponge [158]. Although terrestrial cyanobacteria are well-recognized producers of a wide range of bioactive compounds, marine species have received less attention until recently [159]. One of the most abundant and studied marine cyanobacteria is the pantropic Lyngbya majuscula (Oscillatoriaceae). A prolific producer of metabolites, it has so far yielded more than 110 secondary metabolites including compounds that exhibit antiproliferative, immunosuppressants, antifeedant and moUuscidal activities [159,160]. Shallow water varieties of the cyanophyte contain N-substituted amides of 75'-methoxytetradec-4£'-enoic acid and of 75-methoxy-9-methylhexadec-4£'-enoic acid called malyngamides, a sub-class of which contains the 4-methoxy-3-pyrrolin-2-one system [158]. The structure of the first example of this type, malyngamide A (98), was determined by spectral and chemical means. Hydrolysis led to 4methoxy-A^-pyrrolin-2-one, which is also a component of the alga. Mild acid hydrolysis yielded a P-ketoamide, indicating the presence of an acyclic P-methoxyenamide, the E-configuration of the alkenyl chloride functionality was determined from NOE measurements [161]. Isomalyngamide A (99), from a Hawaiian sample of L. majuscula, differs in the configuration of the chloromethylene group [162]. A number of malyngamides have been identified more recently. A sample of L. majuscula from Madagascar produces malyngamides Q and R (100,101) which differ from other types in that the pyrrolidinone ring is derived from glycine instead of serine [159]. The S-configuration at C-4 in the pyrrolidone ring was established by chiral GC-MS analysis of serine (as the pentafluoropropyl serine methyl ester) released on ozonolysis and acid hydrolysis.
143
CH3{CH2)5
^^
9CH3 100 R = H
101R = CH3
n
0CH3
Y " l
CH3(CH2) R
I)
OCH3
From another shallow-water variety of L majuscula found in Hawaii, a new crystalline metabolite pukeleimide C (102) was isolated. The structure was secured by X-ray studies [163]. Chiroptical studies showed the compound to be racemic and, probably, an artefact. A separate collection of the cyanophyte did not contain pukeleimide C but, instead, yielded a group of related metabolites, e.g. 103, whose structures were assigned from spectral data [164]. Unfortunately, no information is available on the bioactivity of these metabolites. €H3
HaCa
102
Althiomycin (104) was isolated in 1957 from Streptomyces althioticus [165] and showed a broad spectrum of activity towards gram-positive and gram-negative bacteria through inhibition of protein synthesis at the peptidy Itransf erase stage (cf tenuazonic acid) [166]. It has been characterised as an agent with low toxicity and good selectivity towards prokaryotes. Althiomycin has also been isolated from Streptomyces matensis, Cysto-
144
bacter fuscus, Myxococcus xanthus and strains of M. virescens, gramnegative bacteria usually found in soil [167]. The structure of 104 was determined by a combination of spectroscopic methods, partial synthesis and X-ray crystallographic studies [168,169]. X-ray studies yielded the 5configuration of the stereogenic centre in the thiazoline ring [169]. The remaining stereocentre could not be determined since althiomycin is always isolated as a mixture of diastereomers with both R and 5configurations at this carbon. Synthetic approaches to althiomycin and analogues and biological studies of the interaction with prokaryotic ribosomes have been undertaken [170].
104
The terrestrial cyanophyte Scytonema mirabile yielded mirabimide E (105) which possesses an unusual tetrachlorinated ethylene group [171]. Detailed spectral analysis, including NMR analysis of a sample of 105 that had been uniformly enriched in ^^C and ^^N yielded the gross structure. Lemieux degradation, prior to acid hydrolysis, afforded alanine which was shown to have the 5-configuration by chiral GC-MS of the isopropyl ester of N-trifluoroacetylalanine. Reaction of 105 with nitrosylsulfuric acid followed by methanolysis yielded the p-ketol (106) which afforded the methyl ester (107) on treatment with magnesium methoxide.
CH3
107
The stereochemistry of this compound was determined by the Mosher method which, together with the fact that J2.3 = 7 Hz is consistent with an
145
anti-airangement of the P-hydroxy-a-methyl group, indicated the 2J?, 3J?-stereochemistry. Mirabimide E possesses selective toxicity on solid tumours. In contrast, the congeneric mirabimide A-D, e.g. 108, are moderately cytotoxic but did not exhibit selective cytotoxicity [172].
OCH3
The soft-bodied and slow-moving sea hare Dolabella auricularia produces potent defence compounds. This nudibranch has been recognised since 60 AD and its toxic extract has been used by some, particularly in Roman times, to dispatch political rivals [173]. Of the many metabolites produced by this organism, a group of remarkable cytotoxic peptides known as the dolastatins have attracted considerable attention. Of these, dolastatin 10 and 15 (109) are endowed with potent antiproliferative activity with dolastatin 10 having the greater activity. These compounds are present in extremely small quantities; from 1600 kg of £>. auricularia 28.7 mg (1.8.10-6%) of dolastatin 10 and 6.2 mg (3.9.10-7%) of dolastatin 15 were obtained [174]. The exceedingly low yields of dolastatins recovered from D. auricularia have suggested that the moUusk is not the real producer of these compounds [175]. Moreover, some dolastatins and analogues have been found in certain strains of Lyngbya majuscula and assemblages of L. majuscula and Schizothrix calcicola, implying that at least some metabolites isolated from D, auricularia, a generalist herbivore, have a cyanobacterial origin [175]. Dolastatin 15 is a lipophilic pentapeptide esterified with N-acylpyrrolidone containing seven stereogenic centres all with iS-configuration. The structural determination was achieved primarily from NMR studies and comparison with analogues, and the absolute configuration was established from X-ray analysis and by synthesis [176]. Dolastatin 15 is also highly cytostatic affecting dividing cells but does not interfere with the
146
viability of resting cells.and shows low toxicity to proliferating nonleukemic cells. Sorangium cellulosum is an ubiquitous soil bacterium that belongs to the order Myxococcales. It has the ability to glide over solid surfaces, to live in a biofilm and form fruiting bodies. Members of this taxon are a particularly rich source of metabolites with remarkable biological activities [177]. From one strain, another example of N-acyl-4-methoxy-3pyrrolin-2-ones has been isolated. Eliamid (110) has been claimed to have cytostatic, nematocidal and fungicidal activities [178].
109
110 O
CH3
MODIFIED TETRAMIC ACIDS There are several examples of metabolites where the tetramic acid domain is teasingly disguised. In these cases, it is difficult to be definitive about the nature of the apparent modification unless this is substantiated by biosynthetic studies. The case of lactacystin (5), Fig. (2), has already been considered. Another example is presented by the oxazolomycin group of antibiotics, e.g. (Ill), found in strains of Streptomyces [179]. Biosynthetic studies indicate that the carboxylic acid of the amino acid required to form tetramic acids contributes to the formation of the P-lactone [180]. In this section, metabolites that probably have a tetramic acid origin are presented. The neurotoxic lipophilic tripeptide, janolusimide (112), was isolated from the Mediterranean nudibranch Janolus cristatus. The structure was determined by spectroscopic and classical chemical methods [181] and confirmed by synthesis [182]. It has been suggested that the pyrrolidine2,4-dione may arise from condensation of valine and isobutyrate [181].
147
N-CH3
Integramycin (113) is a novel hexacyclic acid isolated from the culture broth of an Actinoplanes sp. It was detected from its activity (IC50 ^ M^^) in an in vitro assay to evaluate inhibition of HIV-integrase (strand transfer) [183]. The structure and relative stereochemistry were deduced from spectral data. In methanol, an epimeric mixture of the C-2 methoxy ethers are formed. Acetylation with acetic anhydride/pyridine generated a separable 1:1 mixture of the C-2 epimeric pyridinium tetracetates (114). Interestingly, both epimers maintained activity in the coupled assay (IC5Q 6 jxM), but were much less active in the strand transfer assays (IC50 30-46 |LlM).
A 3-deoxy tetramic acid heterocyclic system is contained in oteromycin (115), a metabolite produced by an unidentified fungus (possibly an Ascomycete). Oteromycin was uncovered using a bioassay to identify antagonists of the endothelin receptor subtype B that is implicated in both vasoconstriction and vasodilation [184]. Oteromycin showed IC5Q 2.5 |LIM but had little effect in the angiotensin II receptor binding assay (IC50 >25 |LiM). The structure and relative stereochemistry rests on detailed spectral analysis. The obligate marine fungus Ascochyta salicorniae, obtained from the marine alga Ulva sp. from the North sea off Germany, is considered to be an algal endophyte. The fungus was mass cultivated on a solid medium, the ethyl acetate extract of which showed antimicrobial activity [185].
148
Ascosalipyrrolidinone A and B (116,117) were isolated and their structures determined by spectroscopic techniques. The two metabolites differ
N—H
114
115
only in the substituent at C21; butoxy or methoxy. Given that during the isolation n-BuOH and MeOH were used, the possibility that these substituents are artefactual cannot be discounted. Ascosalipyrrolidinone A exhibited antibacterial activity against Bacillus megaterium, and the fungi Mycotypha microsporum and Mycobotrium microsporum at 50 |ig/disk. Moderate activity towards chloroquine-resistant Plasmodium falciparum (IC50 736 ng/ml) and significant activity towards Trypanosoma cruzi and r. brucei subsp. Rhodesiense. The corresponding analogue (118) with tyrosine as the contributed amino acid has been isolated as an inhibitor of platelet-activating factor acetyltransferase from Penicillium rubrum [186].
116R=(CH2)^H3 117R = CI^ H3C
CH3
CH3
118
A diastereoisomer of this compound, its 5-dehydro derivative (119), and two artifacts, 120 and the 4,5-acetonide, have been isolated from the
149
ascomata of Talaromyces convolutus [187]. This fungus produces yellow pigments on the ascomata that contain azaphilones, pre-anthraquinones and the modified tetramic acids. Compounds 118 and its diastereoisomer inhibited growth of the filamentous pathogenic fungi Aspergillus fumigatus, A. niger and Candida albicans, with an activity comparable to that of the known antifungal agent amphotericin B.
HOHQ
Pramanicin (121) was isolated as the antifungal agent from a fungus of the genus Stagonospora [188]. It exhibited modest antifungal activity against a range of microbes including the human opportunistic pathogen Cryptococcus neoformans, the agent responsible for cryptococcal meningitis. It showed an effect on vascular endothelial cells and may act by increasing calcium permeability in cells [189]. The structure and relative configuration were determined by spectroscopic analysis [188]. In later work, pramanicin (121) was found to co-occur with the diene precursor (122) [190].
\
\
OH
121
OH
122
150
In perhaps the most thorough study of its type, the biosynthesis of pramanicin has recently been investigated [190]. A series of experiments using acetate, malonate and serine precursors, labelled with ^H, ^^C, ^^N and ^^O revealed the following set of events. The hydrophobic tail arises from a starter acetate unit that is extended with six malonate units. The possibility of malonate acting as a starter unit was eliminated by the observation that incorporation of diethyl[2-^^C]malonate, which acts as a convenient source of malonyl CoA by hydrolysis and thioesterification, labels the extender units in preference to the stater unit by a factor of two. Moreover the incorporation of [1-^^C, ^H3]acetate occurs with significant retention of three deuterium atoms. A decanoyl chain is elaborated by a fatty acid synthase (FAS), but the transfer of this onto a polyketide synthase could not be demonstrated. Attempts to show the incorporation of decanoyl N-acetylcysteamine failed. In any event, the decanoyl intermediate is further extended by addition of two intact acetate derived malonate units, the extension probably being directed in a PKS-like manner. NHAc
124 The formation of the pyrrolidone group involves condensation of an acetate with L-serine. Since a significant proportion of L-[1,2,3-^^C, ^^N]serine was incorporated into pramanicin, the carbon skeleton of serine is incorporated intact. Acylation with the 14-carbon moiety then ensues leading to the conjugated dieneone tetramic acid intermediate 122. In fact, this compound co-occurs with 121 and, interestingly, is almost exclusively produced when 123 and 124 are used as precursors. The remaining steps involve formation of the trans-diol at C-3 and C-4 and epoxidation of the terminal alkene in the dienone chain. Vancoresmycin (125) is a metabolite extacted from the mycelium of an actinomycete strain of the genus Amycolatopsin sp. It exhibited potent activity against gram-positive bacteria, including vancomycin-resistant strains such as Enterococcus spp. [191], but was inactive towards gramnegative bacteria and showed no fungal activity. The plane structure was derived from MS and NMR studies which also revealed the stereochem-
151
istry of the 4 trisubstituted double bonds. The configurations of the 26 stereocenters remain to be determined [191]. OH OH
HaC-N
HoC
The pseurotins are a class of metabolites that are structurally characterised by an l-oxa-7-aza-spiro[4.4]non-2-ene-4,6-dione skeleton. The first compounds of this type, e.g. 126, were isolated from Pseudeurotium ovalis [192,193], but were also obtained from Cordyceps sophioglossoU des [194], Diheterospora chlamydosporia [195] and Aspergillus flavus [196]. The pseurotins have been shown to exhibit diverse activities, including inhibition of chitin synthase [196], neuritogenic activity by inducing cell differentiation [197] and amorphine antagonism [198]. The biosynthesis of pseurotin A has been studied in some detail [199]. In summary, a propanoyl starter unit is condensed in four successive cycles with the malonyl extender unit. The resulting pentalcetide (127) combines with phenylalanine, probably first with formation of the amide (128) and then with closure of the lactam ring to form the tetramic acid (129) [199].
152
QH
O
O
129
O
O
127
128
CONCLUDING REMARKS The structures of over 120 naturally occurring compounds recognisable as tetramic acid derivatives have been described in the literature so far. Comparable numbers have been derived from fungi (35) and bacteria (34). A similar number have been isolated from cyanobacteria and/or marine sponges. These can be grouped together since, as pointed out previously, marine microorganisms are frequently involved in the production of metabolites isolated from marine macroorganisms. A substantial number of the metabolites isolated still require resolution of stereochemical ambiguities, particularly those examples in which the C-3 acyl groups contain multiple stereogenic centres. A point of interest relates to the isolation of tetramic acid metabolites as salts and whether or not these salts represent the 'natural' form of the metabolites. The role of the tetramic acid group in determining the bioactivity of the metabolites is another question of significant interest. In this respect, it might be important to distinguish between an in vivo role and an in vitro effect. The natural function of the tetramic acid group, particularly as the salt, might be related to the translocation of metal ions or transport of the molecule. Metal ions such as Mg (II) can act as transmembrane transports and influence the fluidity and permeability of membranes [200]. On the other hand, in some bioassays it might not matter whether the compound is present as the salt or as the conjugate acid. The nature of the bioassay might be such that transport of the metabolite is not a problem. The function of the often extravagant acyl substituent is also puzzling. The activity of the ancorinosides, e.g. (27), as membrane type 1 matrix metalloproteinase inhibitors does not seem to depend on the liposaccha-
153
ride substituent at C-3. In fact, tenuazonic acid which does not have fiinctionality apart from the 3-acyltetramic acid is considerably more active [57]. Some of the questions that will require an answer are illustrated by a few observations on the activity of blasticidin (52) and the aflastatins (53, 54). Removal of the tetramic acid group did not eliminate the inhibitory effect on aflatoxin production by Aspergillus flavus or render the modified blasticidins toxic to the fungus [96]. A fragment of aflastatin A (53), containing the tetramic acid portion and an eight carbon chain at C-3, showed no inhibitory activity on production of the toxins [92]. It is clear that tetramic acid metabolites, because of their intricate structures and diverse bioactivity, will continue to attract attention for some time yet.
154
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
165
CHEMISTRY AND BIOLOGICALACnVITIES OF GINKGO BILOBA
K. SASAKI, K. WADA and M HAGA Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan,
ABSTRACT: Ginkgo biloba L is the solo member of the Ginkgoaceae famfly, and its leaf extract (EGb) has been clinically used for the treatment of cerd)ral insufficiency. Several experimental studies have demonstrated that EGb has neuroprotective properties against various age-related physiological dianges and cerebral injuries induced by ischemia, edema and apoptosis. The major bioactive prindples of EGb arc recognized to be flavonoids and terpenoids, ginkgolides and bilobalide. Pharmacological actions of EGb and its constituents indude platelet-activating factor antagonism, anti-oxidant and free radical scavenging properties, as well as modulation of neurotransmission, cerebral glucose and lipid metabolism and cerebral membrane fluidity. These effects are involved in the mechanism related to the protective effect of the extract in the central nervous system. INTRODUCTION Ginkgo biloba L , a dioecious plant, is called a living fossil, because it is the single extant spedes of the family Ginkgoaceae, which made its appearance in the Permian age [1]. Thus far, a great variety of compounds have been isolated from the leaves, seeds and root bark of Ginkgo biloba, induding alcohols, aldehydes, ketones, steroids, catechines, flavonoids, terpenes and organic adds [2, 3]. After the year 1965, for instance, a standardized Gingko biloba leaf extract (EGb) termed EGb761 has been used chiefly in Europe for the treatment of peripheral vascular disease or cerebral insuffidency induding difficulties of concentration and of memory, absent mindedness, confusion, lade of energy, tiredness, decreased physical performance, depressive mood, anxiety, dizziness, tinnitus, and headache [4, 5]. Furthermore, in a placebo-controlled, double-blind randomized trial, it has been suggested that EGb761 is capable of stabilizing and, in a substantial number of cases, improving the cognitive performance and the social functioning of demented patients tested using the Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) and Geriatric Evaluation by Relative's Rating Instrument (GERRI) [6, 7]. A more extensive discussion on dinical trials will be found in the book by van Beek [8], and reviews [9, 10]. Despite the accumulation of considerable evidence in recent years to support the clinical efficacy of EGb [1, 8, 11], the predse medianisms of action of EGb have not yet been fully eluddated; moreover, there is only partial agreement concerning which constituents of EGb are involved in its mechanism of the action in the central nervous system (CNS). Conceivably, however, benefidal effects of EGb could be explained, at least in part, by its neuroprotective and/or neurotrophic properties against various symptoms induced by impaired brain functions in advanced age [12]. EGb761 is made up of 24% flavonoid glycosides and 6% terpenes, which are accepted as active prindples. Many undesirable constituents of Ginkgo biloba leaves (e.g., ginkgoUc add due to its allergic action) are eliminated by the extraction procedures used to prepare the extract
166
The extraction prooedures and oomponents present in £Gb761 are described in detail elsewhere [11, 13]. In this article, we present the principal constituents isolated from leaves and seeds of Ginkgo biloba, and deal with the pharmacological properties of standardized EGb and its constituents mainly in CNS functions. CONSTITUENTS OF GINKGO
BILOBA L. A N D ANALYTICAL METHODS
Tlie represoitative unique constitu^ts so far reported for Ginkgo biloba L. are stated briefly below. Adetailed description of this field has been presented in the book of van Beek [8]. Flavonoids Flavonoids are major constituents of Gingko biloba leaves, and present as biflavones, flavones, flavonols and associated glycosides (Table 1). Biflavones, in particular, are characteristic constituents of Ginkgo biloba. They are of the amentoflavone (1) type, and differ from each other in the number and position of the methoxy groups. Bilobetin (2), ginkgetin (3) and isoginkgetin (4) were diaracterized by Baker et d, [14] and Nakazawa et al. [15]. Moreover, sdadopitysin (5) was isolated by Miura et d, [16] and 5' -methoxybilobetin (6) was isolated by Joly et d, [17]. Briangon-Scheid et d, [18] rqwrted a normal-phase high-performance liquid chromatography (HPLC) system for the identiHcation and the quantitative determination of these biflavones by a two-step gradient of isocratic elution. Furthermore, they also showed the presence of amentoflavone. Pietta et d, [19] reported a reversed-phase HPLC system using tetrahydrofuran-l-piopanol-water as the eluent for the analysis of biflavones.
No.
Ri
R2
R3
R4
(1) (2) (3) (4) (5) (6)
OH OMe OMe OMe OMe OMe
OH OH OMe OH OMe OH
OH OH OH OMe OMe OH
H H H H H OMe
^mmmmmmmmmmmmmmmmmammammmmmmmmmd
167
Table 1. No.
Flavonoids of Ginkgo biloba Leave Compound
Biflavones (1) Amcntoflavone (2) Bilobetin (3) Ginkgetin (4) Isoginkgetin (5) Sciadopitysin (6) 5'-Methoxybilobetin Flavonois (7) Kaempferol (8) Quercetin (9) Isorhamnetin (10) Myricetin (11) Kaempferol-3-O-glucoside (12) Kaempferol-7-O-glucoside (13) Kaempferol-3-O-retinoside (14) Quercetm-3-O-glucoside (15) Quercetin-3-O-rhamnoside (16) Quercetm-3-O-rutinoside (Rutin) (17) Isorhamnetw-3-O-glucoside (18) Isorhamnetin-3-O-rutinoside (19) Myricetin-3-O-rutinoside (20) 3'-0-methylmyricetin-3-0-rutinoside (21) Kaenipferol-3-0-(2"-0-glucosyl)rhaninoside (22) Quercetin-3-0-(2"-0-glucosyl)rhamnoside (23) Kaempferol-3-0-[6'"-0-{p-(7""-0-giucosyl)coumaroyl}-2"-0-glua)syl]rhamnoside (24) Quercetin-3-0-[6"'-0-{p-(7""-0-glucosyl)coumaroyl}-2''-0-glucosyl]rhamnosi^^ (25) Quercetin-3-0-(6"'-0-/7-coumaroyl-2"-0-glucosyl)rhamnosyl-7-0-glucoside (26) Kaempferol-3-0-(6"'-0-/?-coumaroyl-2"-0-glucosyl)rhaninoside (27) Quercetin-3-0-(6"'-0-/>-coumaroyl-2"-0-glucosyl)rhamnoside (28) Kaempferol-3-0-[ a -rhamnosyl-(l-»-2)- a -rhaninosyl-(l--^6)]- P -glucoside (29) Quercctin-3-(9-[ a -rhamnosyl-(l-»-2)- a -rhamnosyl-(l->6)]- j8 -glucoside Flavones (30) Apigenin (31) Delphidenon (32) Luteolin (33) Apigenin-7-O-glucoside (34) Luteolin-3'-0-glucx)side
168
._
c— R3
^OH
RaO^
1 \ r^V"°^ )^} T
'^R4 •
il^^JLj OH
No.
Rl
R2
Fl3
R4
(7) (8) (9) (10)
H H H H glucose H rutinose glucose rhamnose rutinose glucose rutinose rutinose rutinose
H H H H H glucose H H H H H H H H
H OH OMe OH H H H OH OH OH OMe OMe OH OMe
H H H OH H H H H H H H H OH OH
(11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
ORi
0
\,„^
^^^^^u
•••••1 V Ri
f^^^*"
^^0^^'^^^^Y^^
No. Ri 1 (21) H 1 (22) OH 1
>^^^^^>r^ OH 0 H^CT—0
J ^
OH
mmmmmS
More than 20 flavonol glycosides have been isolated from ginkgo leaves. The major aglycones of ginkgo flavonoid glycosides are kaempferol (7), quercetin (8) and isorhamnetin (9), and the minor one is myricetin (10). They were determined by reversed-phase HPLC and diode array detection after hydrolysis of flavonol glycosides [20]. Two new coumaric esters of flavonol diglyoosides were isolated by Nasr et d. [21, 22]. Their structures were determined from the spectrum data to be kaempferol/quercetin-3-0-(6"' -0-/7-coumaroyl-2"-0 glucosyl)rhamnoside (26 and 27). Moreover, Hasler et d, [23] isolated new flavonol diglycosides from the leaves of Ginkgo biloba and determined their structures to be kaempferol/quercetin-3-0-(2"-Oglucosyl)rhaninoside (21 and 22), kaempferol/quercetin-3-0[6"'-0{p-(7""-glucosyl)coumaroyl}-2"-0-glucosyl]rhamnoside(23 and 24), and quercetin-30-(6'"-0-/?-coumaroyl-2"-0-glucosyl)rhanmosyl-7-Ogluooside (25). A procedure that combined counter-current chromatography (CCC) and HPLC was developed for the isolation and the purification of flavonol glycosides, and two novel flavonol triglyoosides (28 and 29)
169
were isolated [24]. Fiavones (30-34) also can be identified in ginkgo leaves [25]. The methods for the analysis of flavonoid glycosides using micellar electrokinetic capillary chromatography [26], HPLC with diode-array UV detection [27] and thermospray liquid chromatography mass spectrometry [28] were developed. Lobstein et al, separated flavonoids using gradient elution with acetonitrile and O.IN phosphoric add within 50 min [29]. Hasler et d, [25] described fingerprint HPLC separation of Ginkgo biloba, and 33 flavonoids were assigned. Recently, a multidimensional counter-current diromatography system for the preparative isolation of isorhamnetin, kaempferol and quercetin from crude extract was described by Yang et d. [30]. Flavonoid metabolites after oral administration of EGb to rats and humans were analyzed by reversed-phase liquid diromatography-
R2O
OR3
OH
o
J
HaC-^O-V
OH
No.
R1
R2
R3
(23) (24) (25) (26) (27)
H OH OH H OH
H H glucose H H
glucose glucose H H H
OH ,CH3
OH
O
I
OH-^^^^^^-V HO
I OH
OH OH
No. (28) (29)
Ri H OH
170
RiO
No.
Ri
R2
R3
(30) (31) (32) (33) (34)
H H H glucose H
OH H OH H OH
OH H H H glucose
Terpenes Ginkgolides, the bitter principles of Ginkgo biloba U were isolated from the leaves by Furukawa [33] for the first time and their chemical structures deteraiined by Maruyama et al. [34] and Nakanishi et d. [35]. The five known ginkgolides, ginkgolides A (35), B (36), C (37), J (38) and M (39) have a cage-like molecule structure with six five-membered rings and a tert'hwtyl group, and differ in the positk)n and number of substituted hydroxyl groups on the spirononane framework. Ginkgolide M is isolated only from the root of the gingko tree. The diemistry and pharmacological properties of ginkgolides were reviewed by Braquet et al. [36]. From molecular electrostatic potential (MEP) and molecular lipophilidty potential (MLP) studies, ginkgolides, are considered to be two-pole molecules. Negative electrostatic MEP
C(CH3)3
CH3
R2
O'
0-^0
-0-
(40) Bilobalide Ri
(35) (36) (37) (38) (39)
Ginkgolide A Gini
R2
R3
H OH H H OH OH OH OH OH OH OH H OH H OH
171
areas are generated around the lactonic cycles. Tliree MEP areas are present in ginkgolldes B, Q and M, while in ginkgolides A and J, diere are two NfEP areas due to the fusion of the negative MEP area as the cage is relatively dosed. Furthermore^ the existence of a marked hydrophobic zone surrounding the tert-hutyl lqx)philic moiety indicated diat it might lodge in a hydrophobic pocket of the membrane. Furthermore, Corey et d. [37, 38] carried out the total syndiesis of ginkgolides. Further experiments demonstrated that ginkgolide M can be syndiesized from ginkgolide C [39]. Extoisive and oonq)lete NMR analysis has been performed [40]. Bitobalkie (40) was isolated from the leaves by Wsinges et d. [41], and is a sesquiterpene lactone, whidi has a tert-bMtyl group and two hydroxyl groups in its chomcal structure [42]. Tlie ginkgo terpenes described above (bilobalide and ginkgolides) seem to be unique constituents to Ginkgo biloha because they have nev^ been found in any other plants. GinkgoUdes A, B, C and bilobalide have b e ^ analyzed by reversed-phase HPLC with dther UVdetectwn at 220 nm [43, 44] or refractive index detection [45]. Furthermore, bilobalide and ginkgohdes A and B were also analyzed by axillary dectrophoresis with U V detection at 1S5 nriL Reasonable separation was accomplished by a phosphate and sodium dodecyl sulfate buffer [46]. Chauret et d. [47] developed an analytical method coupling a gas chromatograph to a high-resolution mass spectrometo: operated in die selected-ion monitoring mode, and confirmed the production of ginkgolides in Gingko biloba cells cultured in vitro. Recently, a method based on liquid chromatography coupled with dectrospray mass spectrometry was developed for the analysis of terpenes in EGb [48]. Total and individual terpene (ginkgoHdes A, B, C, J and bilobalide) contents wore evaluated in leaves, shoots and roots of a young Ginkgo biloba (three years old) cultivated in a greenhouse in natural light [49]. Tlie leaves accumulate more terpenes than the roots and shoots. The contents of ginkgolide A and bilobalide readi a maximum value at the end of summer or at the begiiming of autumn. Recently Carrier et d, [50] detomined terpene contents in leaves of the terminal buds, rosettes and side branches, stem and bark, root and root meristem of the three-year-old Ginkgo biloba. Bilobalide was absent from underground parts, whereas it was the major constituent in aerial parts. Ginkgolide A occurred at the highest concentration, followed by relatively equal amounts of ginkgolides B and C, and a small amount of ginkgolide J. Laurain et d. [51] found that production of ginkgolides A, B, Q and J and bilobalide occurred in two cell cultures derived from a female prothallus and from putativdy transformed embryos, by transfection wiaiAgrobacterium rhizogenes agropine type strain CFBP2409 (A4) Other Constituents Ibataer d. [52] isolated long-chain betulapr^ol-typepolyprenols containing 14 to 22 isoprene units (41) from the ginkgo leaves and determined their structures by mass spectroscopy, ^H-
r
0)-terminal
trans
trans (41)
>
cis
,.
^
"-^ ^^ OH n a-terminal n=10-18
172
NMR and ^^C-NMR. The concentrations of these polypienols increased from 0.04 to 2.0% of dry weight with maturation of the leaves. A supercritical fluid chromatographic procedure for the quantitation of polyprenols in the ginkgo leaves was developed by Huh et d, [53]. 6-Hydroxykynurenic add was isolated from ginkgo leaves; the first time it was found in a gymnosperm. The content reached a maximum value of 0.24% in autumn [54]. To darify the taxonomical dassification, Kraus et d. [55] isolated the water-soluble polysaccharides from Ginkgo biloba leaves. The polysacdiaride mixture could be separated into GFl (MW 23,000), GF2a (MW 500,000), GF2b (MW 24,000) and GF3 (MW 40,000). GFl and GF3 are mainly composed of arabinose and galacturonic add, respectively, and they are common polysacdiarides of higher plants. On the otiier hand, GE2a and GF2b are composed of large amounts of mannose, rhamnose and glucuromc add, and seem to be unique to Ginkgo biloba. Seven k)ng-diain phenols (42a-g) were isolated from the sarootesta of ginkgo seeds, and showed antitumor activity against sarcoma 180 asdtes in mice [56]. They indude anacardic add, bilobol and cardanol, whidi are major allergemc substances of the ginkgo sarcotesta. In additk)n, the structure-activity relationship for antitumor activity against Qiinese hamster V-79 was investigated [57]. A specific method for the quantitative analysis of ginkgolic adds in the leaves and seeds of Ginkgo biloba using the HPLC-electrospray ionization-mass spectrometry technique was developed [58]. Following removal of the outer fleshy layer and roasting or boiling, the albumen of ginkgo seeds is used as a food in Asia. 4-OMethylpyridoxine (MPN) (43), whidi has been known to have antivitamin Be activity was isolated from the seed of Ginkgo biloba (the Js^anese word: gin-nan) as the toxic prindple of gin-nan food poisoning [59]. Synthetic MPN is known to induce oonvulsk)ns through a reduction of tiie brain GABAlevels in humans and in a variety of experimental animals. Furthermore, MPN is present in not only the ginkgo seed but also in its leaves. However, the amount of MPN in the extract of ginkgo leaves is much lower than the concentration that causes the detrimental effects [60]. Fiehe et d. demonstrated biosynthesis of MPN in cell-suspension cultures of Ginkgo biloba [61]. Recentiy, Wada et d, wrote a review of the literature concerning the relationship between gin-nan food poisoning and MPN [62, 63]. Kimura et d, [64] purified and diaracterized a 30 kDa Ginkgo glycoprotein from ginkgo seeds, using an antiserum againstj81-*2 xylose-containiag //-glycans.
Ri
R3 1
R2
R2 HO-1
KJ TR3
(42)
^RiJ; Ginkgoic acid (CH2)12CH3 (CH2)7CH=CH(CH2)5CH3 c. d. e. f. g.
(CH2)9CH=CH(CH2)5CH3
COOH GOGH GOGH
Bilobol (CH2)7CH=CH(CH2)5CH3 (CH2)9CH=CH(CH2)5CH3 Ginkgol (CH2)7CH=CH{CH2)5CH3 (CH2)9GH=CH(CH2)5CH3
H H H H
H 1 H
1
GH GH
1 1
H 1 H
1
H 1
173
CH20CH3 .CH20H
H 3 C ^ N ^
(43) 4-0-Methylpyridoxine
NEUROPROTECTIVE PROPERTIES OF EGB AND ITS CONSTITUENTS ON THE BRAIN There is oonvindng evidence that EGb and its constituents have neuroprotective actions under experimental conditions sudi as hypoxia/isdiemia, seizure activity and nerve damage. Cerebral Hypoxia and Cerebral Ischemia With regard to beneficial effects of EGb in situations such as hypoxia and isdicmia, Karcher et d. [65] reported that the survival time induced by hypobaric hypoxia was gready prolonged in rats treated with EGb (100 mg/kg, Lp.) before 30 min hypoxia. It was shown that the nonflavonefi-actionof EGb was responsible for the prolonged effects on the survival time of mice under lethal hypoxia [66]. EGb also retards the breakdown of the brain energy metabolism in hypoxic artificially ventilated rats [66]. Spinncwyn [67], using a geibil model of bilateral forebrain ischemia induced by occluding the conmion carotid arteries, showed that pretreatmait with EGb (30 and 60 mg/kg/day, p.o., for 14 days) produced an increase in the area of surviving hippocampal CAl neurons. Furthermore, the treatment with EGb reduced ischemic brain damage with middle cerebral artery ocdusion [68]. The teipenefiraction(ginkgolides A and B, and bilobalide) of EGb seems to be related to these effects, Le., ginkgolides A and B, and bilobalide are able to reduce the infarct volume after focal ischemia in mice and rats, and the number of damaged neurons in cultures after glutamate exdtotoxidty and hypoxia is also reduced by ginkgolide B and bilobalide [69]. Cerebral Edema Gabard and Chatterjee [70] were the first to demonstrate the protective and curative effects of EGb (100 mg/kg, p.o., twice a day for 15 days) against cerebral edema induced by triethyltin (TET, 0.002% solution in drinking water) in the rat Subsequently, Otani et d. [71] showed that concurrent administration of EGb (100 mg/kg, p.o.) and TET (0.002% in drinking water) to rats for 14 days reduced the development of a cytotoxic edema in the white matter of the brain, as well as the abnormal levels of water and sodium contents induced by TET alone. Odier experiments by Sancesario and Kreutzberg [72] showed that EGb therapy accelerated the reabsorption of TET-induced cerebral edema and improved the astroglial reaction. As regards
174
active constituents, datterjee et d, [73] indicated that bilobalide might be responsible for the anti-edema effects of EGb. Bilobalide (10 mg/kg p.o., for 6 days) and EGb (100 mg/kg p.o. for 6 days) act protectively and curatively against TET-induced dianges of neuropathobgical parameters, including body weight, consumption of food and water, and pain reaction time in a hot-plate test The TET-induced inhibitory influence on cyclic 3',5'-AMP phosphodiestCTase (PDE) activities precedes edema formation in the rat brain [74]. To darify the medianism of the protective action of EGb against TET-toxidty in rats, in vitro and ex vivo effects of EGb on PDE activities of cerebral tissue were investigated [75]. Higher concentrations of EGb (5-250 mg/L) inhibited the PDE activity in the brain in normal rats, whereas lower concentrations (0.25-4.0 mg/L) of EGb enhanced the activity of the enzyme. The inhibitory effect of TET on the high affinity PDE activity (measured with 0.25 //M cydic AMP) of the brain was diminished in the presence of low EGb concentrations. Furthermore, preventive and curative treatment of TET-poisoned rats with EGb (100 mg/kg, p.o., for 7 days) prevented both the formation of edema and the fall of PDE activity induced by TET alone. These results suggested the antiedema action of EGb might be partiy assodated with its modulating influences on cellular cyclic AMP levels via activation of membrane-bound PDE Neurotrophic Activity in Neural Iiyuiy Barkats et d. [76] showed the effects of dironic treatmoit with EGb on age-dqpendent structural dianges in the hippocampi of three strains of 15-month-old inbred mice (C57BIV6J, BALB/cJ and DBA/2J). Treatment with EGb (50 mg/kg/day, in the drinking water for 7 months) significantly inoreased the projection field of intra- and infira-pyramidal mossy fibers (iipMF) in the CA3 of the hippocampus as compared with control mice, although there was no difference in the sensitivity to EGb among the mouse strains. Since it has been considered that the size of the projection field of iipMF correlates strongly with spatial learning [77], this neuroprotective and/or neurotrophic action of EGb on the hippocampal iipMF might be useful in explainiag the benefidal effect of EGb on spatial learning. This hypothesis was supported by the findings that bilateral frontal cortex-lesioned rats treated with EGb (100 mg/kg, i.p. for 30 days) had improved retention of a delayed-spatial alternation task compared to subjects with lesions treated with saline, and that the treatment with EGb reduced the extent of brain swelling in histological examination [78]. Furthermore, EGb inaeased protein synthesis in many brain regions after unilateral labyrinthectomy in the adult rat, and the most stimulated brain regions were assodated with sensory, behavioral and learning systems [79]. EGb also protects against oxidative damage to DNA and oxidation of mitodiondrial glutathione, and prevents agerelated morphological changes in mitochondria in the brain and liver in old rats [80, 81]. Anumber of studies have been made concerning the effects of EGb on vestibular compensation. Administration of EGb (50 mg/kg, Lp. for 30 days) accelerated vestibular compensation induced by unilateral vestibular neurectomy in rats [82, 83]. The results obtained by perfusion of EGb into vestibular nuclei of alert guinea pigs suggest that EGb has a direct excitatory effect on the neuronal level [84]. Further experiments indicated that the extract without the terpenes was most effective in this experimental model of CNS plastidty involved in vestibular compensation [85]. On the other hand, Sasaki et d, investigated the effects of bilobaUde, a terpene constituent of EGb, fi-om the viewpoint of electrophysiology with the use of a rat hippocampal slice preparation [86]. Bilobalide (10-500 ^M), increases the amplitude of
175
population spikes evoked by dectrical stimuladon, indicating an enhancement of excitability of hippocampal CAl pyramidal neurons. Moreover, in an experimental model of perq)heral neuropathy, it has been shown, by means of electiophysiotogical and histological tedmiques, that bilobalide exerts trophic and protective effects on motor nerves. Hie reinnervation of the extensor digitorum longus musde following traumatk: nerve damage occurs more r^idly in bilobalide-treated rats [87]. Apoptosis Apoptosis is associated with cerd^ral ischemia and scversl neurodegenerative diseases, such as Alzheimo-'s and Parkinson* s diseases [88]. In die study described by Dtdier et al. [89], apoptosis was induced by sectioning of the olfactory nave in adult rats, and was evaluated by measuring either the thidmess of the q)ithelium in relation to neuronal death or DNA fragmentation. These workers showed that pretreatment with EGb (50 or 100 mg/kg/day) reduced the rate of lesion-induced apoptosis of olfactory neurons in the olfactory mucosa of adult rats. In addition, EGb also prevents apoptosis induced by treatmoit with hydrogen peroxide and ferrous sulfate in cerebellar neuronal cells dissociated from rats [90], and protects PC12 nerve cells in a dose-depradent manner against die B amyloid (AB)-induced apoptosis and neurotoxidty measured using the 3-(4,5-dimetiiylthia2Dl-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue assays [91]. With regard to the anti-apoptotic effect of EGb and its terpene constituents, Ahlemeyer et al. [92] compared their effects on apoptosis induced by serum dq)rivation and staurosporine. In cultured chick embryonic neurons, EGb (10 mg/L), ginkgolide B (10 ;
176
measured using 2,7-dicfaloFofluorescem diaceTatc^, and inhibit die AB-induced 4-hydioxy-2nonenal modification of spedfic mitochondrial target proteins [98]. Therefore, there is some disagreement as to which constituents of EGb are diiefly responsible for its anti-apoptotic action. It is probable that tiie animal spedes and the model used for the induction of apoptosis are involved. It needs further study before any condusions can be drawn. Anticonvulsant Activity Bilobalide, a main constituent of the teipene fraction of the EGb, possesses anticonvulsant activity against convulsions induced by pentylenetetrazol, isoniazid, 4-0-methylpyridoxine, and electroshock. Reduced durations of convulsions and prolonged onset time of convulsions induced by chonical oonvulsants and dectroshock were observed in mice treated orally with bilobalide (30 mg/kg/day, for 4 days). However, bilobalide has no protective effect against bicuculline- and strychnine-induced convulsions [99,100]. Learning and Memory Winter [101] demonstrated the effects of EGb in aperitive opoant conditioning of mice. EGb was administered orally at a dose of 100 mg/kg for 4 or 8 weeks prior to the training and was maintained for 10 weeks sftci training. Treatment with EGb increased the number of correct responses and reduced incorrect responses. Furthermore, the learning- and memoryin^roving effects of EGb were confirmed using some conditioned-reflex methods (shutde-box, step-down, stq)-through, and water maze), when the extract was administered orally for 7 days before training attiireedoses of 10, 30, and 90 mg/kg to young and old rats [102]. The radial maze has been widely employed in studies concerning learning and memory, and it has been shown to be sensitive to the aging process. In a trial using an eight-arm radial maze, effects of EGb (30 and 60 mg/kg/day) administered for 3 weeks before testing and throughout the testing period were investigated. Parameters of learning (the number of arms visited, the number of errors and time spent to complete the test) were improved in EGb-treated rats compared to controls [103]. Recendy, anotho^ trial using the eight-arm radial maze was performed in aged male Fisch^ 344 rats (20 months old). Chronic pretreatment with EGb showed a significant positive effect on continuous learning and on delayed nonmatching to position tasks in aged rats [104]. StoU et d. [105] investigated the effects of chrome treatment with EGb on age-related cognitive dianges using passive avoidance learning. Aged nuoe treated daily with 100 mg/kg EGb for three weeks had signifkantly improved short-term memory measured by the avoidance latency 60 seconds after shock, but long-term memory measured by the avoidance latency 24 hours after shodc did not improve. These results were supported by the findings that dironic (30 days) and acute treatments with EGb (60 mg/kg) oihanced short-term memory on olfactory recognition in young and aged rats [106]. Administration of scopolamine to rats induces amnesia in passive avoidance learning. EGb administered intraperitonealiy 30 min before the initial trial at doses of 150-500 mg/kg significantly att^uated the amnesic effects of scopolamine on step-through latendes in a retention trial 4 hours after training for the passive avoidance test in rats [107]. Hoyo* et d, showed that, using an animal model of intrac^ebroventricular streptozotodn treatment, EGb treatment compensated for deterioration in working memory, referrace memory and passive
177
avoidance bdiavior and restored a defikit in co-ebral energy metabolism [108]. Furthennore, pretreatment with EGb (50 and 100 mg/kg, p.o.) ameliorated the impairment of memory induced by bilateral occlusion of the carotid arteries in mice when the passive avoidance test was carried out 48 hours after ischemia [109]. These results lead to the condusion that EGb possesses cognition-enhandng properties. Anti-Stress and Antidepressant Effects Porsolt et al. investigated the effects of repeated oral administration of EGb (SO and 100 mg/kg) on various behavioral models of stress in rodents [110, 111]. The bdiavioral models induded learned he^lessness", shock-suppressed licking (\bgel conflict test), forced swimminginduced immobility C^havioral despair^, shock-suppressed expiration (four-plates test), spontaneous exploration (staircase test) and food consumption (emotional hypophagia). EGb inaeased the amount of food consumption in the emotbnal hypophagia test and reduced the acquisition of behavbral defidts induced by dectric shocks in the learned helplessness" paradigm in both young and old rodents. However, EGb had no marked effects in other models tested. These results indicated that EGb has anti-stress propoties; however, it cannot be assimilated into either classical antidepressant or anxiolytic activity. In addition, it was shown that treatmoit with EGb (100 mg/kg, in 5% ethanol) reduces the development of the polydipsia induced by the stress of daily handling, anesthetization with ether and oral intubation [112]. Rapin et al. [113] have reported that oral treatment with EGb (50 or 100 mg/kg/day, for 20 days) suppresses auditory stress-induced alterations of discrimination learning in both young and old rats; EGb was espedally effective in decreasing the number of inefSdent lever presses and in reducing the reaction time in older animals. Furthermore, treatment with EGb counteracted the auditory stress-induced inaeases in the plasma concentrations of epinq)hrine, norepinephrine and corticosterone in both young and old rats. Wada et al. revealed that feeding a diet containing a 5% powdered dried Ginkgo biloha leaves or an aqueous extract of these leaves to 4-week-old male ddY mice for 7 days shortened the sleeping times induced by anesthetics (hexobarbital, a-chloralose plus urethane). A similar effect was obtained in animals treated orally widi bilobalide or ginkgolide A (both at 10 mg/kg) [114]. Subsequently, Brochet et al. [115] showed effects of single intraperitoneal injections of EGb and ginkgolide B and bilobalide on barbital-induced narcosis in the mouse. Single injections of EGb (25 and 50 mg/kg) ginkgolide B (1 mg/kg) and bilobalide (2 and 5 mg/kg), 60 min prior to sodium barbital (180 mg/kg, i.p.), significantly shortened barbital-induced sleeping time and increased the latency to onset of sleep. From these data, they suggested that EGb indudes constituents that have CNS-stimulatory activity.
MECHANISMS OF ACTION OF EGB AND ITS ACTIVE CONSTITUENTS It has been proposed that possible mechanisms undo'lying the neuroprotective and/or neurotrophic propolies of EGb in the CNS indude influences on neurotransmitters, oiergy metabolism and membrane functions, as well as antioxidant activities and inhibition of plateletactivation factor (PAF).
178
Improvement in Enei^gy Metabolism As shown in Figure (1), injected [**C]2-deoxy-D-glucose ( f ^CJEXj) is transported toward cerebral tissue through the blood braio barrier and then phosphorylated into [^^C]£)G-6phosphate ([^^C]DG-6P), whicfa accumulates in the cells. Measuring radioactivity in brain sections by autoradiography, it is possible to calculate local cerebral glucose utilization (LCGU) [116]. Krieglstein et d. [117] investigated effects of EGb on LCGU by means of the [^^C]DG tedmique and on local cerebral blood flow (LCBF) using [^^Cjiodoantipyrine. Pretreatment with EGb (130 mg/kg, Lv.) significantly increased LCBF and blood glucose levels, although there was no influence of EGb on LCGU. On the other hand, in the awake adult rat, Lamour et d. [118] found significant and widespread decreases (range 3-26% reduction) of LCGU in 21 brain regions of rats injected with EGb (150 mg/kg, i.p.), but not ginkgolide B (10 mg/kg, i.p.). TTic largest dedines of LCGU were observed in the hq)pocampal formation, cerebral cortex, globus pallidus, caudate-putamen, ventral thalamus, infmor oolliculus and superior olive. Similar results were obtained when rats received rq)eated treatment with EGb (50 mg/kg/day, for 15 days) [119]. In regard to LUGU, this discrepancy between these data and those of Krieglstein et d. may be due to different methods of drug administration (L v. vs. Lp., acute vs. dironic).
BBB rPlasman Glu
[^^C]DG
Brain
I - • Glu
^[i4c]DG —•[^^C]DG-6P
Fig. (1). Representative model of c^ebral glucose (Glu) consumption. The labeled 2-deoxyglucose ([^^C]DG) injected enters into competition with the circulating glucose through the blood-brain barrier (BBB), and stops at the 2-deoxygluoose-6-phosphate ([^^C]DG-6P).
When ischemia or hypoxia impairs the oxygen supply, mitochondrial respiration is deaeased and consequently aiergy production is damaged. Glycolysis is enhanced to compensate for the decrease in ATP regeneration. TWo experimental rat ischemia models (normobaric hypoxia and carotid clamping) induced a diminution in glucose uptake and LCGU. EGb (100 mg/kg p.o. for 5 days) administered preventively increased deoxyglucose uptake and
179
LCGU in the cortex, hippocampus, and caudate nudei of rats subjected to normobaric hypoxia. Treatmoit with EGb significantly increased LCGU in rats with bilateral ligature of the carotid arteries, but did not modify the deoxyglucose uptake [120]. Kardier et al. [65] estimated the brain energy metabolism in rats treated with EGb (100 mg/kg Lp. 30 min before hypoxia) under various experimental conditions. Before as well as after hypobaric hypoxia, the brain glucose level and glucose-6-phosphate level were elevated by EGb, probably due to enhanced cerebral blood flow. In the situations of hypobaric or hypoxk hypoxia, the cerebral lactate level of EGb-treated rats was slighdy lower, whereas its pyruvate level was elevated as compared with controls, and tiierefore the lactate^yruvate ratk) was markedly deaeased. In addition, the levels of cerebral aeatine-P and ATP were less severely deaeased by EGb treatment after 7.5 min of hypobaric hypoxia Janssens et d. [121] showed that EGb and bilobalide inhibited the hypoxia-induced decrease in ATP content of endothelial cells in vitro. In addition, these compounds delayed the onset of glycolytic activation, as evidenced by increased glucose transport, as well as by inaeased lactate production under hypoxia. Tbe respiratory control ratio of mitochondria isolated from livers of rats treated orally with EGb and bilobaUde increased, suggesting the protection of ATP content These results indicated that EGb and bilobalide prevented tfie uncoupling of oxidative phosphorylation in mitochondrial respiration. Further investigations showed that in vivo (8 mg/kg) and in vitro (0.8 /nM) treatments with bilobalide markedly inaease the mitochondrial respiratory control ratio, probably by lowaing oxygen consumption during state 4. Bilobalide protects both complexes I and in from inhibition induced by Amytal or antimydn A [122]. Further studies demonstrated that bilobalide prevents the deaease in the state 3 respiration rate induced by ischemia in the liver and brain [123]. Hierefore, bilobalide may protect mitochondrial respiratory activity under ischemic conditions by maintaining complex I and III activities, thus preserving ATP regeneration as long as oxygen is present These medianisms appear useful in explaining the protective effect of EGb on the onset of ischemia-induced damage. Furthermore, Bruel et d. [124] found that EGb (0.25 //g/ml) inaeases glucose transport and glycogen synthesis in cultured vascular smooth musde cells. Essentially similar results were obtained by oral treatment with EGb in liver and skeletal musdes [125], and in erythrocytes [126], Vasseur et d, [127], using intracellular mkaroelectrodes, indksited that administration of EGb (200 mg/kg/day, p.o.) or bilobalide (8 mg/kg day, i.p.) to mice increases the in vitro sensitivity of their pancreatic 6 cells to glucose. In addition, EGb and bilobalide correct the impaired glycogen synthesis in liver and muscle cells of the diabetic rats injected with alloxan or strq)tozotocin [125, 127]. Taken together, these findings indicate that administration of EGb causes an increase in glucose uptake and glycogen synthesis in various tissues in part via its bilobalide constituent, and EGb could be useful in treating non-insulindependent diabetes mellitus. Antioxidant Effects In dissodated rat cerebellar neurons, the effect of EGb on oxidative metabolism was studied using a flow-cytometer and 2',7-dichlorofluorescin (DCFH), which is oxidized to a highly fluorescent compound by intracellular hydrogen peroxide [128]. Preincubation with EGb dose-dependently decreases DCFH fluorescence, and reduces the isonomydn-induced increase of DCFH fluorescence, suggesting that EGb reduces oxidative metabolism in both resting and
180
Ca^Moaded brain neurons [129, 130]. Further experiments indicated that myrioetin and qu^oetin, tiie flavonoid constituents of EGb, reduce oxidation of DCFH in both resting and Ca^Moaded brain neurons [131]. EGb also gready delays the time-dependent increase in the number of dead cerebellar neurons during exposure to hydrogen peroxide [132]. Taken together, these results indicate that EGb and its flavone constituents protect the brain neurons against oxidative stress induced by hydrogen peroxide, whidi is involved in ischemic brain damage. An antioxidant action of EGb has been reported against peroxyl radicals, hydroxyl radicals and superoxide anions [133, 134, 135, 136]. Production of activated oxygen species (Oj*, H2O2, OH') in human neutrophils stimulated with phoibol myristate acetate is significantly decreased in the presence of EGb [134, 135]. The free radical scavenging activities of ginkgo flavonoids against the diphenyl picryl hydrazyl radical are as follows: myricetin > quercetin > kaempferol > luteolin. As for biflavones, the best radical scavengo* is amentoflavone, followed by bilobetin, ginkgetin, isoginkgetin, and sdadopitysin [137]. Recently, free radical scavenging activities of terpene-free EGb and quercetin w^e revealed by means of an in vitro electro-spin resonance assay [138]. Additionally, the in vivo experiments showed that terpene-free EGb inhibits cutaneous blood flux, whidi reflects the skin inflammatory level [138]. In regard to ginkgo terpenes, it has been revealed by means of electron paramagnetic resonance and U\7VIS spectroscopy that ginkgolides B, C, J and M, as well as bilobalide but not ginkgolide A, scavenge superoxide and hydroperoxyl radicals in dimethyl sulfoxide as an aprotic solvent [139]. Akiba et d. showed that EGb prevents the platelet aggregation induced by a combination of 100 f4M terr-butyl hydroperoxide and Fe^*. However, ginkgolides A, B and C, which are known to be PAF-antagonists, have no influence on this aggregation. Therefore, it was suggested that free radicals, but not FAF, might be involved in platelet aggregatk)n induced by oxidative stress [140]. Serotonin (5-HT) produces a rapid elevation of superoxide that stimulates the mitogenesis of bovine pulmonary artery smooth muscle ceUs (SMCs). EGb scavenges superoxide elevated by 5-HT, hence preventing 5-HT-induced mitogenesis on both SMCs and Chinese hamster lung fibroblasts. These results indicate that EGb inhibits the cellular transduction signaling process that leads to mitogenesis, as a result of its antioxidant activity [141]. In addition to radical scavenging properties, it has been reported that EGb reacts with nitric oxide (NO) in in vitro systems [136], and inhibits NO production induced by lipopolysaccharide plus intoferon-Y in maaophage cell Une RAW 264.7 [142]. Fre-treatment with oral administration of EGb reduced nitric oxide overproduction after transient brain ischemia in the MongoHan gerbil [143]. Further experiments showed that EGb inhibits NO production by attenuating the level of iNOS mRNA in a human endothelial cell line (ECV304) [144], also inhibits the activation of protein kinase C (PKC) induced by sodium nitroprusside (SNP), NO generator, and that its flavonoid constituents have protective properties against toxicity induced by SNP on cells of the hippocampus [145]. Recently, it was shown that ginkgolide A, ginkgolide B and bilobalide inhibit NO production in macrophages derived from a human monocytic cell line through attenuation of iNOS mRNA expression. However, these components have no effect on the eNOS-mediated NO production in endothelial ceUs [146].
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Influences on the Neurotransmitters Numerous studies have demonstrated age-related changes in levels of neurotransmitters and their recq>tors in certain areas of the brain. There is a decrease in the levels of acetylcholine and in the numbers of muscarinic receptors and 6-adrenocqptors in the c^ebral cortex and hippocampus of the brain in patients suffering from Alzheimer's disease and in the brains of aging rodents, diaracterized behaviorally by a sevore impairment ia cognitive functions [147, 148, 149]. Numbers of 5-HT recq)tors and levels of dopamine and noradrcnalin and 5-HT have also shown age-related diminution [150, 151, 152], and are known to be involved in the regulation of mood [153]. Furthermore, it has been demonstrated that the activity of monoanune oxidase (NfAO), which r^ulates the brain concentrations of 5-HX norq)inephrine and other biogenic amines, inaeases with advancing age [154]. Hius, the inhibition of NfAO has been shown to produce antidepressant or anxiolytic responses in animal models and in man [155]. Brain Levels of Biogenic Monoamines Nforier-Teissier et d. [156] determined that administration of EGb alters the levels of catecholamines, indolamines and their metabolites in some brain areas of young rats and mice. Marked changes in the EGb-treated brain were found for norepinephrine, 5-HT, and its metabotite, 5-hydroxyindole-3-acetic add, whereas it was less effective for dopamine and its m^abolite 3,4-dihydroxy-phenylacetic add. EGb-induced changes depend on the route of administration (p. o. or L p.), dose and duration of treatment (acute or dironic). In old rats (26 months old), oral administration of EGb (10 mg/kg and 30 mg/kg, for 7 days) produces elevations of 5-HT in the frontal cortex, hippocampus, striatum and hypothalamus, and of dopamine levels in the hippocampus and hypothalamus compared with controls. On the other hand, EGb decreases the 5-HT level in the pons, and those of norepinephrine in the hippocampus and hypothalamus [157]. In this connection, Racagni et al, [158] showed that the O-methylated amine metaboUte of norepinephnne, normetanq)hrine, was markedly elevated (+500%) in the cerdjral cortex by du:onic oral administration of EGb (100 mg/kg, for 14 days), suggesting an increase of norq)inephrine turnover. In additbn, treatment with EGb (50 or 100 mg/kg/day, for 20 days) diminished the inareased plasma levels of epiDq)hrine, norepinephrine, and corticosterone induced by acute auditory stress in young and old rats [113]. GABA is the major inhibitory neurotransmitter in the CNS and acts to counter glutamateinduced exdtatk)n. Bilobalide (30 mg/kg/day, p.o., for 4 days) elevates GABA levels in the hippocampus and cerebral cortex in mice. These effects of bilobalide are due to a potentiation in glutamic add decarboxylase activity and an enhancement in the protein amount of 67 kDa glutamate decarboxylase. Furthermore, isoniazid and 4-O-methylpyridoxine, pot^t convulsants, induce reductk)ns in brain GABA levels, whereas bilobalide counteracts these effects. These results indicate that potentiation of GABAergic transmission induced by bilobalide might explain its anticonvulsant activity against isoniazid and 4-Omethylpyridoxine [159,160]. Monoamine Oxidase Activity White et al, [161] explored in rat brain mitodiondrial extracts the effect of EGb on MAO activity in vitro. MAOA and MAOB activities wore assayed using [^H]5-HT and [^*C]B-
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phenetfaylamme as substrates, respectively. EGb inhibited both NfAOAand MAOB activities of rats and mice in vitro to similar extents. These results have suggested that the inhibition of MAO may be a mechanism underlying antidq)ressant or anxiolytk: responses of this extract obtained in animal models and man. Similar observations using a fluorimetric method were shown for EGb, but not for ginkgolide A and ginkgolide B [162]. Sloley et d. showed that kaempferol is a primal in vivo, but not ex vivo, rat brain MAQ-inhibitor in EGb [163]. Besides, the effects of long-term treatment with EGb (500 mg^g/day, for 7 months) on c^ebral MAO activity were investigated in mice subjected to a chronic mild stress. EGb induced reductions in basal MAO activity in 18-month-old mice. Hie age-related inaease in brain MAO activity was lower in die untreated mice subjected to stress and EGb potentiated this effect [164]. Recently, the effects of EGb on aggression woe investigated using MAO-A knockout nuce. EGb reduced their aggressive behavior in resident-intruder confrontations to levels seen in wild types, and decreased their [^H]ketanserin binding to 5-HT2A reoq)tors in the frontal cortex [165]. On the other hand. Fowler et d, recently measured MAO-A and MAO-B activities in the human brain using positron emission tomography and ["C]dorgyline and ["C]Ir
Receptors
Taylor [168] examined the effects of chronic administration of EGb (100 mg^g/day, for 28 days in drinking water) on the binding of [^H]quinudyidinyl benzilate ([^H]QNB) to the muscarinic cholinergic receptors of die hippocampus of young (3 months old) and old (24 months old) male Fisher 344 rats. Asignificant diminution in the number (B^^) of muscarinic receptors was observed in the old rats compared to the young rats. In contrast, EGb produced a maiiced iacrease of the B,^ value io the hippocampus of old rats in comparison to controls of the same age, and also a slight increase of the B,^ in the young rats. Rapin et d, [11] have reported an increase of the acetylcholine synthesis rate constant evaluated by a bolus injection of [^H]choline in the hqipocampus of 4-month-old rats after acute administration of EGb (100 mg/kg Lp.). Similar resuhs were obtained in the frontal cortex, hippocampus and corpus striatum after dironic treatment with EGb (100 mg^g/day p.o. for 21 days). On the other hand, the acetylcholine turnover rate was not modified by either acute or chronic administration of EGb. These results indicate that EGb might increase acetylcholine release. In the cholinergic nerve terminals of the hippocampus of 24-month-old Wistar rats, Kristofikova et d, [169] showed that both in vivo administration with EGb (50 mg/kg/day for 30 days in drinking water) and in vitro applicatbn of EGb to synaptosomes (15-30 /^g/ml, i.e. 50-100 //g/mg proteio) cause significant increases in high-affinity dioline uptake (HACU) levels. Subsequently, these workers showed that EGb (100 figfal) markedly elevates the specific binding of [^H]hemidiolinium-3 ([^H]HCh-3) (to 306%) in hippocampal synaptosomes from young Wistar rats, suggesting that EGb causes an ino-ease in the number of dioline
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cairiers [170]. Taken together, these results suggest tiiat EGb might enhance the cholinergic neurotransmitter syston on presynaptic nerve terminals.
Adrenoceptors With regard to the number ( B ^ ) of 6-adr^oceptors measured with pH]dihydroa^)renolol ([^HjDHA) in rat oerd)ral cortex, Taylor [168] demonstrated that chronic administration of EGb (100 mg/kg/d for 28 days) had no effect on fi-adroiergic binding in either young or old rats, although tiie B ^ value for B-adrenocq)tors was significantly reduced with advance of age. However, Brunello et d. [171] and Racagni et d. [158] rq)orted that the afGnity (K^) and B ^ of [^H]ra[A binding to B-adrenocqptors decreased with diromc treatment with EGb (lOOmg/kg/day) for 2 months or 30 days. In aged rats (24 months old), the B ^ of [^H]rauwolscinc binding to aa-adrenoceptors in the hippocampus and cerebral cortex is significantly reduced as compared with young rats (4 months old). Chronic treatment with EGb (5 mg/kg/day, Lp. for 21 days) does not alter the BB„ value for Cj-adrenoccptors binding measured with [^HJrauwolsdne in the hippocampus of young rats, but significantly increases the B , ^ of [^H]rauwolscine binding in aged rats [172]. These results suggest that a^-adrenoceptors involved in the control of noradrenergic activity are more susceptible to EGb treatment in aged rats. [^HJAmine
Uptake
EGb inhibits the uptake of [^H]norepinq)hrine ([^H]NE) and [^H]dopamine and [^H]5hydroxytryptamine^^[^H]5-HT) into in vitro synaptosomes prepared from the striatum and cortex in a concentration-dq)endent mann^. Tlie rank order of potency for the inhibition of amine uptake is NE > dopamine > 5-HT [173]. Similar results were obtained by Ramassamy er d, [174]. These workers showed that EGb deaeased the specific uptakes of [^H]dopamine, [^H]5-HT and [^H]choline by synaptosomes prepared from tiie striatum of mice in a concentration-dependent manner. Tlie IQ^ values were 637 figfiol for [^H]dopamine uptake, 803 /ig/ml for [^H]5-HT uptake, >2000 //gAnl for [^H]choline uptake. However, they conduded that the inhibition of amine uptake caused by EGb appears to be non-specific, since EGb also prevents the specific binding of the dopamine uptake inhibitor [^H]GBR12783 to membranes prq)ared from striatum. EGb in vitro modifies the [^H]5-HT uptake by synaptosomes prepared firom nuce cerebral cortex in a biphasic manner. As mentbned above, the uptake of [^H]5-HT is inhibited by a high concentration of EGb [174]. On the other hand, low concentrations of EGb (4-16 //g/ml) A similar inaease was also obtained when significantly inaease [^H]5-HT uptake. synaptosomes were prepared from the cortk:es of mice treated orally with EGb, either acutely (100 mg/kg, 14 hours and 2 hours before death) or semi-dironically (2 x 100 mg/kg/day, for 4 days). Furthermore, such an inaement in the [^H]5-HT uptake is attributed to the flavonoid constituents of EGb [175], and may be associated with the mechanism of its antidq)ressant activity.
5'HT Receptors Adeaeased (22%) number of 5-HTi^ recq)tor binding sites labeled by [^H]8-hydroxy-2(di-/i-
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propylainino)tetralin (pHJS-OH-DPAT), a S-HTi^ receptor agonist, in cerebral cortex membranes of Wistar rats was observed in aged (24 months old) rats as compared with young (4 months old) animals. Chronic treatment with EGb (5 mg/kg/day, for 21 days) did not alter the B ^ value in young rats, whereas it significantly inaeased it in aged rats (33%) [176]. On the other hand, Bolanos-Jim^iez et d, showed that chronic treatment with EGb (50 mg/kg/day, 14 days) produced a relatively small diminution in pHJS-OH-DPAT binding to hippocampal 5-HTi;^ receptors in 18-month-old rats [177]. There is at the moment no dear explanation for this disaepancy. An inhibitory effect of S-OH-DPAT on forskolin-stimulated adenylyl cyclase activity is observed in hippocampal membranes of the guinea pig and rat, and has been used as an index of the functional activities of S-HTj^ receptors [178]. Q)ld stress induces a reduction of the inhibitory effect of S-OH-DPAT in the hippocampus isolated from 18-month-old rats, although it has no influence on either the affinity or number of [^H]8-0H-DPAr binding sites. The administration of EGb (50 mg/kg p.o. for 14 days) prevents the cold stress-induced reduction in the inhibitory effect of 8-(Xl-DPAr on forskolin-stimulated adenylyl cyclase activity in old rats. These results indicate that EGb prevents the stress-induced desensitization of hippocampal 5-HTu^ receptors; thus, its effects might explain anti-stress and antidepressant properties of EGb [177].
NMDA Receptor Taylor [173] showed that EGb acts in vitro as an inhibitor of radioligand binding to the competitive and non-competitive sites of ^-methyl-i>-aspartate (NMDA) receptors. In addition, the most potent inhibition (K^ = 0.5 mg/ml) is observed for non-competitive NMDAsites labeled by ['H]MK-801. MPTP-Induced Dopaminergic Neurotoxicity It is known that l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) selectively causes degeneration of the nigrostriatal dopaminergic neuronal pathway in several animal species, which is considered an animal model of Parkinson's disease [179]. As shown in Figure (2), MPTP administered systematically crosses the blood brain barrier, and is oxidized by MAOB into MPP*. This metaboUte is concentrated into dopaminergic neurons and consequently destroys such neurons by generation of ifree radicals [180, 181]. In mice implanted subcutaneously with osmotic minipumps releasing MPTP for 7 days (105 /^g/h/mouse) (approximately 100 mg/kg/day), a decrease in [^H]dopamine uptake by a synaptosomal fraction prepared from striatum was observed. This neurotoxic effect was prevented by the chronic injection of EGb (approximately 100 mg/kg/day in drinking tap water) for 17 days. Such a protective activity of EGb against MPTP neurotoxicity is unlikely to depend on inhibition of the MPTP uptake by dopamine neurons, since the concentration at which EGb prevents [^H]dopamine uptake is too high to reach the brain under in vivo experimental conditions [174, 182]. Therefore, it appears likely that a possible explanation lies in thefree-radicalscavenging property of EGb, which neutralizes free radicals generated from MPP* in the dopaminergic neurons. In this regard, another study showed that protective and curative treatments with EGb prevented the reduction of striatal dopamine levels induced by MPTP. MPTP (30 mg/kg/day.
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BBB I i^Astrocyte
Nigrostriatal dopamine neuron
MPTP • ' - > MPTP injection
Fig. (2). Hypothesized mechanisms of neurotoxicity of MPTP. After injection of MPTP, its native fomi crosses the blood brain barrier (BBB) and is oxidized by monoamine oxidase B (MAO-B) into MPP^. This metabolite is transported and concentrated into nigrostriatal dopamine and exerts a neurotoxic effect
i.p. for 6 days) sigtdficantly reduced striatal dopamine levels in C57 mice. On the other hand, when C57 mice were pretreated with EGb (20, 50, 100 mg/kg/day, Lp.) for 7 days and then treated with the same extract 30 min before MPTP injection for 6 days, the neurotoxic effect of MPTP was antagonized in a dose-dependent manner. Moreover, in mice treated with EGb (50 mg/kg/day, Lp.) for 2 weeks after MPTP-lesion, the recovery of striatal dopamine levels was accelerated. MPTP is oxidized by MAO-B into MPP^ a positively charged species, whereas EGb, but not ginkgolides A and B, inhibits MAO-B activity. Therefore, another possible explanation for this protectk)n might lie in the inhibition of MAO activity caused by EGb to prevent the oxidization of MPTP into MPP* [162]. Effect of EGb on the Neuroendocrine System It is well known that neuro^docrine dianges with advancing age provide information about CNS functions [183]. The serum prolactin (PRL) level inaeases in old rats (26 months old) compared with young rats (3 months old). Administration of EGb (10 mg/kg/day, p.o., for 7 days) deaeases the blood PRL level, while greatly inaeasing adrenocorticotrophic hormone (ACTH) in old rats compared with age-matched controls. On the other hand, EGb, at a dose of 30 mg/kg, deaeases the serum level of growth hormone (GH) and ACTH in young rats compared with age-matdied controls [157]. Glucocorticoids are also essential for many aspects of normal brain development; however, hyperseaetion induces pathological states such as damage to the hippocampus [184]. Treatment with EGb (100 mg/kg/day, for 8 days) causes a 50% reduction of the plasma corticosterone level This reduction is probably due to deaeases in the number (B,^), mRNA
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expression and protein levels of adrenal mitochondrial peripheral-type benzodiazepine receptors (PBR), whidi are a key element in the r^ulation of cholesterol transport Similar results have been observed in the chronic administration of ginkgolides A and B (2 mg/kg/day, i.p. for 8 days)[185]. Further study demonstrated that EGb and ginkgolide B also decreased PBR expression and cell proliferatk)n in the highly aggressive human breast cancer cell line MDA-231, which is ridi in PBR [186]. With regard to steroidogenesis, in addition to PBR, an ACTH-dependent process is also responsible for its regulatfon. Using cultured adrenocortical cells, Amri et al, [187] have shown that ex vivo treatment with EGb (100 mg/kg/day, p.o., for 8 days) and ginkgolide B (2 mg/kg/day, i p . , for 8 days) reduces ACTHstimulated cortkx)Sterone production by 50% and 80%, respectively. Moreover, Mardlhac et al. [188] showed that administration of EGb (50 or 100 mg/kg p.o., for 14 days) reduces basal cortkx)sterone seaetion and the subsequent inaease in oortkx>tropin-releasing hormone (CRH) and arginine vasopressin {/^/?) gene expression. Ginkgolide B (2 mg/kg/day i.p., for 14 days) reduces basal corticosterone seaetion without alteration in the subsequent CRH and /WF inorease. However, the stimulation of CRH gene expression by insulin-induced hypoglycemia is attenuated by ginkgolide B. These results indkrate that EGb and ginkgolide B are also able to affect the hypothalamic-pituitary-adrenal axis at the hypothalamic level Corticosteroids play a pivotal role for the development of behavbral sensitization to an^hetamine through the type II glucocorticoid receptor [189], Trovero et d. [190] showed that EGb reduces D-amphetamine (0.5 mg/kg, i.p., for 12-24 days)-induced behavioral sensitization as estimated by increasing values of locomotor activity, although EGb itself has no locomotor effect Furthermore, chronic administration of D-amphetamine reduces the density of [^H]dexamethasone binding sites of type II glucocorticoid recq)tors in the dentate gyrus and the CAl hippocampal regions of D-amphetamine-treated animals, indkating down-regulation of type II glucocorticoid receptors. On the other hand, pretreatment with EGb (50 or 100 mg/kg/day, p.o., for 20-24 days) prevents this down-regulation of type II glucocorticoid receptors, suggesting that EGb restores the density of these receptors. Taken together, these studies indicate that EGb is able to modukte both stress-induced and age-related behavioral sensitizatk>n by regulating alterations of the neuroendocrine system. Effect of EGb on the Phospholipid Metabolism Effects of EGb on ischemia-induced principal changes in the cerebral lipid metabolism were reviewed by Robin et al. [191]. Figure (3) shows diagram of cerebral lipid metabolism following ischemia. Pretreatment witii EGb could normalize the increased mitodiondrial lipid peroxide content and cytosolic lactase dehydrogoiase activity and the deaeased mitochondrial phospholipid contents and superoxide dismutase activity in rat brain after occlusion of common carotid arteries [192]. Rogue et d. [193] have shown that EGb is a potent inhibitor of phospholipase C (PKQ in vitro (ICJQ: 82 //g/ml). Furthermore, in isdiemic rats pretreated with a single injection of EGb (100 mg/kg, i.p.), a deaease in PKC activity is observed as compared with untreated ischemic animals. Impairment of membrane-bound Na,K-ArPase, whidi is responsible for maintaining and restoring membrane potential, and an increased level of malondialdehyde (MDA), which is a known as an index of lipid peroxidation, are seen aft^ unilateral focal cerebral ischemia in the mouse. Pretreatment witii EGb (100 mg/kg/day, p.o. for 10 days) preserves the Na,K-ArPase activity during cerebral ischemia and prev«its the kiaeased MDA levels caused by cerebral
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Hydroperoxide Prostaglandin Leukotriene
Fig. (3). Diagram of cerebral lipid metabolism following ischemia. During ischemia, AIT-synthesis is disturbed by deficiencies in oxygen and glucose supplies. The subsequent energy failure stimulates phospholipase C (PLC) and Aj/Aj (PIAj/Ai), and thereby leads to formation of diacylglycerol (DAG), which is converted to free fatty adds (FFA) by Upasc, and leads to accumulation FFA and lysophospholipids. Among FFA, especially, the peroxidation of arachidonic add (AA) initiates a cascade leading to lq)oxygenase and cydooxygenase metabolites (prostaglandins and leukotrienes) and hydroperoxide, which are augmented during reperfusion following ischemia. Aoetylation of lyso-platelet-activating factor (lyso-PAF) leads to PAF, a mediator of inflammation.
ischemia [194]. Similar inhibitory effects of EGb on MDA production induced by hydrogen peroxide have been shown in erythrocyte membranes [195, 196]. Electroconvulsive shock (ECS), as well as ischemia, induces inaeases in free fatty add (FFA) and diacylglycerol (DAG) in the rat brain, probably due to the breakdown of membrane phospholipids through the activation of phospholipases (PLC, PLAj/Aj). EGb treatment (100 mg/kg/day, p.o. for 14 days) selectively decreases endogenous FFA levels and increases endogenous DAG levels in the hippocampus. Therefore, ECS-induced accumulation of FFAis prevented in the hippocampus of EGb-treated rats during clonic seizures (30 sec to 2 min after
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ECS). Furthermore, the inaeased DAG levels induced by ECS are delayed by EGb treatment and the subsequent decrease in DAG levels is accelerated by EGb treatment in both the hippocampus and cortex [197]. Hypoxic or ischemic conditions led to an immediate release of free dioline via the breakdown of choline-containing phospholipids in rat hippocampus slices. Klein et d. [198] showed that bilobalide inhibited the hypoxia-induced dioline release in a dose-dependent manner both in vitro (ECjo*. 0.38 /^M) and ex vivo (2-20 mgAcg, p.o.). Asimilar reduction of dioline release was confirmed after administration of EGb (200 mg/kg, p.o.). Bilobalide also inhibits the ^-methyl-D-aspartate-induced, PLAj-dependent release of dioline from hippocampal phosphol4)ids both in vitro (10-100;^M) and in vivo (20 mg/kg, Lp.) [199]. Rabin et d. [200] investigated the effect of EGb on the rate of FFAreincorporation into brain phospholipids during reperfiision following ischemia in the gerbil brain by means of quantitative autoradiography and biodiemical analysis. Isdiemia-reperfusk>n selectively reincorporated arachidonic add (AA) into brain phospholipids. Pretreatmait with EGb (50 or 150 mg/kg/day, for 14 days) accelerated AA reincorporation following isdiemia, suggesting that EGb ameliorates the neurotoxic reaction caused by prolonged ^posure of the brain to high concentrations of AAand its metabolites, and stabilizes the membrane bilayer. Taken together, these results showed that EGb can prevent isdiemia-induced Na,K-ArPase injury, and suppress hypoxia- and ECS-induced membrane phospholipid breakdown in the brain, and bilobalide might be associated with its protective action. In addition, EGb reduces AA-induced neuronal damage as a consequence of the increase in reincorporation of A A Therefore, these medianisms might provide a possible explanation for neuroprotective properties of EGb and bilobalide against oxidative damage. Anti-PAF Platelet-activating factor (PAF), a potent phospholipid inflammatory mediator, enhances glutamatergic exdtatory synaptic transmission in the h^>pocampus [201]. Braquet et d, [202, 203] showed that ginkgolides, mainly ginkgolide B, act as potent antagonists of PAF in various cell types. Pretreatment of ginkgolides (10 mg/kg/day, p.o., for 7 days) ameliorated behavioral impairments assessed by the MacGraw stroke index, and inq)roved the mitodiondrial respiration evaluated by the respiratory control ratio (RCR) following c^ebral ischemia obtained by bilat^al ligature of the common carotid arteries in Mongolian g^bils [204]. The order of these effects in cerebral isdiemia was ginkgolide B > ginkgolide A > ginkgolide C > ginkgolide J, and was correlated with that of their PAF antagonistic properties described by Braquet et d, [203]. Consistent with this finding, liu et d. showed that both preand post-hypoxic treatment with ginkgolide B (25 mg/kg/dose, two serial doses) decreased the inddence of cerebral infarction in hypoxic ischemic brain injury of immature rats [205]. Moreover, Akisu et d, [206] found that endogenous PAF concentrations in brain tissue markedly inaeased in the hypoxic-ischemic brain in immature rats. Pretreatment with EGb reduces endogenous PAF concentrations in cerebral hypoxic-ischemic brain injury of immature rats as compared with controls. These results indicate that the PAF-antagonistic activity of the ginkgolides contributes to the neuroprotective effect against brain injury associated with an episode of the post-isdiemic phase. This idea was supported further by the observation that in primary neuronal cultures isolated from onbryonic rat cerebral cortex, ginkgolide B demonstrates protective effects against glutamate neurotoxidty involving PAF [207].
189
It has been demonstrated that ginkgolide B prevents bng-tenn potentiation (LIP) induced by PAF in the h^}pocampus [208] and in the voitral part of the medial vestibular nudei [209]. These results suggest that PAF might act as a retrograde messenger in ITP, which activates the presynaptic mechanisms enhancing the glutamate release. Izqui^do et d, found that pre- or immediate post-training intrahippocampai or intraamygdala infusion of the PAF antagonist, ginkgolide B, produces amnesia for avoidance tasks. These results support the idea that PAF may play a role in memory formatfon [210]. Amine Uptake and Membrane Fluidity Protonged incubatbn of synaptosomes prqiared from the striatum in the presence of ascorbk: add (10^ M) decreases the ability of synaptosomes to take up [^H]dopamiae. Similar inhibition is also observed in the ability of cortical synaptosomes to take up [^H]5-HT. Furthermore, this decrease is potaitiated by addition of Fe^* tons. EGb (4-16 /igAni), in particular its flavonoid fraction, prevents the reduction in the ability of synaptosomes to take up either 5-HT or dopamine [211]. Moreover, EGb (10 //g/ml) prevents a decrease in binding of [^H]GBR12783, the dopamine uptake inhibitor, to dopamine recq)tors in the presence of the combination of ascorbic add/Fe^* ions. These results suggest that EGb-induced prevention of the impainnent of the ability of synaptosomes to take up [^H]amine by the ascorbic add/Fe^* ions is related its inhibitory properties in generation of free radicals. However, EGb does not modify the inaeased [^H]dopamine release that is triggered by high potassium concentrations in the presence of the combination of ascorbate/Fe^*, suggesting that the vesicular exocytotic dopamine release does not seem to depend upon peroxidation [212]. Furthermore, Ramassamy et d. [213] showed that the combination of ascorbic acid/Fe^* ions could deaease synaptosomal membrane fluidity measured by fluorescence polarization using 1,6-diphenyl 1,3,5-hexatriene in a concentration-dq)endent manner. Free radical generation by ascorbic add/Fe^* results in a decrease of membrane fluidity through the peroxidation of neuronal membrane Upids. These membrane altCTations were prevented by either EGb (2-16 ;
190
adreooo^tors and 5-HT,A leocptois, increases the uptake of cboiine, and improves deterioration in cognitive functions, induding learning and memory, with advancing age. It is worth noting that EGb demonstrates evid^ce of neuroprotective effects against various agerelated physiological changes and injuries, although EGb has hardly any influence under normal conditions; that is, EGb may fadlitate a spontaneous cure of damage occurring in the brain with advancing age. Hierefore, such attributes appear to be associated with the observation that EGb is effective in mild to moderately affected patients with ALdieimer's disease and multi-^nfarct donentia. It is accepted that flavonoids and teq)enes, composing 24% and 6% of the standardized extract, respectively, contribute to the pharmacological activities of EGb as active constituents. Ginkgolides and bilobalide are especially unique constituents in Ginkgo biloba^ and have been found in no other plants. It has been generally considered diat the anti-oxidant activity of flavonoids and the platelet-activating factor antagonism of teipenes are usefiil for explaining the greater part of the restorative and/or neuroprotective properties of EGb as described above. On the other hand, as far as we know, there seem to be no publications concerning clinical trials demonstrating the effectiveness of other antioxidants or anti-PAF antagonists on dementia. Therefore, the possibility of a separate yet unidentified mechanism remains to be clarified. For example the activities of bilobalide on membrane phospholipids and mitochondria may contribute to other defense medianisms of EGb. Furthermore, a comprehensive understanding of the greater part of EGb constituents, Le., those other than flavonoids and terpenes, is still lacking. FurthCT elucidation of the constituents of EGb is thus necessary. REFERENCES [I]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 28 © 2003 Elsevier Science B.V. All rights reserved.
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CHEMISTRY AND BIOLOGICAL ACTIVITIES OF ISOPRENYLATED FLAVONOIDS FROM MEDICINAL PLANTS (MORACEOUS PLANTS AND GLYCYRRHIZA SPECIES) TARO NOMURA, TOSHIO FUKAI, and YOSHIO HANO School of Pharmaceutical ScienceSy Toho University, 2-2-1 Miyama, Funabashiy Chiba 274-8510, Japan
ABSTRACT: Among a large number of phenolic compounds isolated from natural source, various isoprenoid-substituted phenolic compounds have often been found in plants. Moraceous plants and licorice (Glycyrrhiza species) are rich sources of the isoprenoid-substituted phenolic compounds, including flavonoids. Some of the Morus flavonoids, such as kuwanons G and H, have been regarded as optically active Diels-Alder type adducts. Furthermore, some of the isoprenylated-flavonoids from the moraceous plants and licorice showed the interesting biological activities. This article reviews the biological activities of the isoprenylated-flavonoids from the root barks and/or barks of moraceous plants and from Glycyrrhiza species by our group. The chemical studies conceming the biological activities of these compounds are also described briefly.
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I.
INTRODUCTION (SURVEY OF ISOPRENYLATED FLAVONOIDS FROM THE MORACEOUS PLANTS AND GLYCYRRHIZA SPECIES)
Moraceous plants Moraceae comprise a large family of sixty genera and nearly 1400 species, including popular species such as Artocarpus, Morus, and Ficus, which are found in temperate, subtropical, and tropical regions of the world. Mulberry tree, a typical plant of genus Morus, has been widely cultivated for its leaves, which serve as indispensable food for silkworm. In addition, the root bark of the mulberry tree (Mori Cortex, Morus alba L. and other of genus Morus, "Sang-Bai-Pi" in Chinese, "Sohakuhi" in Japanese) has been used as a material of traditional Chinese medicine for an anti-inflammatory, diuretic, antitussive, expectorant, and antipyretic purposes [1-3]. The earliest written reference to the use of Mori Cortex is contained in the "Shen Nong Ben Cao Jing" (SNBCJ, shin-no hon-zo kyo in Japanese), the first Chinese dispensatory whose original anonymous volumes probably appeared by the end of the third century [4,5]. In the Chinese book, 365 crude drugs are classified into three classes (upper: plants with lowest side-effects and nontoxic, useful for health care; middle: plats that are nontoxic or possess only weak toxicity in whose use care must be exercised; lower: toxic and only for clinical use. Mori Cortex is described as belonging to the middle class. The crude drug is used as a component in traditional Chinese medicinal prescriptions, such as "Wuhu Tang (Gokotou in Japanese)" and "Mahuang Lianqiao Chixiaodou Tang (Maou-rensho-shakushozu-tou)", which are applied clinically as a therapy for bronchitis and for nephritis, respectively [6]. On the other hand, a few pharmacological studies on the mulberry tree had demonstrated a hypotensive effect of the extract in rodents [7,8]. Considering the above and reports described later, it was suggested that the hypotensive constituents would be a mixture of many phenolic compounds. Our interests were focused on the phenolic constituents of the mulberry tree. So, we have studied phenolic compounds of the mulberry tree and the related plants [8]. About seventy kinds of new phenolic compounds could be isolated from Japanese cultivated mulberry tree {Morus alba, M. bombycis, and M, Ihou) and Chinese crude drug "Sang-Bai-Pi" (the root bark of Chinese mulberry tree). Most of them are isoprenylated flavonoids. Among them, kuwanon G (1) was the first isolation of the active substance exhibiting the hypotensive effect firom the Japanese Morus alba root bark [9]. Furthermore, kuwanon G (1) and its isoprenylated derivative kuwanon H (2) [10] are considered to be formed through an enzymatic Diels-Alder type reaction of a chalcone and a dehydrokuwanon C or its
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morusin (3) : R = H artonin E (7): R = OH
sanggenon A (4): Ri = CH2CH=CMe2. R2 = OH (4'): Ri = OH, R2 = CH2CH=CMe2
kuwanonG(1):R = H kuwanon H (2): R = CH2CH=CMe2
OH O artobiloxanthone (8)
sanggenon C (5): Ri = CH2CH=CMe2, R2 = OH (5'): Ri = OH, R2 = CH2CH=CMe2
OH sanggenon 0 (6)
OH 0 cycloartobiloxanthone (9)
OH brosimoneA(13)
Fig. (1).
Structures of compounds 1 - 1 5 from moraceous plants.
soroceal (15)
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equivalent, Fig. (1). Since that time, about forty kinds of Diels-Alder type adducts, structurally similar to that of 1 have been isolated from Morus species. These Diels-Alder type flavonoids are characteristic constituents oiMorus species [8,11-14]. Morusin (3), a flavone derivative, isolated from the root bark oi Morus alba L., as a main isoprenylated flavonoid, has a structure bearing an isoprenoid moiety at the C-3 position and a 2',4'-dioxygenated pattem in the B ring [15]. These features are one of the characteristics of the isoprenylated flavonoids of Morus root bark. Furthermore, from the Chinese crude drug "Sang-Bai-Pi" purchased in Japanese market, our group reported a series of isoprenylated flavonoids, such as sanggenons A (4) [16] and C (5) [17]. Recently, the structure of sanggenons A and C were revised from 4' and 5' to 4 and 5, respectively, [18], Fig. (1). Sanggenon C (5) seems to be a Diels-Alder type adduct of a chalcone derivative and a dehydroprenyl (=3-methyl-1,3-butadienyl)phenol having a sanggenon A type partial structure. From the root bark of one of the Chinese mulberry tree, Morus cathayana, a series of isoprenylated flavonoids could be isolated [19-21]. Some of the flavonoids are the sanggenon A type flavanones (SATF), 3-hydroxyflavanone having a prenyl (=3-methyl-2-butenyl) group at 2 position and an ether linkage between C-3 and C-2' positions, such as 4 and 5. Most SATFs are (27?,55)-flavanones, sanggenons A (4), C (5), L, M (100), sanggenols F, G, and J, and soroceins D and F [22], but sanggenon O (6) is (2iS',ii?)-flavanone [23]. The stereochemistry at C-2 and C-3 of sanggenons B (45), Fig. (6), D (33), E, P (sorocein H), S, sorocein E, and sanggenols H and I is still unclear. Earlier studies of flavonoids and stilbenes with one or more isoprenoid groups (prenyl group, 2,2-dimethylpyran ring, geranyl group, famesyl group, etc.) from Morus species have been summarized in review articles [8,11-13,24-26]. On the other hand, the plants of Artocarpus species distribute over the tropical and subtropical regions, and have been used as traditional folk medicine so called "Jamu" in Indonesia against inflammation, malarial fever and so on. Many kinds of isoprenylated flavonoids have also been isolated from Artocarpus species by Venkataraman's group and other several groups [27-30]. Our group also studied the constituents of Indonesian Artocarpus species, such as A. heterophyllus, A, communis, A. rigida, A. venenosa (^Paratocarpus venenosa), and A. altilis, a moraceous plant from Sri Lanka [30-32]. About seventy kinds of isoprenylated flavonoids have been isolated from these Artocarpus species. The compounds, except some ones, have a characteristic structure bearing an isoprenoid side chain at the C-3 position of flavone skeleton, and the B ring has a 2',4',5'-trioxygenated pattem, such as artonin E (7) corresponds to 5'-hydroxymorusin [31]. In addition to the feature, some of the flavone, such as artobiloxanthone (8) and
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cycloartobiloxanthone (9) have a unique structure having the C-C linkage between an isoprenoid side chain at the C-3 position and the 6'-carbon of the B ring of flavone skeleton. This C-C linkage was considered to be synthesized biogenetically through a phenol oxidation in the plants [29,30]. These flavonoids are characteristic constituents oi Artocarpus species. Some of the Artocarpus flavonoids, such as artonin I (10), have been regarded as intermolecular [4+2] cyclo-addition product from the isoprenyl portion of a dehydroprenylphenol, as a diene, and the a,punsubstituted bond of a chalcone skeleton, as a dienophile. As artonin I (10) was considered to be formed through the Diels-Alder type reaction of a chalcone derivative, morachalcone A (11) and artocarpesin (12), the precursor artocarpesin (12) was added to the Morus alba cell cultures to produce artonin I (10) [32]. Earlier studies of the isoprenylated phenols from Artocarpus species have been summarized in review articles [27,28,30]. Most Diels-Alder type adducts with a dehydroprenylflavonoid and a chalcone (DADCs) have been isolated from Asian Morus and Artocarpus species. However, some DADCs have also isolated from American moraceous plants. From Brosimopsis oblongifolia, a Brazilian moraceous plant, a series of DADCs, brosimone-family, were isolated. One of them, brosimone A (13) is a unique adduct, and it is likely to form through an intra-molecular [4+2] cycloaddition reaction between the dehydroprenyl moiety at the A' ring, as the diene, and the a,P-double bond of the chalcone skeleton (A ring-Ca-B ring), as the dienophile, of brosimone D (14) [33], Fig. (1). From a Brazilian moraceous plant, Sorocea bonplandii, ketalized Diels-Alder type adducts, such as soroceal (15), have been isolated [34]. On the other hand, from Paraguayan moraceous plants, Sorocea bonplandii, our group isolated the similar ketalized Diels-Alder type adducts along with a unique adducts, sorocenol B (16) [35], Fig. (2), which may be a derivative induced from the Diels-Alder type adducts between a chalcone derivative and a dehydroprenylated resorcinol through the oxidative reaction. A series of isoprenylated flavonoids has been isolated from the following moraceous plants: Brosimopsis oblongifolia, a Brazilian plant [36], Chinese Cudrania tricuspidata [37], and Taiwanese and Chinese Cudrania cochinchinensis [38]. Many xanthones with one or two isoprenoid groups have also been isolated from these Cudrania species [11]. Latex from the wood of Antiaris toxicaria, a toxic Indonesian plant, has been used for arrow poison. From the root bark of the plants, antiarones A (17) and B (18) [39], the first examples of isoprenylated aurone derivative, have been isolated along with the unique dihydrochalcone derivatives, antiarones J (19) and K (20) [40], Fig. (2). These compounds (19 and 20) are biogenetically considered to be formed
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through a cyclization accompanied by hydration of the isoprenyl group attached to the B ring of chalcone derivatives such as antiarone E (21) [41]. Two new cyclomonoterpene-substituted isoflavones, ficusins A (22) and B were isolated from the Indonesian moraceous plant, Ficus septica Barm F [42]. Earlier studies of phenolic compounds (flavonoids, xanthones, benzaldehydes) have been summarized in review articles [8,11,30].
OH sorocenol B (16)
antiarone A (17)
antiarone J (19): Ri = OH. R2 = CH2CH=CMe2 antiarone K (20): Ri = OMe, R2 = H
Fig. (2).
0 antiarone B (18)
OH O
antiarone E (21)
Structures of compounds 1 6 - 2 2 from moraceous plants.
Glycyrrhiza species Licorice (liquorice, kanzoh in Japanese, gancao in Chinese) is the name applied to the roots and stolons of some Glycyrrhiza species (Leguminosae or Fabaceae) and has been used by human beings from ancient times. The genus Glycyrrhiza consists of about 30 species and chemical studies have so far been carried out on 15 of them. Glycyrrhizic acid (110) is the major triterpenoid saponin in licorice root and the main sweetener of the herb. The saponin has been isolated from G. glabra^ G. uralensis, G. inflata, G. aspera, G. korshinskyi, and G. eurycarpa, and thus, these plants are generally accepted as licorice. In European countries, G. glabra is chiefly used as licorice. On the other hand, in Asian countries, G. glabra, G. uralensis, G. inflata, and G. eurycarpa are used as licorice. Following extraction, the herb yields the licorice products of commerce which are used as sweetening agents, flavoring for American type tobaccos, chewing gums, candies, etc., as a depigmentation agent in cosmetic, and as pharmaceutical products, e.g., anti-ulcer.
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anti-hepatitis medicines, antitussives, etc. Among them the most important industrial use of the herb is in the production of additives as flavor and sweetening agents [43]. In SNBCJ, licorice is described as belonging to the upper class and is recommended for lengthen one's life span, for improving health, for cures for injury and swelling, and for its detoxification effect. One hundred ten prescriptions are recorded in the earlier Chinese medicinal book "Shang Han Lun", where seventy prescriptions include licorice [43]. In the Japanese market, Chinese licorice is classified by its place of production, e.g., Northeastem licorice (Touhoku kanzoh in Japanese), Northwestem licorice (Seihoku kanzoh), Xinjiang licorice (Shinkyou kanzoh), etc. Among these licorice, Northeastem licorice had been identified as G. uralensis, but the original plants of the others had been unidentified. We investigated the phenolic constituents of certain Glycyrrhiza species identified by authorities, and many phenolic compounds were isolated from these plants [43]. The main phenols of licorice are glycosides of liquiritigenin (100) and isoliquiritigenin (70), e.g., liquiritin (113), isoliquiritin, liquiritin apioside, licuraside, etc. [43]. As minor phenolic compounds, many isoprenoidsubstituted flavonoids, chromenes, dihydrophenanthrenes, and dihydrostilbenes were isolated from Glycyrrhiza species. Some of them characterized each plant [43^5]. The 5- and 6-positions of most flavonoids from European licorice are unsubstituted, but the 5-position of flavonoids from Chinese licorices is generally substituted with a methoxyl group or a hydroxy1 group (the 5-OH of some compounds forms ether linkage with an isoprenoid group at the 6-position). For example, the main isoprenoid-substituted flavonoid of G. glabra var. typica, Russian licorice, is a pyranoisoflavan, glabridin (23). The 5-position of most flavonoids from the plants is unsubstituted, e.g., 23, glabrene (24), glabrol (25), 3-hydroxyglabrol (26), etc.. Fig. (3).
glabridin (23)
Fig. (3).
giabrene (24)
glabrol (25): R = H 3-hydroxyglabrol (26): R = OH
Structures of compounds 23 - 26 from licorice {Glycyrrhiza glabra).
3*-(Y,y-dimethylallyl)kievitone (27)
206
On the other hand, from Chinese and Kyrghiz G. glabra, both 5-unsubstituted flavonoids (e.g., 24) and 5-oxygenated flavonoids, e.g., 3'-(y,y-diniethylallyl)-kievitone (27), have been isolated. Nevertheless, 5-position of the most flavonoids with one or two isoprenoid groups from these plants is substituted with a hydroxyl or a methoxyl group. The main isoprenoid-substituted flavonoid of the Kirghiz licorice is compound 27, but the isoflavan (23) has not been isolated [46-49], Isoprenoid-substituted flavonoids isolated from commercial Kyrghiz licorice and European licorice that was cultivated in Japan were summarized in Table 1 [49-51]. Table 1,
Flavonoids isolated from Kyrgyz and European Glycyrrhiza glabra Kirghiz [49]
3'-(Y,Y-Dimethylallyl)-kievitone (27) Glisoflavanone (3',6-diprenyl-2',4',5,7-tetrahydroxyisoflavanone) Glyasperin A (77) Glyasperin C (61) Glyasperin D (62) Isoderone(DMP;4',3']-5,7-dihydroxyisoflavone)'' Semilicoisoflavone B (68) 8-(Y,Y-Dimethylallyl)-wighteone (58) Gancaonin G (60) Gancaonin H (DMP;4',5']-6-prenyl-3',5,7-trihydroxyisoflavone) 1-Methoxyphaseollidin (125) Edudiol (3,9-dihydroxy-l-methoxy-2-prenylpterocarpan) Glabrene (24) Glabridin (23) 4'-C>-Methylglabridin (123) Hispaglabridin A (3'-prenylglabridin) Glabrol (25) 3-Hydroxyglabrol (26)* Glabrone (DMP;4',3']-2',7-dihydroxyisoflavone) Medicarpin (3-hydroxy-9-niethoxypterocarpan) Shinpterocarpin (DMP;3,4]-9-hydroxypterocarpan) Euchrenone as (DMP;4',3']-7-hydroxy-8-prenylflavanone) Glyinflanin K (2DMP;7,8, ;2',3']-isoflavan) Glyinflanin G (2DMP;4,5, ;4',3']-2',3-dihydroxychalcone) Kanzonol U (DMP;2',3']-4',6-dihydroxy-2-arylbenzofuran] Kanzonol V (DMP;2',3']-4',6-dihydroxy5-prenyl-2-arylbenzofuran) Kanzonol W (DMP;7,8]-2',4'-dihydroxy-3-arylcoumarin) Kanzonol X (3',8-diprenyl-2',4',7-trihydroxyisoflavan) Kanzonol Y (3,5'-diprenyl-a,2',4,4'-tetrahydroxy-dihydrochalcone) Kanzonol Z (DMP;7,8]-3,4'-dihydroxy-3'-prenylflavanone) 3-Hydroxyparatocarpin C**
+-H-¥ +++ ++ ++ +++ + ++ ++ + ++ +++ ++ ++ -
European [50,51 ] -
+++ ++++ ++ ++-H+++ +++ ++ ++++ +++ ++ ++ ++ ++ +++ +++ +++ +++ +++ +++
Yields from dried licorice roots: -H-i-H=more than 0.01%; +-H-=between 0.01 and 0.001%; ++=0.001-0.0001%; +=1-0.1 ppm. • The compound was obtained from the stolons. ° 2,2-dimethylpyrano[b=DMP. ** Tentative name used here (DMP;4,5]-3'-prenyl-2',3,4'-trihydroxychalcone).
The difference of the substituents at C-5 is expected that European and Chinese licorices exhibit different actions in therapeutically use. For example, 5,6-disubstituted isoflavans do not showed a potency of
207
anti-HIV activity in vitro, but two isoflavans with no substituent at both 5- and 6-positions obtained from Erythrina lysistemon (Leguminosae) have the activity as described later [52]. As described the above, moraceous plants and Glycyrrhiza species are rich sources of isoprenylated phenolic compounds. The phenolic nuclei having the isoprenoid-derived substituents, e.g., simple isoprene or a monoterpenoid, vary over a wide range from a simple phenol to complicated ones. Some of the moraceous plants studied by our group have been used as traditional herbal medicines in the native countries. It is interesting to clarify the relationship between the usage and biological activities of the isoprenylated phenolic compounds. So we studied some of the biological activities of these compounds. This article reviews the biological activities of the isoprenylated flavonoids isolated from the moraceous plants and isoprenoid-substituted phenols (flavonoids, xanthones, dihydrostilbenes, and dihydrophenanthrenes) from Glycyrrhiza species by our group and other several groups. 11. HYPOTENSIVE ACTIVITY OF ISOPRENYLATED FLAVONOIDS FROM THE ROOT BARK OF MORUS SPECIES The first report for the hypotensive effect of the mulberry tree was presented by Fukutome in 1938, who asserted that oral administration of the hot water extract of the mulberry tree showed a remarkable hypotensive effect in rabbits [53]. Ohishi reported the hypotensive effect of the ethanol extract of mulberry root bark [54]. Suzuki and Sakuma reported that the hypotensive activity seemed to be due to phenolic substances, and that the effect disappeared on acetylation [55]. Later, Katayanagi, et aL reported that the ether extract of the root bark gives to rabbit (6 mg/kg, i.v.) showed a marked hypotensive effect and that the active constituents seemed to be a mixture of unstable phenolic compounds [56]. Tanemura ascribed the activity of mulberry root bark to acetylcholine and its analogous presumably contained in the alcohol soluble fraction, and that the hypotensive constituents produced a yellowish-brown precipitate on treatment with Dragendorff reagent [57]. Yamatake, et al reported that n-butanol- and water-soluble fractions of mulberry root bark had similar effect except for those on the cardiovascular system. Both fractions showed cathartic, analgesic, diuretic, antitussive, anti-edema, sedative, anticonvulsant, and hypotensive actions in mice, rats, guinea pigs and dogs [7]. On the beginning of our study of mulberry tree, the hypotensive constituents had not been identified. In view of the reports, we assumed that the hypotensive compounds of the plant would be a mixture of unstable
208
phenolic compounds and therefore undertook a study of the phenolic constituents of the root bark of the cultivated mulberry tree. The root bark of the cultivated mulberry tree was extracted successively with n-hexane, benzene, and methanol. The methanol extract, 1-20 mg, showed a dose-dependent decrease in arterial blood pressure in pentobarbital-anesthetized rabbit, Fig. (4). The extract was fractionated successively by silica gel column chromatography (C.C.), polyamide C.C, silica gel preparative (p.) TLC, and p. HPLC leading to isolated of kuwanons G (1, 0.2% yield) [9] and H (2, 0.13% yield) [10]. The root bark of Moms alba n-Hexane Residue Benzene
Extract
Residue I Methanol Residue
Extract -T extract Ethyl acetate soluble portion I C.C, p. TLC, p. HPLC
C.C, p. TLC Morusin (3), kuwanons C (42), D, E (43), F, oxydihydromorusin (46), mulberroftiran A (47)
Kbwanons G (1), H (2), L (44), M (35), albanol B (97) mulberrofurans C (28), F (29), and G (30) Fig. (4). Isolation procedure of flavonoids from the root bark of Morus alba.
mmHg
PN n > < l i < M H H H H M M ( H i t i i kuwanon G 1 mg/kg i.v. mmHg
tsmmm |ioo 50
iilSliSirJ'^^
kuwanon H I mg/kg i.v.
'
10 s
Fig. (5). Effects of kuwanon G (1) and kuwanon H (2) on blood pressure. Electrocardiogram (ECG), phrenic nerve discharge (PN), and electroencephalogram (EEG) in a gallamine-immobilized rabbit.
209
Both compounds (1 and 2) almost equally caused decrease of arterial blood pressure in a dose dependent and reversible manner at the dose of between 0.1 and 3 mg/kg, i.v. in pentobarbital-anesthetized as well as in un-anesthetized, gallamine-immobilized rabbits. Fig. (5), [58]. These hypotensive actions of kuwanons G (1) and H (2) were not modified by atropine or eserine, suggesting the non-cholinergic nature origin. Furthermore, neither propranol nor diphenhydramine affected their actions on the arterial blood pressure. Although they produced no significant change in both electrocardiogram (ECG) and respiration when administered intravenously in rabbits. The hypotensive effects of kuwanon G (1) and H (2) did not accompany with heart rate change [58]. In pentobarbital-anesthetized pithed dogs, kuwanons G (1) and H (2) also significantly decrees of femoral arterial blood pressure. These effects suggested that mechanism of hypotensive effects of kuwanons G (1) and H (2) mediated through peripheral system. Mulberrofurans C (28) [59], F (29) [60], and G (30) [60], Fig. (6), were also isolated as hypotensive components from the mulberry tree. Mulberrofuran C (28) is considered to be formed by a Diels-Alder type of enzymatic reaction process of a chalcone derivative and dehydromoracin C (31) or its equivalent. Furthermore, mulberrofurans F (29) and G (30) seems to be Diels-Alder type adducts derived from chalcomoracin (32) and mulberrofuran C (28), respectively, by the intra-molecular ketalization reaction of the carbonyl group with the two adjoining hydroxyl groups, 3'(5')-OH and 2"-0H. Intravenous injection of mulberrofuran C (28, 1 mg/kg) produced a significant hypotension (37 mmHg fall) in rabbit (male, 3.3 kg) anesthetized with pentabarbital sodium (30 mg/kg). Single intravenous injection of mulberrofurans F (29) and G (30) (both 0.1 mg/kg) caused a marked depressor effect in rabbit by 26 mm Hg and 16 mm Hg, respectively. On the other hand, in Japan, "Sang-Bai-Pi" (the root bark of Chinese mulberry tree) imported from China has been used as an herbal medicine, hence a study of the components of this crude drug purchased in the Japanese market was undertaken. Its phenolic components are different from those of Japanese mulberry tree. For example, morusin (3) and kuwanon G (1) are the main phenolic components of Japanese mulberry tree, in the case of "Sang-Bai-Pi", these components are minor ones, while sanggenons A (4) [16], C (5) [17], and D (33) [61] are the main components [24]. Sanggenons C (5) and D (33) showed the hypotensive effects as follows: Sanggenon C (5) caused transient decrease in arterial blood pressure at the doses of 1 mg/kg in pentobarbital-anesthetized rabbit by 15 mm Hg, while at the doses of 5 mg/kg the compound (5) caused a transient decrease by 100 mm Hg, which continued for more
210
mulberrofuran C (28): R = H chalcomoracin (32): R = CH2CH=CMe2
mulberrofuran F (29): R = CH2CH=CMe2 mulberrofuran G (30): R = H
kuwanon E (43) sanggenon B (45)
Fig. (6).
Structures of flavonoids (28 - 44) from moraceous plants.
211
than one hour by 15 mm Hg [17,62]. Sanggenon D (33) caused a transient decrease at the dose of 1 mg/kg in pentobarbital and urethane anesthetized male Wister strain rat by 35 mm Hg, while the compound (33) caused a decrease by 80 mmHg at the doses of 1 mg^g in spontaneously hypertensive rat [61,63]. III. ANTI-TUMOR PROMOTING ACTIVITY OF MORUSIN (3) Cancer chemoprevention is the most important subjects in cancer research at present and is a new medical strategy for cancer prevention, which was established by recent understanding of molecular multistage carcinogenesis in humans. To find nontoxic cancer preventive agents, Fujiki and his coworker studied natural products derived from marine and plant sources [64,65]. In 1987, Yoshizawa, et aL reported that (-)epigallocatechin gallate (EGCG), which is a main constituent of green tea, inhibited tumor promotion by teleocidin in mouse skin [66]. In 1988, Fujita, et aL reported the inhibitory effect of EGCG on carcinogenesis with 7V-ethyl-A^-nitro-A^-nitrosoguanidine in mouse duodenum [67]. On the other hand, in the course of our examination the constituents of the Morus root bark, we found the following novel photo-oxidative cyclization. When a solution of morusin (3) in chloroform (CHCI3) was irradiated using high-pressure mercury lamp, morusin hydroperoxide (34), Fig. (6), was obtained in ca, 80% yield [68]. The reaction did not occur in the dark and was depend on the solvent; the reaction occurred in low polar or nonpolar solvent such as CHCI3 and benzene, but not in protic solvent. The reaction mechanism was suggested as follows [69]: morusin (3) in the ground state interacts with an oxygen molecular to form a contact charge transfer complex [3 O2] (CCTC). On irradiation, the CCTC gives an excited charge transfer state that presumably leads to reactive species such as free radicals as described in Fig. (7). Recently, the proof of presence of the CCTC was provided by laser desorption/ionization time-of-flight mass spectrometry of 3 [70]. The hydroperoxide (34) was also obtained with the oxidation of morusin (3) with singlet oxygen or radical initiator [71]. HO^^s^^OH hv 34 •OOH
Fig. (7). Reaction mechanism of photo-oxidative cyclization of morusin (3).
212
This photoreaction and the relative reaction of morusin (3) along with the anti-tumor promoting activity of EGCG encouraged us to examine the anti-tumor promoting activities of a series of isoprenylated flavonoids isolated from Morus species. First we examined the inhibition against three biochemical effects; the specific binding of ^H-12-O-tetradecanolylphorbol-13-acetate (TPA) to mouse particulate fraction, the activation of Ca^'^-activated phospholipid-dependent protein kinase (protein kinase C) with teleocidin, and induction of ornithine decarboxylase (ODC) with teleocidin in mouse skin [72]. Interestingly, of the eight isoprenylated flavonoids, morusin (3), kuwanons G (1) and M (35), mulberroforan G (30), and sanggenon D (33) gave similar results in these biochemical tests as described in Table 2. Table 2.
Effects oi Morus flavonoids on biological and biochemical activities Inhibiting of specific [^H]TPA binding (ED50 jimol/L)
57 99 100 85 34 62 48 60
Morusin (3) Kuwanon G (1) Kuwanon H (2) Kuwanon M (35) Mulberrofuran G (30) Sanggenon A (4) Sanggenon C (5) Sanggenon D (33)
Inhibition of activation of protein kinase C (ED50 fimol/L)
80 40 80 22 46 80 46 42
Inhibition of ODC induction
(%) 43 34 -35 25 10 -62 -17 17
100
^
o
o
Concentration (mol/L) of morusin (3) Fig. (8). Effects of morusin (3) on specific binding of [^H]TPA to a mouse skin particulate fi-action. Various concentrations of morusin (•) or TPA (o) were incubated with a particulate fi-action of mouse skin in the presence of 4 nmol/L [^H]TPA for 2 h at 4°C, and the assay mixture was filtered on glass filter membrane with acetone cooled in a dry ice-ethanol bath. Non-specific bindings were measured in the presence of 500-fold excess of unlabelled TPA.
213
Of these five compounds, morusin (3) is the least toxic and can be isolated as one of the main phenolic compounds from the root bark. The more detailed data for the above these biochemical tests of morusin (3) were as follows [73]. As shown in Fig. (8), morusin (3) caused dose-dependent inhibition of the specific binding pHJTPA to a mouse skin particulate fraction. The concentration of morusin (3) for 50% inhibition (ED50) was 57 |amol/L, whereas that of unlabelled TPA was 4 nmol/L. As morusin (3) was assumed to interact with the phorbol ester receptor, we examined whether it inhibited the activation of protein kinase C by teleocidin in vitro [73]. Fig. (9) shows that morusin (3) inhibited the phosphorylation of histone type III-S by protein kinase C dose-dependent and that 80 |imol/L morusin caused 50% inhibition. 100
o
VA
50
a
0.
VA
10-
\o-
10-*
Concentration (mol/L) of morusin (3) Fig. (9). Inhibition by morusin (3) of activation of protein kinase C by teleocidin in vitro. The assay mixture (0.25 mL) contained 20 jimol/L CaCh, 7.5 |ag of phosphatidylserine, 2.3 (^mol/L teleocidin, and various concentrations of morusin (3) with 0.05 units of partially purified enzyme. Enzyme activity was measured as the incorporation of ^^P from [7-^^P]ATP into histone type III-S during incubation for 3 min. at 30^.
Furthermore, we examined the inhibition of the induction of ODC induction by teleocidin in mouse skin. Application of 11.4 nmol morusin (3) caused 43% inhibition of the induction of ODC by 11.4 nmol teleocidin [73]. From the results of these three tests, morusin (3) might inhibit the tumor-promoting activity of teleocidin on mouse skin. As shown in Figs. (10) and (11), the percentage of tumor bearing mice in the group treated with 7,12-dimethylbenz[a]anthracene (DMBA) plus teleocidin reached 100% by week 15, o in Fig. (10). In contrast, the onset of tumor formation was delayed 5 weeks by treatment with morusin (3), • in Fig. (10), and the percentage of tumor-bearing mice in the group treated with DMBA plus teleocidin and morusin (3) was 60% at week 20. The average number of tumors per mouse in week 20 was also reduced from 5.3, o in Fig. (11), to 1.1, • in Fig. (11), by morusin (3) treatment.
214
On the other hand, morusin (3) itself did not show a tumor promoting activity on mouse skin, x in Figs. (10) and (11). From these results, morusin (3) is an anti-tumor promoter judging from its ability to inhibit the short-term effects induced by tumor promoters.
100
to
10
20
Weeks of promotion
10
20
Weeks of promotion
Figs. (10) and (11). Inhibition by morusin (3) of tumor promotion by teleocidin in a two-stage carcinogenesis experiment on mouse skin. Inhibition was achieved by a single application of 100 ^g of DMBA, and teleocidin (2.5 }ig) and morusin (1 mg) were applied twice a week throughout the experiments.
As mentioned the above, morusin (3), kuwanon G (1), kuwanon M (35), mulberroforan G (30), and sanggenon D (33) showed inhibitory effects in the three biochemical tests. The anti-tumor promoting activities of later four flavonoids with one or two isoprenoid groups have not been tested in a two-stage carcinogenesis experiments, due to limitations of their amounts available, but their inhibitory potencies to the three biochemical tests were almost similar to that of morusin (3). Furthermore, the twelve isoprenylated flavonoids from the moraceous plants and two flavonol glycosides (48 and 49) from Epimedium species (Berberidacaceae) [74] along with quercetin (50) were tested for inhibitory effects on carcinogenesis by a test for inhibition of specific binding of [^H]TPA to a mouse skin particulate fraction. While the other biochemical tests and the inhibition of tumor promotion of teleocidin in a two-stage carcinogenesis experiment have not been carried out, due to limitation in their amounts available, some of isoprenylated flavonoids from the moraceous plants showed the similar inhibitory potencies to those of morusin (3) and the related compounds, Figs. (6) and (12), as shown in Table 3. On the other hand, EGCG and green tea extract are acknowledged cancer-preventive agents in Japan [75,76]. Natural products with antitumor promotion activity isolated from foodstuff and medicinal plants have been summarized by Konoshima and his co-worker and Akihisa and
215
his co-worker [77,78]. Considering these results as well as the results of biochemical tests and anti-tumor promoting activity of the isoprenylated flavonoids from the moraceous plants in a two-stage carcinogenesis experiment with teleocidin, the isoprenylated poly-phenolic compound seems to be interesting compounds for finding cancer preventive agents and the more detailed experiments should be carried out. Table 3.
Effects of the isoprenylated flavonoids on inhibition of specific [^H]TPA binding (ID50, ^mol/L)
Kazinol C (36) Kazinol E (37) Kazinol F (38) Kazinol J (39) Kazinol M (40) Kazinol N (41) Kuwanon C (42) Kuwanon E (43)
Kuwanon L (44) Sanggenon B(45) Oxydihydromorusin (46) Mulberroftiran A (47) Ikarisoside A (48) Ikarisoside B (49) Quercetin (50)
80 70 98 90 100 >100 80 83
80 95 95 >100 >100 >100 >100
OMe oxydihydromorusin (46) mulberrofuran A (47)
ikarisoside A (48): R = Rha ikarisoside B (49): R = Glu(1 ^ 2)Rha
OCH3 OH
O
quercetin (50): Ri = OH,R2=R3 = H cirsilioi (51): Ri = H, R2 = 0Me, R3 = Me
antiarone L (57) artonin H (56)
Fig. (12). Structures of flavonoids (46 - 57) from moraceous plants, Epimedium species, and test reagents (50 and 51).
IV. INHIBITION OF ARTONIN E (7) AND RELATED COMPOUNDS ON 5-LIPOXYGENASE Previously, we reported the effects of Morus flavonoids on arachidonate metabolism in rat platelet homogenates, such as inhibition of 12-hydroxy5,8,10-heptadecatrienoic acid (HHT), thromboxane B2, and 12-hydroxy5,8,10,14-eicosatetraenoic acid (12-HETE) [79,80]. As described in the
216
introduction, Artocarpus plants (Moraceae) have been used as traditional medicine in Indonesia for swelling and malarial fever. This usage seems to be expecting for effect of anti inflammation. As leukotrienes are known to be chemical mediators of anaphylaxis and inflammation, a number of compounds have been studied and developed as selective inhibitors of 5-lipoxygenase, the enzyme initiating leukotriene biosynthesis from arachidonic acid. So the inhibitory effect of the Artocarpus flavonoids against arachidonate 5-lipoxygenase was examined [81]. Yamamoto, et aL screened various flavonoids, and found that cirsiliol (51), Fig. (12), potently inhibited 5-lipoxygenase and proposed two structural factors of the flavonoids for the specific inhibitory activity, one is catechol type of the B ring and the other is the presence of an alkyl-like side chain at the C-3 position [82,83]. We had interesting for the inhibitory effects of a series of Artocarpus flavones on the 5-lipoxygenase activity. Seven Artocarpus flavonoids and morusin (3) were tested for their inhibitory actions on arachidonate-5-lipoxygenase purified from porcine leukocyte [84]. As shown in Fig. (13), the IC50 values varied depending on the structural modification of the compound. The compounds having three hydroxyl groups at positions 2\ 4\ and 5' on the B ring (compounds 7, 8, 52 and 55) were more potent inhibitors. Thus, the vicinal diol partial structure was important for 5-lipoxygenase inhibition.
OH o heterophyllin (52)
OH
0
artonin A (54)
cycloheterophyllin (53)
Inhibitory effects (IC50 ± SD, N=3, ^imol/L) on arachidonate 5-lipoxygenase activity
OH
0
artonin B (55)
Morusin (3) Artonin E (7) Artobiloxanthone (8) j Cycloartobiloxanthone (9) Heterophyllin (52) Cycloheterophyllin (53) Artonin A (54) Artonin B (55)
2.9 ± 0.4 0.36 db 0.03 0.55 db 0.20 1.3 ±0.2 0.73 ±0.21 1.6±1.0 4.3 ± 0.5 1.0 ±0.1
1
'
Fig. (13). The inhibitory effect (IC50 ± SD) on arachidonate 5-lipoxygenase activity.
As shown in Fig. (14), 5-lipoxygenase was inhibited depending on the concentration of artonin E (7), which gave the lowest IC50 (0.36 |Limol/L) of all the eight compounds. On the other hand, morusin (3), which
217
lacked the 5'-hydroxyl group of artonin E (7), was a less potent 5lipoxygenase inhibitor (IC5o=2.9 |Limol/L). Artonin E (7) was significantly more potent than cirsiliol (51, Fig. (12), IC5o=1.3 |Limol/L), which was reported as a 5-lipoxygenase inhibitor. This finding was consistent with the report that the inhibitory activity of cirsiliol (51) with 5-lipoxygenase was enhanced by introducing a lipophilic alkyl group at the C-3 position of theflavoneskeleton. Inhibitory actions of artonin E (7) and morusin (3) on other mammalian arachidonate oxygenases were examined. Artonin E (7) inhibited two 12-lipoxygenase from porcine leukocytes and human platelets, 15-lipoxygenase from rabbit reticulocytes, and fatty acid cyclooxygenase from bovine vesicular glands (IC5o=2.3, 11, 5.2, and 2.5 |amol/L, respectively). However, IC50 values for these oxygenases were higher by one order of magnitude than that for 5-lipoxygenase. Morusin (3) also inhibited these enzymes (except for human platelet 12lipoxygenase) with IC50 values of micro molar order as follows: two 12lipoxygenase from porcine leukocytes and human platelets, 15lipoxygenase from rabbit reticulocytes, and fatty acid cyclooxygenase from bovine vesicular glands; IC5o=3.4, > 30, 3.3 and 1.6 |imol/l, respectively. These results indicated that artonin E (7) was a relatively specific inhibitor for 5-lipoxygenase. Thus, which the selectivity for 5-lipoxygenase was not observed with morusin (3). Significant differences of IC50 values of artonin E (7) and morusin (3) between porcine leukocyte 12-lipoxygenase and the human platelet 12-lipoxygenase should be noted since the leukocyte and platelet 12-lipoxygenase were distinct both catalytically and immunologically.
Concentration (|imol/L) Fig. (14). Dose-dependent inhibition of 5-lipoxygenase by artonin E (7, • ) , morusin (3, o), and cirsiliol (51, A).
V. INHIBITION OF ARTONIN E (7) AND RELATED COMPOUNDS ON MOUSE TNF-a RELEASE AND THEIR CYTOTOXIC ACTIVITIES
218
As described in Chapter III, morusin (3) has been found to be anti-tumor promoter in a two-stage carcinogenesis experiment with teleocidin. Considering the similarity of the structures between morusin (3) and artonin E (7), artonin E (7) was expected to be an anti-tumor promoter. Furthermore we found a novel photo-oxidative cyclization of artonin E (7) as follow: photo-reaction of artonin E (7) in CHCI3 containing 4% ethanol solution with high-pressure mercury lamp produced artobiloxanthone (8) and cycloartobiloxanthone (9), and the treatment of artonin E (7) with radical reagent (2,2-diphenyl-l-picrylhydrazyl: DPPH) resulted in the same products, Fig. (15), [84].
(±)-artobiloxanthone (8)
artonin E (7)
hv, 24 h. CHCI3 DPPH, 24 h, CHCI3 (in the dark)
Fig. (15).
8
9
34% 70%
3% 4%
OH 0 (±) -cycloartobitoxanthone (9)
Photoreaction of artonin E (7) and the reaction with radical reagent.
As described in Chapter III, we have reported the photo-oxidative cyclization on morusin (3). These results suggested that the photo-
OH 0 (±) -cycloartobiloxanthone (9)
OH 0 (±)-artobiloxanthone (8)
Fig. (16). Plausible mechanism for the formation of artobiloxanthone (8) and cycloartobiloxanthone (9) from artonin E (7).
219
oxidative cyciization of artonin E (7) may proceed through phenol oxidation via the semiquinone radicals described in Fig. (16). This chemical reactivity and the similarity of the structures between morusin (3) and artonin E (7) encourage us to examine the anti-tumor promoting activity of artonin E (7). Recently, Fujiki, et al. proposed a new tumor promotion mechanism applicable to human cancer development on the basis of experiment with okadaic acid. They described that tumor necrosis factor-a (TNF-a) induced by okadaic acid acts as a mediator of human carcinogenesis [65]. As briefly summarized in Fig. (17), okadaic acid inhibits the action of protein phosphatase type 1 and 2A, resulting in the accumulation of phosphorylated protein. Fujiki's group has shown that TNF-a acts as a timior promoter in BALB/3T3 cell transformation in vitro. The results of the studies on the okadaic acid class tumor promoters suggest that inflammatory stimuli or chemical tumor promoters induce TNF-a release from target tissues, and TNF-a gene expression in the initiated cells. This released TNF-a acts as a tumor promoter in the autocrine and paracrine system. According to the assumption that TNF-a is an endogenous tumor promoter associated with inflammatory potential, many historical puzzles of tumor promotion, such as its relationship to inflammation, can be solved. Based on this new tumor-promotion pathway, inhibition of TNF-a production leads to inhibition of tumor promotion. Furthermore, recent investigation has revealed that TNF-a is involved in various diseased, such as rheumatoid arthritis, Crohn's disease, multiple sclerosis, graft-versus-host disease, HIV, malaria, sepsis, and cachexia associated with cancer [85-90]. So, specific inhibitions of TNF-a production will almost certainly be effective not only in cancer prevention but also in the therapy and prevention of these other diseases.
—1 protein i j — ' phosphatase 1
okadac acid
^ phosphorylated proteins
t 1
{jene expression
- c-fos ojun
NF-KB
TNF-a - •
p
phosporylated proteins
~3
V
t ' _
ODC TNF-a
— •
_
Fig. (17). Mechanism of tumor promotion with okadaic acid.
Based on the above descriptions, we examined the inhibitory effect of the Artocarpus flavonoids on TNF-a release stimulated by okadaic acid using BALB/3T3 cells. This experiment was carried out in co-operation with Dr. Fujiki's group (Saitama Cancer Center Research Institute, Japan). All the compounds tested inhibit the TNF-a release stimulated by
220
okadaic acid at suitable lower concentration. This result suggests that several Artocarpus flavonoids act as anti-tumor promoter against to the okadaic acid type promotion. However, the detail mechanism is not clear at present, Fig. (17). The comparison of the inhibitory effects of the Artocarpus flavonoids against the TNF-a release (Table 4) and arachidonate 5-lipoxygenase, Fig. (13), was carried out. Artonin E (7) was the most potent inhibitor on both tests and the other compounds, artobiloxanthone (8) and heterophyllin (52), inhibited stronger than cycloartobiloxanthone (9), cycloheterophyllin (53), and morusin (3). The compounds showing stronger activity, all have three hydroxyl groups in the B ring. This characteristic feature might be important factor for both biological activities [91,92]. It is also noteworthy that the bioactivities of these flavonoids may reflect the use of Artocarpus species to the treatment for inflammation and malarial fever in Jamu medicines as is stated above. Table 4. Inhibitory effects (IC50, Mmol/L) of six flavonoids for the release of TNF-a from BALB/3T3 cells by treatment of okadaic acid Morusin (3) Artobiloxanthone (8) Heterophyllin (52)
1.76 0.94 0.48
Artonin E (7) Cycloartobiloxanthone (9) Cycloheterophyllin (53)
0.43 1.94 7.8
We also examined the cytotoxic activities of the Artocarpus flavonoids, artonins A (54), B (55), E (7), H (56), heterophyllin (52), and cycloheterophyllin (53), against cancer cells, mouse L-1210 and colon 38. All compounds tested showed the cytotoxic activities against both cancer cells (Table 5) [93]. Among them, cytotoxicity of heterophyllin (52), artonins B (55) and E (7) w^ere stronger than critical drug, l-(2-tetrahydrofuryl)-5-fluorouracil (TFFU). While we examined the cytotoxic activities of three dihydrochalcone derivatives isolated from Antiaris toxicaria (Moraceae), antiarones J (19), K (20), and L (57), against the two cancer cells [94]. All the compounds showed the weak cytotoxic activities against both cancer cells. Artonin E (7) also exhibited Table 5. Cytotoxic activities (IC50, |ig/mL) of Artocarpus and Antiaris flavonoids against L-12i0 and Colon 38 cells
Artonin A (54) Artonin B (55) Artonin E (7) Artonin H (56) Heterophyllin (52) ' Positive control.
L-1210
Colon 38
8.8 23 2.2 8.8 2.3
14.3
1.4 1.9 3.5 1.3
L-1210 Cycloheterophyllin (53) Antiarone J (19) Antiarone K (20) Antiarone L (57) TFFU*
Colon 38
4.7
4.6
77.0 81.3 80.4
70.4 46.3 >100
2.9
3.9
221
cytotoxic activities against human oral cells and MT4-cells as shown in Chapter VII (Table 7). VI.
BOMBESIN RECEPTOR ANTAGONISTS, KUWANONS G (1) AND H (2), ISOLATED FROM MORUS SPECIES
Bombesin and its mammalian counterparts, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to have a wide range of physiological and pharmacological functions [95]. Ligand-binding and molecular cloning studies have revealed two pharmacologically distinct G-protein-coupled receptor subtypes for mammalian bombesinlike peptides; a GRP-preferring (GRP-R) and an NMB-preferring bombesin receptor (NMB-R) [96]. A series of observations indicates that the mammalian bombesin-like peptides may act autocrine growth factors in human small cell lung carcinoma (SCLC) and other cancers. First, many human SCLC cell lines have been shown to express bombesin-like peptides [97]. Second, peptide bombesin receptor antagonists or anti-bombesin antibodies inhibit SCLC cell growth in vitro and in vivo [98,99]. These data suggested that the bombesin receptor antagonists might be useful for the treatment of some kinds of SCLC and other cancers. Because most antagonists reported thus far are peptides except for CP-70,030 and CP-75,998 (first synthetic non-peptide antagonists) [100-102], so, Fujimoto's group (Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan) screened the four hundred plant extract samples to search for non-peptide bombesin receptor antagonists. The methanol extract of the underground part of cultivated mulberry tree, Morus bombycis, was found to potently inhibit [^^^I]GRP binding to Swiss 3T3 cells. Bioassay-directed fractionation led to the isolation of two known flavone derivatives, kuwanons G (1) and H (2), which were identified by direct comparison with the authentic samples [103]. The antagonistic profiles of kuwanons G (1) and H (2) were characterized from the following results [103]. Kuwanon H (2) inhibited specific binding of [^^^I]GRP to GRP-referring receptors in murine Swiss 3T3 fibroblasts with K{ value of 290±50 nmol/L, which is more potent than that of kuwanon G (1), K\ value=470±60 nmol/L. The Ki value of 2 was about one order of magnitude more potent than those of CP-70,030 and CP-75,998, but had no effect on endothelin-1 or neuropeptide Y binding. While kuwanon H (2) inhibited specific binding of [^^^I]bombesin to rat esophagus membranes, the Ki value was about one order of magnitude less potent, Ki value of 2=6,500±2,000, than that of [^^^I]GRP toSwiss 3T3 cells. While bombesin (10 ^ mol/L) increased intracellular Ca^"^ levels in Swiss 3T3 cells, kuwanon H (2, 500 nmol/L) attenuated the bombesin-
222
induced increase in cytosolic free Ca^"^ concentration ([Ca^"^]!) by 60%, but not bradykinin- or endothelin-1-induced increase in [Ca^"^]}, Fig. (18).
r\
r
808
215
t\
< \
f t V
BOM
t
t
S
BOM
t
BK
t
S
t
BK
A^ Y1 t t t t V
ET-1
S
ET-l
Fig. (18). Effect of kuwanon H (2) on agonist-induced increases in [Ca^^\ in Swiss 3T3 cells. Cells were stimulated by 10"* mol/L bombesin (BOM), 10"* mol/L endothelin-1 (ET) or 10"* mol/L bradykinin (BK). Kuwanon H (S, 500 nmol/L at the final concentration) or dimethyl sulfoxide (V) was added 1 min before stimulation.
In Swiss 3T3 cells, GRP stimulates ["^H]thymidine incorporation in a concentration-dependent manner. Kuwanon H (2) inhibited GRPinduced DNA synthesis in Swiss 3T3 cells. The IC50 value was around 100 nmol/L, close to its K, value for [^^^I]GRP binding to Swiss 3T3 cells, Fig. (19). Kuwanon H (2) demonstrated selectivity toward GRP, as concentration of 10"^ mol/L uninfluenced basal and 5% serum-induced [ HJthymidine incorporation. From above results, kuwanon H (2) appears to be a selective antagonist for GRP-R.
B o.
B o
35000
25000
15000
-log (2) mol/L Fig. (19). Dose-dependent effects of kuwanon H (2) on basal (o) and GRP (10" mol/L)-induced DNA syntheses in Swiss 3T3 cells (•). Values are the mean ± S.E. for four determinations.
As bombesin family peptides are thought to be autocrine growth factors for SCLC, the results described above suggested that kuwanon H
223
(2) might be useful against SCLC. Unfortunately, however, kuwanon H (2) had no effect on the growth of two human SCLC lines, Lu-134 and NCI-HI 28. At the time, kuwanon H (2) was the most potent of non-peptide bombesin receptor antagonists (NPBRA) that had been reported. Its affinity might be too low to determine whether the non-peptide antagonist is effective against human lung cancers. However, kuwanon H (2), and possibly kuwanon G (1) also, can serve as lead compounds for more rational drug design in the synthesis of more potent antagonists. Furthermore, these compounds may be useful tools on the study of GRP-R. Recently, it was reported that NPBRA, PD 176252, with high binding affinity which was developed via the application of a peptoid drug design strategy [104]. VIL
EFFECTS OF PHENOLS AGAINST BACILLUS SUBTILIS (M45) (REC-ASSAY), HUMAN ORAL CELLS, AND HIV-INFECTED MT-4 CELLS
Rec-assay was developed by Kada et al. for screenings chemical and enveloped mutagens. Recombination less mutant strain of Bacillus subtilis (M45) is more sensitive to the cell-killing action of chemical mutagens, e.g., mytomycin C, A^-nitroso-A/-methylurethane, etc., than the wild-type bacteria (HI7) [105]. This assay was also useful for prescreening of anticancer drugs, such as enediyne-family antibiotics [106]. For the constituents of plants, the assay was modified and used exclusively for the detection of anti-mutagen compounds [107]. Since the sensitivity of the rec-assay to chemicals having induction activity of DNA damage is higher than from other screening technique, such as Ames test, this method may be useful for pre-screening of anticancer agents in crude drugs. Furthermore, the antibacterial compounds against the wild-type strain (HI7) may be expected that these antibacterial compounds have another bioactive potency. We tried the application of the rec-assay (unmodified) for the detection of bioactive phenolic compounds obtained from Glycyrrhiza species [51], and spore rec-assay [108,109] was used for moraceous flavonoids as shown in Table 7. Sixty-nine Glycyrrhiza phenols out of a total 108 compounds showed inhibitory activity against the growth of both HI7 and M45 strains. Cytotoxic activities of these antibacterial compounds {Glycyrrhiza phenols and moraceous phenols) against human oral squamous cell carcinoma (HSC-2) and human T-lymphoblastoid cell line MT-4 cells were also shown in Table 7 [110-113] along with other biological activities reported until the middle of 2002. In the Table, relatively strong-cytotoxic compounds against HSC-2 (CC5o<25 |ig/mL)
224
that showed no activity against these B. subtilis strains were also shown in Table 7. On the comparison between these bioactivities of the phenolic compounds, relationship between the antibacterial activity against B, subtilis and cytotoxicity against HSC-2 or MT-4 cells was not found. Hatano et al. reported antibacterial effect of licorice flavonoids against methicillin-resistant Staphylococcus aureus (MRSA) [114]. 8-(y,Y-Di-methylallyl)-wighteone (58) and 3'-(Y,Y-dimethylallyl)-kievitone (27) showed relatively strong activity against clinically isolated MRSAs (MIC=8 lag/mL). Licochalcone A (59), gancaonin G (60), glyasperins C (61) and D (62), glabridin (23), licoricidin (63), licocoumarone (64), and isoangustone A (65), Fig. (20), showed slightly weak activity against the bacteria (MIC=16 |ig/mL) [114].
8-(Y,Y-dimethylallyl)-wighteone (58): Ri = R3 = R4 = H, R2 = CH2CH=CMe2 gancaonin G (60): Ri = Me, R2 = R3 = R4 = H isoangustone A (65): Ri = R2 = H, R3 = 0H, R4 =CH2CH=CMe2
glyasperin C (61): Ri = R2 = H glyasperin D (62): Ri = Me, R2 = H licoricidin (63): Ri = H, R2 = CH2CH=CMe2
licochalcone A (59) ^^^
i
licocoumarone (64)
Fig. (20). Structures of licorice flavonoids (58 - 65). Table 6.
Antimicrobial activity of licorice flavonoids (MIC, ng/mL) MSSA FDA 209P
Glabridin (23) Glabrene (24) Licochalcone A (59) Licochalcone B (82) Liquiritigenin (101) Liquiritin (113) Licoisoflavone B (67) Formononetin (116) Licoricidin (63) Glycyrol (76) Isoglycyrol (117) 3-0-Methylglycyrol(118) Vestitol(119) Licoricone (120) Glycyrin (121), Isolicoflavonol (122) GancaonolB(123) Glyasperin D (62) Gancaonin I (126) AMOX
12.5 12.5 3.13 25 >100 >50 12.5 >25 3.13 >100 >25 >16 >50 25 >50 12.5 >32 6.25 3.13 0.1-0.2
MSSA Smith
MRSA K3
MRSA ST 28
M luteus ATCC9341
12.5 12.5 12.5 12.5 6.25 6.25 100 100 >100 >100 >50 >50 12.5 12.5 >25 >25 3.13 6.25 >100 >100 >25 >25 >16 >16 >50 >50 >50 >50 >50 >50 12.5 25 >32 >32 6.25 6.25 1.56 1.56 0.20 25-50
12.5 12.5 6.25 100 >100 >50 12.5 >25 6.25 >100 >25 >16 >50 >50 >50 25 >32 6.25 3.13 50
12.5 25 6.25 >100 >100 >50 ND >100 6.25 >100 >100 >16 >50 >50 >50 25 >32 12.5 3.13 0.025
E. coli NIHJ JC-2 >100 >100 >100 >100 >100 >50 ND >100 >100 >100 >100 >16 >50 >50 >50 >100 >32 >50 >50 3.13
MSSA means methicillin-sensitive Staphylococcus aureus. M. and E. mean Micrococcus and Escherichia, respectively. A positive control used was amoxicillin (AMOX). ND, not determined.
225
We also screened anti-MRSA flavonoids [115] in the course of the study of anti'Helicobacter pylori flavonoids from licorice described in Chapter X. In our screening (Table 6), glabrene (24), licoisoflavone B (67), and gancaonin I (126) also exhibited anti-MRSA effect. Among these compounds, 23, 24, 27, 59, 61, 62, 63, 67, and 126 exhibited relatively high anti-bacterial activities against B. subtilis (HI7), Table 7. Rec-assay Seven compounds, licoisoflavanone (66), licoisoflavone B (67), semilicoisoflavone B (68), gancaonin C (69), isoliquiritigenin (70), 6prenyleriodictyol (71), and 8-prenyleriodictyol (72), Fig. (21), showed positive results in the rec-assay. Isoliquiritigenin (70) was most potent of the seven compounds (Table 7). The simple chalcone 70 has distributed widely in Glycyrrhizin plants as a minor constituent, and its glycosides are mainflavonoidsof licorice as described in Chapter 1. Recently, Okuyama et al. reported inhibition effect of the chalcone (70) on azoxymethane-induced murine colon aberrant crypt foucus formation and carcinogenesis [116].
licoisoflavanone (66) ""OH
gancaonin C (69)
Fig. (21).
isoliquiritigenin (70)
6-prenyleriodictyol (71): Ri = CH2CH=CMe2. R2 =H 8-prenyleriodictyol (72): Ri = H, R2 = CH2CH=CMe2
Structures o f licorice flavonoids (66 - 72).
Anti-HIV activity Anti-HIV activity of prenylflavones from mulberry tree, kuwanon H (2), morusin (3) and its derivatives, was reported by Luo et al. [117]. We studied the effect of Morus flavones on HIV-1 me infected MT-4 cells, but no flavone showed anti-HIV activity in our screening system [111]. These discrepant results might be due to multiple acting sites of
226
flavonoids. We also screened anti-HIV flavonoids from moraceous plants and Glycyrrhiza species, however, only two flavonoids from Glycyrrhiza species and a 2-arylbenzofuran from Morus species showed weak anti-HIV activity with selective index (SI=50% cytotoxic concentration (CC50) for MT-4 cells / 50% effective concentration (EC50) for HIV-infected MT-4cells): 3-hydroxyglabrol (26, SI=10), kumatakenin (73, SI=20), and moracin C (74, SI=12), Figs. (3) and (22), [111]. Manfredi et al. reported the isolation of an anti-HIV diprenylated bibenzyl from G. lepidota, but its therapeutic index (TI=EC5o/CC5o) was small [118]. McKee et al. investigated anti-HIV activity of isoprenoidsubstituted isoflavans from Erythrina lysistemon (Leguminosae). They concluded that both a free 4'-hydroxy1 group and a lack of substituents at positions C-5 and C-6 are necessary for even minimal in vitro anti-HIV activity [52]. MeO.
OMe
norartocarpetin (83)
Fig. (22).
wjghteone (84)
Structures offlavonoids73 - 84 from Glycyrrhiza species and Moraceous plants.
227
Cytotoxic activity against human oral squamous cell carcinoma (HSC-2) Most potent isoprenoid-substituted phenols against HSC-2 cells (CC5o< 10 |ig/mL) were gancaonin R (75), glycyrol (76), glyasperin A (77), licoricidin (63), antiarone I (78), artonin E (7), broussoflavonols B (79) and C (80), kazinol B (81), and morusin (3), Table 7. Structure-activity relationship of isoprenoid-substituted phenols for the cytotoxic activity against HSC-2 cells could not be clear. Nevertheless, most compounds having higher cytotoxic activity have two sets of a hydrophilic group (hydroxyl group) in the vicinity of hydrophobic group (isoprenoid group) at two different sites on a same plain in the molecule. We also investigated the cytotoxic activity of these compounds against normal human gingival fibroblasts (HGF) and compared with activity against HSC-2 cells. Licochalcone B (82) exhibited significant tumor selectivity: index of tumor specificity (ITS=CC5o for HGF/CC50 for HSC-2) was 42 [111]. Norartocarpetin (83) was also showed tumor selectivity (ITS=11) [99]. ITSs of 23 compounds of 63 isoprenoidsubstituted phenols were 2'-^4 [110-112]. Apoptosis is a normal physiological process that occurs during embryonic development as well as during the maintenance of tissue homeostasis. It can be induced by a variety of treatments, such as UV irradiation, cytotoxic chemotherapy, etc. Cells, which die by apoptosis usually, suffer similar morphological change, including nuclear condensation, cytoplasmic blebbing, and DNA fragmentation. Wang et al. reported induction of apoptosis by apigenin (4',5,7-trihydroxyflavone) and related flavonoids through cytochrom c release and activation caspase-9 and caspase-3 in leukaemia HL-60 cells [119]. We found that glabrol (25) and wighteone (84) induced intemucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 cells [111]. This is consistent with the report by Yanagisawa-Shirota et al.; induction of intemucleosomal DNA fragmentation depends on the cell types, rather than apoptosis inducers (ascorbic acid, H2O2, tumor necrosis factor, etoposide, hyperthermia, and UV irradiation) [120]. VIII. ESTROGEN-LIKE ACTIVITY OF PHENOLS FROM THE MORACEOUS PLANTS AND GLYCYRRHIZA SPECIES Traditional Japanese women with their high soy intake, a rich source of
228
Table 7. Inhibitory activity against Bacillus subtilis HI7 and rec-assay (disk division method) of isoprenoid-substituted phenols (75 |ig/disk), their cytotoxic activities against HSC-2 and MT-4 cells, and other biological activities Trivial name
Inhibition forHH"
Rec-assay*
HSC-2^
MT-4''
CC50
CC50
(Hg/mL)
(Hg/mL)
other activity^
(Licorice phenols/ Angustone B Bavachalcone (broussochalcone B) Dehydroglyasperin C 3'-(y,y-Dimethylallyl)kievitone (27) 8-(Y,Y-Dimethylallyl)wighteone (58) Edudiol Echinatin Formononetin (116) Gancaonin C (69) Gancaonin E Gancaonin G (60) Gancaonin H Gancaonin I (126) Gancaonin O Gancaonin P Gancaonin Q Gancaonin R (75) Gancaonin S Gancaonin U Gancaonin V Gancaonin Y Glabranin (glabranine) Glabrene (24)
+
+
-H-
-
14 12
6 2
-H-
-
31
10
++
-
20
10
+
-
22 ND 45 20 125 13 19 97 42 20 14 11 8 10 12 34 ND 14
+-I-
_ +
-
+ + ++ + ND
-
++ -H-
+ -H-
+ ND
± ±
-
+
-
ND
-
± ± ±
-
ND
11 ND 12 100 9 55 28 14 >50 55 22 6 18 2 2 10 ND >100
-H-
±
12
10
Glabridin (23)
+++
±
13
4
Glabrol (25) Glycycoumarin Glycyrin Glycyrol (76) (neoglycyrol) Glyasperin A (77) Glyasperin B Glyasperin C (61) Glyasperin D (62) Glyasperin J Glyasperin K Glisoflavanone Glyinflanin A (glycyrdione A) Glyinflanin B Glyinflanin C (glycyrdione C) Hispaglabridin A (124) 3-Hydroxyglabrol (26) 3-Hydroxyparatocharpin C^ Isoderrone Isoglycyrol(117) Isoliquiritigenin (70)
4-+ -f+
±
18 32 14 <4
>100 13 11 14
-
-
AFE, FDC
ABM ABM, EBV, LAT ABE ABH, APA, EBV, ODD ALR, 5LG ABM ABH, ABM
ALR, ICO, 5LG, NKA, IC0,5LG,NKA ALR, IOC, 5LG, NKA AIA, IPP ABE, ABH, ABM, AFE, AMA, AOA, EAA ABE, ABH, ABM, AFE, AOA, EAA, EAB, ETA, ICO, IMI, PSO, SAE ABE ABC, ABE, AOA, CPH ABC, ABE, ABH ABC, 5LG
-
<8 46 31 48 ND ND 21 ND
53 9 34 >10 ND ND 46 ND
++ ++
-
31 19
27 8
+
-
14 31
>100 12
ABE, AOA, MOS ABE
-
38 ND 16 22
12 ND >100 14
ALA
+ -H-H-H-
++ + ++ ++
-H-
+ ++
-
++
-
± ±
++
ABM ABM, ABH
ABC, ALR ABE, ATP, EAA, MCC, MGA, TFV, UFI
229
Table 7 (continued) Kanzonol B (81) Kanzonol G Kanzonol H Kanzonol P Kanzonol R Kanzonol S Kanzonol U (glabrocoumarone A) Kanzonol V Kanzonol W Kanzonol X (tenuifolin B) Kanzonol Y Kumatakenin (73)
+ + + + ++
—
Licochalcone A (59) Licochalcone B (82) Licoflavonol Licoisoflavanone (66) Licoisoflavone A (phaseoluteone) Licoisoflavone B (67) Licoricidin (63) Licoricone (120) Licorisoflavan A Medicarpin 1-Methoxyphaseollidin (125) 1 -Methoxyficifolinol 3-(9-Methylgancaonin P 4'-(9-Methylglabridin (123) Naringenin Paratocarpin L (macarangaflavanone B) Pinocembrin 6-PrenyleriodictyoF (71) S-PrenyleriodictyoF (72) 6-Prenylnaringenin (90) Semilicoisoflavone B (68) Shinpterocarpin Sigmoidin A Sigmoidin B (99) Topazolin Wighteone (84) (erythrinin B)
±
-
ND ND 46 ND ND ND ND
ND ND >10 ND ND ND ND
-
ND ND ND
ND ND ND
—
ND 375
-H-
—
20
ND 51 TCD 15
-
-
4 22 72 55
16 13 40 21
43 8
7 15
45 14 45 28 11 ND ND ND 24
64 47 53 12 8 ND ND ND 14
105 ND 35 29 78 ND 20 43 19 20
>100 ND 60 32 22 ND 29 26 4 12
+
17 <8 9 ND 30 13 20 <8 ND ND
>8 9 ND ND >100 11 >100 11 ND ND
-hH-
±
<8
4
ND
ND
-
-
<8 <8
>100 20
-I-+
+ + + -H-
+
-H-
++ + -H-H-
+
-
+ -H-
+ ++ -H-
+ ++ -I-+
++ ++ ++ ++ -H-
ND ++ + ++
+
+
~ -
± ±
± + +
-
+
-
ND
-
SOA
AFE, APV, AVC, SST, ABE, ABH, ABM, ALA, ATP, CPH, LFP ABE, CPH, LFP ABE AFE, AOA, TD ABM, AFE, TD ABE, ABH, ABM, AOA, 5LG, NKA, LAT ABH, BDB AOA, FDC, QRI ABH, LAT ABE, ABH, AOA, MOS ARI AIA
ABE, AFE, ESEC, IMS ABE, AFE, ESEC, IMS ABE, AFE, CTK, FDC, HPE
(moraceous phenols)* Albanin D Albanol B (97) Alvaxanthone Antiarone B (18) Antiarone F Antiarone G Antiarone H Antiarone I (78) Antiarone J (19) Artobiloxanthone (8) (KB-l) Artonin E (7) (KB 3) Broussoflavonol B (79) Broussoflavonol C (80)
_
_
ND ND ++ ++ ++
ND ND
-
-H-
++ +-I-+
-
CTC, CTL 5LG, TAR CTC, CTL, 5LG, RAR
230
Table 7 (c ontinued) Broussoflavonol E Cudraphenone B Cudraphenone D Cycloartobiloxanthone (9) Heterophyllin Isoalvaxanthone Kazinol B (81) Kazinol E (37) Kazinol F (38) Kazinol N (41) Kuwanon C (42) (mulberrin)
ND ND ND
Kuwanon G (1) (albanin F, moracein B) Kuwanon M (30) Kuwanon R Moracin C (74) Morusin (3) (mulberrochromene)
ND ND ND ±
10 ND
-HH-
~ -
12 13 20 ND ND 14 19 <8 10 20 15
-H-
-
54
66
-
-
-
24 ND 17 <8
7 ND 19 9
23 <8
ND 2
-H-
+ ND +
-
-H-
++
+ ND ++
-
ND
ND
ND ND ND 12 9 9 10 44
Mulberrofiiran B Mulberrofliran G (25) (Albanol A) Norartocarpetin (83) Oxydihydromorusin (46) (morusinol) Sanggenol C Sanggenol M Sanggenon A (4) Sanggenon B (45)
ND ND
ND ND
-
-
+
45 12
ND ND ND -hH-
ND ND ND ±
25 10 23 22
>10 ND ND ND
Sanggenon C (5)
+++
±
13
ND
Sanggenon M (100) Sorocein F
ND ND
ND ND
21 24
ND ND
++
15 >100
CTC, CTL, 5LG, TAR CTC, CTL, 5LG, TAR ARP,ICO,5LG,NOP AOA, ETA, TPA ETA, TPA ABE, AFE, ARP, CTM, 5LG, 12LG, NOP, SPB, TPA BRA, FHA, FTB, HPA, ODC, PKC, TPA HPA, ODC, PKC, TPA AFA ABE, AFE, ANC, AOA, ARI, ARP, ATP, CTM, FEA, FHA, FTB, ICO, 5LG, NOP, ODC, PKC, PSO, SPB, TPA, TAR ARI, FEA, FHA, FTB, ODC, PA, PKC, TPA, ETA, ICO AVR, FEA, FHA, FTB, SPB, TPA
AFE, ABE, ICO, 5LG, NOP, TPA ABC, ABE, AFE, FHA, FTB, HPA, PKC, TPA
"Diameter of inhibition zone (8 mm paper disk was used), +=less than 11 mm, -H-=between 11 and 18 mm, -H-+=more than 18 mm. Diameter of inhibition zone for kanamicin (10 |ig/disk) is 22 mm. * Difference in diameter of inhibition zone between Bacillus subtilis M45 (rec: - ) and HI 7 (rec: +), diameter of inhibition zone on M45 minus that on HI7; -^diameters are same, ±=less than 2 mm, +=between 2 and 5 mm, -H-=more than 5 mm. A positive control is mitomycin C (0.75 ^ig/disk): the difference of inhibition zone is 6 mm. Inhibition zones of a negative control (kanamicin) were same for the both strains. ^CCso of doxorubicin was 2 |ag/mL. '^ 50% Cytotoxic concentration against human T-lymphoblastoid cell line MT-4 cells without HIV infection. " ABC = antibacterial action against a cariogenic bacterium, Streptococcus mutans [121,122]; ABE = antibacterial effect [8,123-132]; ABH = antibacterial effect against Helicobacter pylori [see the text, 126,127], ABM = antibacterial effect against MRSA [see the text, 114, 115,133]; AFA = anti-feedant activity against silkworm [134]; AFE = antifungal effect [112,134-140]; AIA = anti-inflammatory activity [141,142]; ALA = anti-Leishmania activity [143,144]; ALR = inhibition on aldose reductase [43]; AMA = anti-mutagenic activity [145]; ANE = anti-nociceptive effects in mice [146]; AOA = antioxidant activity [123,125,147-154]; APA = anti-protozoa activity [155]; APV = anti-picomavirus activity [156]; ARI = aromatase inhibitory activity [157]; ARP = inhibition of aggregation of rabbit platelets [158,159]; ATP = anti-tumor promoting activity [see the text, 160,161]; AVC = antiviral activity against Coxsackie virus [162];
231
AVR = antiviral activity against rhinovirus type 2 [8]; BDB = benzodiazepin-binding stimulator [163]; BRA = bombesin receptor antagonist [see the text]; CPH = inhibition of the cytopathic activity of HIV [164]; CTC = cytotoxic activity against colon 38 [see the text]; CTK = cytotoxic activity against KB cells [165]; CTL = cytotoxic activity against L1210 [see the text, 166]; CTM = cytotoxic activity against mouse macrophage cell line (RAW 264.7) [167]; EAA = estrogen agonist activity [168]; EAB = estrogenic and anti-proliferate properties in human breast cancer cell [169]; EBV = inhibitory effect on TPA-induced Epstein-Barr virus early antigen [170]; ESEC = effect on sea-urchin egg cleavage [171]; ETA = effect on tyrosinase activity [153,172-174]; FDC = feeding deterrents for Costelytra zealandica (white) [135,175]; FEA = inhibition of formation of 12-HET from [l-'*C]arachidonic acid [see the text, 79,80]; FHA = inhibition of formation of 12-HHT from [l-^'^CJarachidonic acid [see the text, 79,80]; FTB = inhibition of formation of thromboxane Bi from [l-''*C]arachidonic acid [79,80]; HPA = hypotensive activity [see the text]; HFE = hepato-protective effect [176]; ICO = inhibition on cyclooxygenase [43,172,177,178]; IFF = inhibition of photo-phosphorylation [179]; IMI = inhibitory effect on melano-genesis and inflammation [172]; IMS = inhibition of macrophage superoxide production [180]; LAT = inhibitory effect on lysoFAF (platelet-activating factor) acetyltransferase [181]; LFF = effect on leukotriene formation in human polymorpho-nuclear neutrophils [182]; 5LG = inhibition on 5-lipoxygenase [43,177], 12LG = inhibition on 12-lipoxygenase [177]; MCC = inhibition effect on murine colon carcinogenesis [116]; MGA = mutagenic activity [183]; MOS = protection of mitocondrial fractions against oxidative stresses [184]; NKA = inhibition on Na^ K^-ATPase [43]; NOP = inhibitory activity on NO production from lipopolysaccharide-induced nitric oxide (NO) production from mouse macrophage cell line (RAW 264.7) [167]; ODC = inhibitory activity on induction of ODC [see the text]; ODD = reducing of endogenous oxidative DNA damage [185]; PKC = inhibition of activation of protein kinase C [see the text]; PSO == inhibitory effect on production of superoxide anions [172]; QRI = quinone reductase-inducing activity [186]; SAE = synergistic anti-oxidative effect with lycopene [187]; SPB = substrate for PCB-degrading bacterium, Burkholderia sp. [188]; SOA = stimulation of superoxide anion generation in rat neutrophils [189,190]; SST = suppression of SOS-inducing activity of Trp-P-l {umu test) [191]; TAR = inhibitory effect on TNF-a release [see the text]; TCD = inhibitory activity on TNF-a-induced cell death in mouse hepatocytes [192]; TFV = tube formation from vascular endothelial cells of rats [193]; TPA = inhibiting of ^H-TFA binding [see the text]; UFI = preventive effect on ulcer formation induced by severe necrotizing agents in rats [194]. /Eriodictyole, liquiritigenin (101), sophoraflavanone B (86, AFA), isobavachin (94), kanzonol Z, euchrenone as, ovaliflavanone B, prenyllicoflavone A, glepidotin A, 3,3'-di-0-methylquercetin, 3,4'-di-0-methylquercetin, paratocarpins B and C, glyinflanin G, and licuraside exhibited no effect against both M45 and HI7 strains. ^ No name: names in the table are tentative name used here. * Antiarones A and K, brosimone L, cudraflavanone A, and cyclohetrophyllin (53) showed no effect against both M45 and HI 7 strains. These data were obtained with spore rec-assay (38 ^ig of sample/disk). ND means not determined. The structures of all phenolic compounds isolated from Gfycyrrhiza species until 1996 were reviewed in the reference [43], new compounds from the plants since the time to 2000 were summarized in the proceeding [44].
plant-derived estrogens (phytoestrogens [195]), have a low incidence of breast cancer and few menopausal symptoms. This has led to the hypothesis that at the menopause phytoestrogens might act as natural selective estrogen receptor modulators, tweaking estrogenic responses in the cardiovascular system, bone, and brain, but dampening responses in
232
the breast and uterus. The finding that the soy-derived phytoestrogen genistein (85), Fig. (23), preferentially binds to the form of the estrogen receptor found mainly in the cardiovascular system lends some credence to that belief [196]. It is expected by recent many evidence that phytoestrogens exert beneficent actions to chronic diseases, e.g., heartattacks and other cardiovascular problems, osteoporosis, Alzheimer's disease, etc. [197]. Nevertheless, the isoflavonoids and Ugnans bind w^ith low^ affinity to estrogen receptors, and thus, it is also suggested that they may induce production of sex hormone binding globulin in the liver and in this way influence sex hormone metabolism and biological effects [198]. Recently, Cooke et al. reported that genistein (85) decreased mouse thymocyte numbers and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis [199]. In addition, genistein (85) produced suppression of humoral immunity. These data indicate that use of soybased infant formulas and soy/isoflavone supplements has aroused concern: genistein (85) and daidzein (102) may be capable of producing thymic and immune abnormalities. Therefore, the screening of phytoestrogen from medicinal plants may be important.
genistein ( 8 5 ) : R = OH daidzein ( 1 0 2 ) : R = H
OH sophorafiavanone B ( 8 6 ) : R = OH isobavachin ( 9 4 ) : R = H
0
licoflavone C (87): R^ = R2 = H 8-prenylquercetin (88): R i = R2 = OH noranhydroicaritin (93): Ri = OH, R2 = H
lupiwighteone (89) 6-prenylnaringenin ( 9 0 ) : R = H lonchocarpd A ( 9 1 ) : R = CH2CH=CMe2
17p-estradiol(92)
Fig. (23). Structures of phytoestrogens (85, 87, 88, 93, and 102) and related compounds having no estrogenic effect (89-91).
Numerous phytoestrogens with a diversity of structures have now been recognized [168,169,200-210]. Akiyama et al. reported that sophorafiavanone B (86), Fig. (23), isolated from a Thai crude drug, Anaxagorea luzonensis (Annonaceae), showed about two-fold higher affinity for the bovine uterine estrogen receptor than that of genistein (85), Table 8 [197]. They also reported that synthetic 8-prenylated flavonoids, licoflavone C (87) and 8-prenylquercetin (88), also exhibited the binding
233
affinity but their binding affinities were weaker than that of 86. In the report [197], it was also reported that a synthetic 6-prenylated isoflavone, lupiwighteone (89), and 6-prenylated and 6,8-diprenylated flavanones, 6prenylnaringenin (90) and lonchocarpol A (91), Fig. (23), did not exhibit the binding affinity against the receptor. This study indicated that the position of the steric hydrophobic bulky group (prenyl group) is important for the binding affinity of prenylated flavonoids. We examined whether other types of phenols with two or more aromatic rings bind to the estrogen receptor [45,211]. About one hundred phenols with isoprenoid groups or without side chains from moraceous plants and Glycyrrhiza species and synthetic flavonoids were evaluated with the estradiol receptor-binding assay [204]. Among them, 13 compoimds exhibited weak binding affinities (1C50<1 |ig/mL) in which three compounds were isolated from moraceous plants (95, 97, and 100), and six were constituents oi Glycyrrhiza species (24, 75, 76, 94, 99, and 101), Fig. (24).
HOJ tetrahydrogiabrene (96)
* bH alband B (97)
OH 8-geranylapigenin (98)
sigmoidin B (99): Ri = OH, R2 = CH2CH=CMe2 liquiritigenin (101): Ri = R2 = H
sanggenon M (100)
Fig. (24). Estrogenic compounds from moraceous plants (95, 97, and 100) and Glycyrrhiza species (99 and 101) and synthetic estrogenic flavonoids with a isoprenoid group (96 and 98).
Relative binding affinities of these compounds against 17p-estradiol (92) (RBA=[IC5o of 92 (nmol/L)] / [IC50 of test sample (nmol/L)]) values are shown in Table 8. The affinity of gancaonin R (75) was stronger than those of genistein (85, RBA=0.004) and daidzein (102, RBA=0.00035) [205]. The affinities of the other 12 compounds were similar to those of the isoflavones (85 and 102) in dietary foods. Recently, Vaya et al. reported binding affinities of glabrene (24), isoliquiritigenin (70), and glabridin (23) for human estrogen receptor [168].
234
Table 8. Relative binding affinities of phenolic compounds for the bovine uterine estrogen receptor Compound
RBA
Sources
Gancaonin R (75) Noranhydroicaritin (93) 8-Prenylquercetin (88) Isobavachin (94) Isobavachalcone (95) Glabrene (24) Tetrahydroglabrene (96) Glycyrol (76) Albanol B (97) 8-Geranylapigenin (98) Sigmoidin B (99) Sanggenon M (100) Liquiritigenin (101)
0.016 0.0064 0.0057 0.0054 0.0045 0.0022 0.0016 0.0012 0.0011 0.00095 0.00077 0.00048 0.00038
Compound
RBA
Sources
[ref.]
Sophoraflavanone B (86) Sophoraflavanone B (86) Licoflavone C (87) 8-Prenylquercetin (88) Hinokiresinol Nyasol (c/5-hinokiresinol) (3i?)-nyasol (35)-nyasol Neoflavone dimer T Neoflavone dimer 2° Dihydrochalcone* Homoisoflavone*" 10-Hydroxycoronaridine Coronaridin
0.0071 0.0091 0.0063 0.0015 0.00083 0.00017 0.0010 0.0067 (0.028)* (0.0028)* 0.0010 0.0024 (0.003)* (0.0005)*
Anaxagorea luzonensis synthesis synthesis synthesis Chamaecyparis obtusa Anemarrhena asphodeloides Anemarrhena asphodeloides Anemarrhena asphodeloides Pistacia chinensis Pistacia chinensis Dracaena loureiri Draceana loureiri Tabernaemontana penduliflora (not reported)
[197] [197] [197] [197] [209] [209] [209] [209] [210] [210] [204] [204] [208] [208]
G. uralensis (aerial part) synthesis synthesis G. pallidiflora (root) M cathayana (root) G. glabra (root) synthesis G. uralensis (underground part), G. aspera (root) M alba (root bark) synthesis G. uralensis (aerial part), G. eurycarpa (aerial part) M cathayana (root bark) Glycyrrhiza species (root and aerial part)
" 3,3"-Dimer of 3,4-dihydro-4-(4'-hydroxyphenyl)-7-hydroxycoumarin. * No data of the positive control (92) was reported. * 4,4'-Dihydroxy-2,6-dimethoxydhihydrochalcone. *" 5,7-Dihydroxy-3-(4-hydroxybenzyl)-4chromone.
On the molecular modeling examination of gancaonin R (75), the prenyl groups did not lie into the lipophilic pocket of estrogen receptor that is illustrated in Fig. (27). These groups existed near the B and C rings of 17p-estradiol (92) as shown in Fig. (25). On the molecular models, the volume of 17p-estradiol (92) was 266.9 A^ (based on van der Waals radius of atoms) and that of compound 75 was 374.5 A^, and the distance between 3-0- and 17-0- of 92 was 10.9 A and 4'-0- and 3(5)-0of 75 was 11.0 A. The overlapping volume of these two models of 75 and 92 was 204.2 A l We also studied the structure-activity relationships with computation modeling of these estrogenic phenols (Table 8) and non-estrogenic phenols [45]. For the examination of molecular modeling of the isoprenoid-substituted phenols, we made a space model of estrogen receptor (SMER) with an imaginary compound (103), Fig. (26), that built up with
235
estrogenic steroids (104-108) having higher binding affinities than that of 17p.estradiol(92)[212].
Fig. (25). Molecular models of gancaonin R (75, ball and stick) and 17p-estradiol (92, stick): overlay of B These molecular models were minimized with KfM2, and then calculated with ring of 75 and A ring of 92. Mopac6.
104 SMERmodd(103)
106
107
108
Fig. (26). Structures of an imaginary compound (103) that built up with estrogenic steroids (104 - 108) having higher binding affmities than that of 17p-estradiol (92).
The SMER, Fig. (27), is similar model to estrogen receptor excluded volume (RExV) postulated by Kym et al. [212]. The volume of the SMER was 424.7 A^. Most flavonoid skeletons (A-C rings) lay into the SMER, however, the prenyl (geranyl) groups existed out of the SMER as shown in Fig. (27). By our examination of the modeling as illustrated in Fig. (27), it was indicated that orientations of these estrogenic compounds in tiie binding site of the estrogen receptor relative to 17P-estradiol (92) depend on tiieir
236
SMER A ring of 76 and 103
Prenyl group of 76
Lipophilic pocket
Fig. (27). Overlapping of the SMER and molecular model of glycyrol (76).
skeletal structures as shown in Fig. (28). The binding sites of 17pestradiol in estrogen receptor-a and -(5 have been determined by X-ray crystallographic study [213,214]. In Fig. (28), the amino acid residues beside the phenols mean hydrogen-bonding binding site of the estrogen receptor-a [213]. The orientations of these prenylated phenols against binding site of estrogen receptor-p [214] may be similar to against estrogen receptor-a. Fig. (28). The binding sites of albanol B (97), a 2arylbenzofuran derivative w^ith three additional aromatic rings, in estrogen receptor-a may be different from those of prenylated phenols. Fig. (28): the binding sites of 97 may be similar to those of raloxifene. Asp 351, Glu 353, Arg 394, and His 524 [213], but the binding of 97 is not completely because of lacking of the binding to His 524, and the A, B, and C rings of 97 may lie in the lipophilic pocket of the receptor as shown in Fig. (29). A and E rings of sanggenon M (100) may also locate in the lipophilic pocket. Fig. (29). From the molecular modeling analyses of isoprenoid-substituted phenols that did not showed binding affinity for the estrogen receptor, it was indicated that the binding sites at C-3 and C-17 of 17p-estradiol are rigid, but the lipophilic pocket near C-4~C-7 is flexible. IX.
AKll'HELICOBACTER FLAVONOIDS
PYLORI ACTIVITY OF LICORICE
Helicobacter pylori is a bacterium that lives in the human stomach and duodenum. The bacterium is generally recognized as one of the etiological agents of peptic ulcer. Therefore, it is generally accepted that ulcer patients with H. pylori infection require treatment with antimicrobial agents in addition to anti-secretory drugs, whether on first
237
H----[His 524]
[Glu 353]
[Glu 3531
[H2O]IH2O] [Arg394]
[Arg 394]
17p-estradioi(92)
isoflavones (Ri = O. A^-^ : 85 (R2 = OH), 102 (R2 = H) lsoflav-3-ene (R, = R2 = H, A^"*): 24 Isoflavan (Ri = H2. R2 = H, no A^'^= ^•^): 96 H - - . [His 524]
[Glu 3531
....-[His 524]
[Glu 353]., [H2O]''' [H2O]-
[Arg 394] dihydrostilbene :75
[Arg 394] coumestan: 76
[His 524]
[Glu 353]»
[Asp 351],^
[Glu 353],
[H2O][Arg 394]
[H2O]-' [Arg 394]
flavones (R = H, A^^): 87,98 flavonols (R = OH. A^^): 88,93 flavanones (R = H, no A^'^): 86,94,99,101
albanol B (97)
[His 524] [Glu 353]
[H2O]''
/
[Arg 394]
[Arg 394] isobavachalcone (95)
sanggenon M (100)
Fig. (28). Orientations that flavonoids and a dihydrostilbene (75) may adopt in the binding site of the estrogen receptor relative to 17p-eatradiol (92): the amino acid residues mean binding site of estrogen receptor-a. A-D mean the positions of the A-D rings of 17p-estradiol but not the rings of these phenols.
presentation with the illness or on recurrence [215]. On the other hand, gastric cancer is one of the most frequent cancers on a worMwide and the leading cause of death from cancer. Since the discovery of H. pylori [216,217], an association between the bacterium and gastric cancer has
238
A ring Ertng
(SMERandlOO) (SMERand97) Fig. (29). Overiapping of the SMER (stick) and molecular model of albanol B (97, ball and stick) and sanggenon M (100, ball and stick).
been suspected. Descriptive epidemiological data indicate that gastric cancer occxirs morefrequentlyin some populations that have higher rates of H. pylori infection. Furtheraiore, rates of both K pylori infection and gastric cancer are correlated inversely with socioeconomic status and increase as a ftmction of age and/or intake of dietary salt [218,219]. Recently, Uemura et al. reported the first evidence of the association between K pylori and gastric cancer with a long-term study [220]. They studied a large group of Japanese patients with duodenal ulcer, gastric hyperplasia, or non-ulcer dyspepsia. During the study, gastric cancer developed in 2.9% of the patients infected with K pylori but in none of the uninfected patients. However, most people with chronic H. pylori infection have no symptoms of peptic ulcer or gastric cancer, which raises questions regarding preventive agents against these diseases; infected individuals without disease symptoms may be protected by anti-bacterial compounds in the diet and/or medicinal plants used frequently [221-239]. Many anti-//. pylori agents with a diversity of structures have been isolated from plant sources [240-252]. However, their antibacterial activities against the bacterium in stomach are unclear [253] as the bacterium in the narrow interface between the gastric epithelial cell surface and the overlying mucus gel [215,217]. Among these antibacterial compounds, flavonoids could be expected to show anti-//. pylori effects in vivo, because kaempferol (109) exhibited antibacterial action in K pyloriinfected Mongolian gerbils [243]. As the biological activities of
239
flavonoids are generally weak, the phenolic compounds may act as bacterial suppressors in the stomach. Most elderly Japanese favor Kampo-medicines (traditional Chinese medicines modified in Japan) rather than synthetic medicines. Generally, traditional Chinese medicines consist of mixtures of crude drugs and require extraction with boiling water for lengthy periods. Sasaki et al. reported that aqueous solutions of some kinds of licorice saponins solubilize water-insoluble substances such as a-tocopherol and oleanolic acid [254]. The solubilizing effect of the saponins is expected to result in licorice extract containing lipophilic compounds such as the isoprenoid-substituted flavonoids. Licorice extract is frequently used in Japanese over-the-counter (OTC) drugs that can be purchased without a doctor's prescription, e.g. stomachic, cough medicines etc. [255]. Therefore, we studied anti-K pylori activities of the flavonoids from licorice [256]. Licorice is for the most part derived from Glycyrrhiza glabra, G. uralensis and G. inflata [43]. G. glabra is used worldwide but the other two species are mainly consumed in Asian countries as described in Chapter 1. The common constituents of these licorices are triterpenoid saponins, glycyrrhizic acid (110) and licorice-saponin G2 (112), Fig. (30), a flavanone, liquiritigenin (101), Fig. (24), its 4'-0-glucoside, liquiritin (113), and an isoflavone, formononetin (116). These saponins (110 and 112) exhibited no anti-//. pylori activity and the aglycone of 110, glycyrrhetic acid (111), showed weak activity (Table 9) as reported previously [257]. The flavanone glucoside (113) also exhibited no inhibitory activity against the bacterium, and its aglycone (101) showed weak activity (Table 9). The main flavanone of the licorices is compound 113 but the aglycone (101) is a minor component in these plants. Kim et al. reported the anti-//. pylori activity of the metabolites of poncirin (114) from Poncirus trifoliate (Rutaceae) by human intestinal bacteria [246]. They speculated that one of the metabolites, isosakuranetin (115), contributes to the prevention of gastritis to some degree because of its anti-if. pylori activity (MIC=10 |ig/mL against H, pylori ATCC 43504, 5x10^ cfu). In the case of licorice, the intestinal bacteria would be expected to hydrolyze these glycosides (110 and 113). However, it may be difficult that these hydrolysates (111 and 101) to be transferred from the intestine to the stomach. Formononetin (116) is frequently isolated from leguminous plants. The compound exhibited weak anti-//. pylori activity when bacterial concentration was 2x10^ cfu. The compound also showed weak antibacterial activity against a clarithromycin (CLAR, a macrocyclic antibiotic)-resistant strain, GP98, at higher bacterial concentration (2x10^ cfu) but not against CLAR-sensitive strains (Table 9). Strain GP98 is also an amoxicillin (AMOX, a penicillin class antibiotic)-resistant strain. The compound
240
may be a bacteriostatic agent for these CLAR (AMOX)-sensitive strains. Nevertheless, the isoflavone (116) is a minor constituent of licorice. These observations suggested that anti-//. pylori agents in licorice are isoprenoid-substituted flavonoids OOH
H00<
= A OH
0
kaempferoi (109) glycyrrtiizic acid (110): Ri = A, R2 = CH3 glycyrrtietlc acid (111): Ri = H, R2 = CH3 licorice-saponin G2 (112): Ri = A, R2 = CH2OH
a
OCHa
OH liquiritin (113): R = p-D-gtucopyranosyl
Fig. (30).
0
OMe
poncirin(114): R = 2-0-a-L-rhamnopyranosylp-D-glucopyranosyl isosakuranetin (115): R = H
Structures of compounds 103 - 109.
We selected eight flavonoids from licorices (G. glabra^ G, inflata, and G, uralensis) to test for anti-//. pylori activity (Table 9). A pyranoisoflavan, glabridin (23), and a pyranoisoflav-3-ene, glabrene (24), are characteristic flavonoids of European G. glabra as described in Chapter 1. These compounds exhibited weak anti-//. pylori activity against these four strains. The characteristic flavonoids of G. inflata are licochalcones A (59) and B (82). Compound 82 did not exhibit anti-^. pylori activity. However, compound 59 showed weak bioactivity. The characteristic flavonoids of G. uralensis are an isoflavan with two prenyl groups, licoricidin (63), a prenylated coumestan, glycyrol (76), a coumestan with a dihydropyran ring, isoglycyrol (117), and a pyranoisoflavone, licoisoflavone B (67). These compounds were also isolated from G aspera but this licorice is commercially unimportant because of its small plant size [43]. No anti-//. pylori activity of the coumestans (76 and 117) could be detected against the strains examined, but the isoflavan (63) and the isoflavone (67) exhibited antibacterial activity. The inhibitory activity of 67 against the growth of CLAR and AMOXresistant strain GP98 is of considerable value: its minimum inhibitory concentration (MIC) was 3.13 |ig/mL against 2x10^ cfu of this strain. Nevertheless, this compound is a minor component of licorice, and thus
241
the main anti-//. pylori agent of G. uralensis may be licoricidin (63). Tabic 9. AnXi'Helicobacter pylori activities (MIC, ng/mL) of the characteristic compounds of licorices (Glycyrrhiza glabra, G. inflata, and G. uralensis) ATCC 43504 Glycyrrhizic acid (110) Glycyrrhetic acid (111) Licorice-saponin G2 (112) Liquiritigenin (101) Liquiritin (113) Formononetin (116) Glabridin (23) Glabrene (24) Licochalcone A (59) Licochalcone B (82) Licoricidin (63) Licoisoflavone B (67) Glycyrol (76) Isoglycyrol (117) AMOX**
>100 >100 50 25 >100 >100 50 50 >50 >50 >100 12.5 12.5 12.5 12.5 12.5 25 25 >50 >50 12.5 12.5 6.25 6.25 >50 >50 >100 >100 0.05 0.025
ATCC 43526 >100 >100 50 25 >100 >100 50 50 >50 >50 >100 12.5 12.5 12.5 12.5 12.5 25 25 >50 >50 12.5 12.5 6.25 6.25 >50 >50 >100 >100 0.05 0.025
ZLM 1007
GP98
>100 >100 >100 >100 50 50 25 25 >100 >100 >100 >100 50 50 50 50 >50 >50 >50 >50 12.5 >100 12.5 12.5 12.5 25 12.5 12.5 12.5 12.5 12.5 12.5 25 25 12.5 12.5 >50 >50 >50 >50 12.5 6.25 6.25 6.25 6.25 6.25 3.13 6.25 >50 >50 >50 >50 >100 >100 >100 >100 0.20 0.05 0.025; 0.10
(cfii)'* (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b)
source
G. glabra G. glabra G. inflata G. inflata G. uralensis G. uralensis G. uralensis G. uralensis
* (a): 2x10^ colony forming units (=cfti), (b): 2x10^ cfii. ** Positive control; amoxicillin (=AMOX). ATCC 43504, ATCC 43526, and ZLM 1007 are CLAR-sensitive strains.
Next, we attempted to isolate further flavonoids exhibiting anti-^. pylori activity from the extract of G. uralensis. In 1967, Takagi and Ishii reported that one of the flavonoid-rich fractions of G. uralensis (FMIOO), which also included about 15% glycyrrhizic acid (110), is effective in prevention of digestive gastric ulcer by suppressing gastric secretion [258,259]. The fraction was developed as an anti-ulcer drug and ten similar medicines containing licorice extract have been also supplied as prescribed drugs for treatment of gastric ulcer, duodenal ulcer, and gastritis [255]. Our study of FMIOO showed that the medicine exhibited anti-/f. pylori activity but did not contain licoricidin (63), which is the main isoprenoid-substituted flavonoid in G. uralensis [259,260] and exhibited anti-//. pylori activity as described above. The other antibacterial agent 67 was not detected in FMIOO on TLC analysis. The above investigations indicated strongly that licorice extract contains some anti-//. pylori flavonoids.
242
H3CO.
3-0-methylglycyrol (118)
OH glycyrin (121)
6,8-diprenylorobol (124)
0
isdicofiavonol (122)
1-methoxyphaseollidin (125) HO^ ^..^ ^ 0 ^
OH O
0CH3 gancaonin I (126)
gancaond C (127)
_ . ^ ^
dihydroisoflavone A (128)
OCH3 4'-0-methylglabriclin (129)
hispagiabridin A (130)
shinflavanone(131)
Fig. (31). Structures of compounds 117-128 isolated from the active fractions of the methanol extract of G. uralensis and compounds 129 -131 from the dichloromethane extract of G. glabra (Russian licorice).
The isolation of flavonoids from the methanol extract of G. uralensis was carried out under non-basic conditions, because some flavonoids isomerize under basic conditions, e.g. racemization of flavanones and isoflavanones, ring-open reaction of flavanones etc. Bioactive fractions were separated by some chromatographic methods and each step was monitored with anti-//. pylori activity with the paper disk method. Eighteen compounds were isolated from these bioactive fractions and
243
their anti-H. pylori activities were shown in Table 10. The MICs of the growth of H. pylori of vestitol (119), licoricone (120), 1-methoxyphaseollidin (125), and gancaonol C (127), Fig. (31), were similar to that of licoricidin (63). The activities of the other flavonoids were weak and similar to those of glycyrrhetic acid (111) and liquiritigenin (101). All the compounds investigated here had weaker anti-//. pylori activity; however, these compounds may be chemopreventive agent agents the H. pylori infection. Furthermore, these compounds may be bacteriostatic agents for the bacteria in the stomach and prevent peptic ulcer or gastric cancer disease in H. pylori-mfQCtcd people. However, further pharmacological and clinical studies including the antibacterial effect in liquid medium are required for confirmation of this hypothesis. Imakiire et al. also reported antibacterial activities of compound 23, 4'-0-methylglabridin (129), hispaglabridin A (130), glabrol (25) and shinflavanone (131), Fig. (31), from the lipophilic extract of Russian licorice, G. glabra; Maruzen P-TH® that is a material of medicines and cosmetics [126,127]. Table 10. Anti-Helicobacter pylori activities (MIC, |ig/mL) of the flavonoids from Glycyrrhiza uralensis ATCC 43504 ATCC 43526 ZLM 1007 Glyasperin D (62) 3-0-Methylglycyrol (118) Vestitol (119) Licoricone (120) Glycyrin (121) Isolicoflavonol (122) Gancaonol B (123) 6,8-Diprenylorobol (124) l-MethoxyphaseoUidin (125) Gancaonin I (126) Gancaonol C (127) DihydrolicoisoflavoneA(128)^ CLAR** AMOX**
25 25 >16 >16 12.5 12.5 12.5 12.5 50 50 50 25 >32 32 >50 50 16 16 50 50 16 16 >25 >25 0.025 0.0125 0.05 0.025
25 25 >16 >16 12.5 12.5 12.5 12.5 50 50 25 25 32 16 >50 50 16 8 50 50 16 8 >25 >25 0.0125 < 0.0063 0.05 0.025
12.5 12.5 >16 >16 12.5 12.5 12.5 12.5 50 25 25 25 32 32 50 50 16 16 50 50 32 16 25 25 < 0.0063 < 0.0063 0.05 0.025
* (a): 2x10^ cfu, (b): 2x10^ cfu. ^ Tentative name used here. ** Positive control; clarithromycin (=CLAR) and amoxicillin (=AMOX).
ZLM 1200 25 25 >16 >16 12.5 12.5 25 12.5 50 50 50 25 32 16 >50 50 16 16 >50 50 32 16 >25 >25 0.0125 < 0.0063 0.025 0.125
GP98
(cfu)*
12.5 6.25 >16 >16 12.5 6.25 12.5 12.5 25 25 25 12.5 16 16 50 50 16 8 50 50 16 16 25 25 50 12.5 0.2 0.1
(a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b)
244
Protonpump inhibitor-based triple therapy is now the most commonly accepted eradication regimen for peptic ulcer patients with H. pylori infection. However, CLAR resistance is an increasing problem as its use has become more common in recent years [234,261]. It is interesting that licorice flavonoids exhibited anti-//. pylori activity against not only CLAR and AMOX-sensitive strains but also CLAR and AMOX-resistant strain CP98: Although licorice has been used as a crude drug in Japan from more than 1200 years [262], these strains have not developed resistance to the licorice flavonoids. These compounds may be useful as lead compounds in the development of a new class of anti-//. pylori agents. X. EFFECTS OF ISOPRENYLATED FLAVONOIDS FROM MORUS SPECIES ON TESTOSTERONE 5a-REDUCTASE In Japan, the extracts of mulberry tree have been used for promotion of hair growth and prevention of baldness [263]. Testosterone 5a-reductase catalyses the reduction of testosterone to its active form, 5a-dihydrotestosterone (5a-DHT). 5a-DHT has been implicated in certain androgen-dependent conditions such as benign prostatic hyperplasia, acne, and male pattern boldness [264]. And 5a-reductase activity is high in situ. Inhibitions of 5a-reductase may be usefuU for the treatment of these diseases. Therefore we studied on 5a-reductase inhibitory activity of some isoprenylated flavonoids isolated from the root bark of Japanese mulberry tree [265]. Table 11 shows the 5a-reductase inhibitory activity of flavonoids isolated from the root bark of Morus species. Most of the flavonoids had inhibitory activities against 5a-redactase, and showed the activity in the range of 10^ - lO"'' mol/L. Kuwanon E (43) had the most potent activity of these compounds and its IC50 value is 6.9x10"^ mol/L, while kuwanon G (1) had no effect at 10"^ mol/L. Fig. (32) shows the effects of kuwanon E (43) on the Lineweaver-Burk plots of rat prostate 5a-reductase activity using testosterone as a substrate. The addition of 3x10"^ mol/L kuwanon E (43) produced a parallel shift indicating un-competitive inhibitor. And the apparent K\ value is 7.6x 10~^ mol/L. Enzyme kinetic studies of inhibitor are very important for considering as a therapeutic agent. It is interesting to note that isoprenoid-substituted flavonoids having non-steroidal structures are potent un-competitive inhibitors of 5a-reductase. So, it would be expected that the isoprenoid-substituted flavonoid derivertive would be an interesting lead compounds for testosterone 5a-reductase inhibitor.
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Table 11. Effects of Morus flavonoids on testosterone 5a-reductase Inhibition (%)* 59.6 35.6 63.0 94.0
Morusin (3) Oxydihydromorusin (46) Kuwanon C (42) Kuwanon E (43) Kuwanon G (1) Kuwanon H (2) Kuwanon L (44) Mulberrofiiran A (47) Mulberrofuran G (30)
0 100 53.0 24.2 37.0
IC50 (mol/L)
8.2x10"^ 6.9x10"' 1.8x10"^ 4.4x10"^
' Final concentration at 100 |imol/L.
lA'estosterone (1/10^ mol/L) Fig. (32). Lineweaver-Burk plots of inhibition of prostatic 5a-reductase by kuwanon E (43). The assay was carried out at varied concentration of [4-*'*C]testosterone in the absence (o) or in the presence of 0.3 Hmol/L kuwanon E (•).
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
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PLANT POLYPHENOLS: STRUCTURE, OCCURRENCE AND BIOACTIVITY PffiRGIORGIO PIETTA, MARKUS MINOGGIO, LORENZO BRAMATI ITB-CNR, Via FJli Cervi, 93- 20090 (MI), Italy ABSTRACT: Main dietary plant polyphenols are grouped into structural types and their occurrence in most common foods and beverages is briefly described. The major groups of plant polyphenols which are examined include flavonols, flavones, flavanones, isoflavones, anthocyanins, proanthocyanidins and flavanols. The current evidence on the absorption and the metabolism of each group is discussed. The biochemical and pharmacological activities of plant polyphenols are summarized, including antioxidant and anti-radical activity, chelation of metal ions, modulation of some enzymes activity, anticarcinogenic, antiatherosclerotic, anti-inflammatory, spasmolytic, hepatoprotective, antiviral, antimicrobial and oestrogenic activity, and inhibition of histamine release. An overview on the epidemiological evidence linking the intake of plant polyphenols and diminished risk of chronic diseases is also included.
INTRODUCTION Polyphenols constitute one of the most and widely distributed groups of substances in the plant kingdom, with more than 8000 phenoUc structures currently knovra. They can be divided into at least 10 different classes based upon their chemical structure, ranging from simple molecules, such as phenolic acids, to highly polymerized compounds, such as taimins. Flavonoids constitute the most important group with a common structure of diphenylpropanes (C6-C3-C6), consisting of two aromatic rings linked through three carbons that usually form an oxygenated heterocycle. Based upon the variations in the heterocyclic ring , flavonoids can be subdivided into eight major subclasses, including flavonols, flavones, flavanones, isoflavones, flavanols, anthocyanins, proanthocyanidins and tannins. Plant polyphenols have been of interest for long time owing to their role in plant pigmentation, reproduction and protection against predators and pathogens. Recently, interest in the biological effects of plant polyphenols (particularly flavonoids) has increased, because of the the potential health benefits associated with some dietary polyphenols. This
258
contribution provides a brief description of the chemistry of plant polyphenols, their occurrence, bioavailability and bioactivity. CLASSES OF PLANT POLYPHENOLS I. Hydroxy benzoic acids derivatives The hydroxybenzoic acid derivatives (HBAs) are phenolic compounds with a general structure Ce-Ci. The related C6-C2 acids (phenylacetic acids) occur occasionally as minor components of foods. Variations in the basic structure of HBAs include hydroxylation and methoxylation of the aromatic ring, Fig. (1), Table 1.
Fig. (1). Basic structure of hydroxybenzoic acids
Although HBAs can be detected as free acids in some fruits (e.g., gallic acid in persimmons) or after being released during fruit and vegetable processing, they occur mainly as conjugates. For example, gallic acid and its dimer ellagic acid may be esterified with a sugar, usually glucose to produce the so-called hydrolysable tannins. In addition, gallic acid may esterify condensed tannins (i.e., derivatives of flavanols like those present in tea) or quinic acid [1,2]. Four HBAs, namely 4hydroxybenzoic acid (4-HBA), vanillic acid (3-methoxy-4-hydroxy), syringic acid (3,5-dimethoxy-4-hydroxy) and protocatechuic acid (3,4dihydroxy), are constituents of lignin [3]. These acids occur also as esters of glucose.
259
Table 1.
Structure of the most common hydroxybenzoic acids
Trivialname
MW (Da)
Position of OH groups
Benzoic acid
122
/
Salicylic acid (2-Hydroxybenzoic acid)
138
2
4-Hydroxybenzoic acid
138
3
Protocatecliuic acid (3,4-Dihydroxybenzoic)
154
3,4
Gallic acid (3,4,5-Trihydroxy benzoic)
170
3,4,5
Vanillic acid (4-Hydroxy-3-methoxybenzoic
168
4
3
Isovanillic acid (3-Hydroxy-4-methoxybenzoic)
168
3
4
Syringic acid (4-Hydroxy-3,5-dimethoxybenzoic)
198
4
3,5
Position of OCH3 groups
Dietary occurrence Some herbs and spices are comparatively rich in various HBAs. After hydrolysis, protocatechuic acid is the dominant HBA in cinnamon bark (23-27 mg/kg), accompanied by saUcylic acid (7 mg/kg) and syringic acid (8 mg/kg). Gallic acid dominates in clove buds (175 mg/kg) and is accompanied by protocatechuic acid (10 mg/kg), genistic acid/4-HBA (7 mg/kg) and syringic acid (8 mg/kg) [4]. The fruit of anise {Pimpinella anisum) contains 730-1080 mg/kg of the glucoside of 4-HBA [3]. The skin of potato tubers contains protocatechuic acid (100-400 mg/kg FW) and vanillic acid (20-200 mg/kg) along with up to 30 mg/kg of gallic, syringic and salicylic acids [5]. Cereals contain also different HBAs. Canadian wheat flours were found to contain vanillic acid (up to 16 mg/kg) and syringic acid (up to 7 mg/kg). Oats contain vanillic acid, 4-HBA and salicylic acid, particularly in the hulls [6]. Alcoholic beverages (wine and beer) have a different content of HBAs. The gaUic acid content of French wines and spirits can reach 31-38 mg/1
260
[7]. White wines contain less HBAs than red wines, namely 16-46 and 65-126 mg/1 for white and red Califomian wines [8]. Barley contains vanillic acid (6-17 mg/kg) and syringic acid (1-22 mg/kg), and both are found in malt (12 mg/kg each) and hops (59 and 30 mg/kg). These two acids are foimd in stout, ale and lager beers in the range from 0-2 mg/1) accompanied by gaUic, protocatechuic and 4-HBA (0.1-1.8 mg/1 each) [9]. Table 2 gives an overview of the occurrence of some HBAs in foods [10,11] and Table 3 shows the content of the major HBAs in selected foods [12]. Table 2.
Occurrence of some HBAs in different dietary sources (10,11]
HBAs Benzoic acid Salicylic acid 4-Hydroxy benzoic acid Vanillic acid Syringic acid Protocatechuic acid Gallic acid Table 3.
Dietary source universal component of angiosperms, csp. of berries anise, dill, white mustard, allspice,rosemary,thyme, majoram raspbcny, gooseberry, pecans, anise, fennel vanilla, garden cress, paprika rosemary, basil, thyme, garden cress tarragon, clove, anise, cinnamon, blackberry, blueberry tea, nuts, olive oil
Content of some HBAs in fruits (mg/kg) [12] 4-Hydroxy benzoic acid Food Blackberry Blackcurrant Raspberry Redcurrant Strawberry White currant
6-16
0-6 15-27 10-23 10-36 5-19
Protocatechuic acid 68-189 10-52 25-37
Gallic acid 8-67 30-62 19-38
3-8
-
-
11-44 3-38
2. Hydroxycinnamic acid derivatives Cinnamic acids are ^ra«5'-phenyl-3-propenoic acids differing in their ring substitution, Fig. (2). COOH
I
%^ Fig. (2). Basic structure of cinnamic acids
II
261
The most common cimiamic acids are caffeic (3,4-dihydroxycimiamic acid), ferulic (3-methoxy-4-hydroxy), sinapic (3,5-dimethoxy-4-hydroxy) and p-coimiaric (4-hydroxy) acid, Table 4 [13]. These compoimds are widely distributed as conjugates, mainly as esters of quinic acid (chlorogenic acids, CGA). Table 4.
Structure of the most common cinnamic acids
Trivialname
MW (Da)
Position of OH groups
Cinnamic acid
148
/
p-Coumaric acid (4-Hydroxycinnamic)
164
4
Cafleic acid (3,4-Dihydroxycinnamic)
180
3,4
Ferulic acid (4-Hydroxy-3-metlioxycinnamic)
194
4
3
Sinapic acid (3,5-Dimetlioxy-4-iiydroxycinnamic)
224
4
3,5
Position of OCH3 groups
Depending on the identity, number and position of the acyl residues, these acids may be divided into the following groups: mono-esters of caffeic, p-coumaric and ferulic acid; di-, tri- and tetra-esters of caffeic acid [14,15]; mixed di-esters of caffeic and ferulic acid or caffeic and sinapic acid [16]; mixed esters of caffeic acid with dibasic aliphatic acids (e.g., oxalic, succinic) [17]. Cinnamic acids may condense with molecules other than quinic acid, including rosmarinic, malic and tartaric acid, aromatic amino acids, choline, mono- and polysaccharides, glycerol, myo-inositol, and different glycosides (anthocyanins, flavonols and diterpenes) [13]. Dietary occurrence There is no doubt that caffeic acid is the cinnamate that occurs most extensively, and the various caffeoylquinic acids (CQA) and dicaffeoylqumic acids (diCQA) are the most ubiquitous conjugates. Usually the 5-isomers dominates, but in some fruit and brassicas the 3isomer is prevalent. Because of the quantity commonly consumed, coffee
262
beverage must top the list, with 200 ml instant brew (2% w/v) supplying 50-150 mg CQA, mg. Blueberries, aubergines, apples, cider and green mate are good sources in some populations [13]. However, in view of quantities consumed by other populations, wines could make a significant contribution for tartaric acid conjugates and grapes and grape juice for caftaric acid, respectively. Lettuce is the major source of chicoric (dicaffeoyltartaric) and caffeoylmalic acid (up to 3 mg/100 g), but endive may have twice the concentration. Spinach is ahnost certainly therichestsource of conjugated p-coumaric acid at some 30-35 mg/100 g [18]. Broccoli florets and leafy cruciferous vegetables will be the major source of sugar esters and of conjugated sinapic acid (10 mg/100 g). Tomato and tomato products are likely to be the major source of glucosides at up to 13 mg/100 g in total, and possibly the second richest source of conjugate/7-coumaric acid (3 mg/100 g). Cereal bran and bran-enriched products are the most important source of wall-bound cinnamates with up to 30 and 7 mg ferulate/10 g in maize and wheat bran, respectively. This would make these products the richest dietary source of ferulic acid. However, coffee brew could supply up to 10 mg ferulate (as feruloylquinic acid, FQA) per 200 ml cup [13], and it is the first for conjugated ferulic acid , followed by Citrus juices. Table 5.
Dietary sources of individual cinnamates and each major class of conjugate [13,18]
Name Dietary source Cinnamates CafTeic acid Coffee beverage, blueberries, apples, ciders p-Coumaric acid Spinach, sugar beet fibre, cereals brans Ferulic acid Coffee, citrus juices, sugar beet fibre, cereal brans Broccoli, kale, other leafy brassicas, citrus juices Sinapic acid Conjugates Coffee beverages, blueberries, apples, ciders Caffeoylquinic acids Sweet cherries p-Coumaroylquinic acids Coffee Tartaric conjugates Spinach, lettuce, grapes, wines Malic conjugates 1 Culinary herbs, mixed herbs, possibly stuffing Rosmarinic acid Spinach, sugar beet fibre, cereal brans Cell wall conjugates
263
3. 4-oxo-flavonoids: flavonols, flavones, isoflavones, flavanones, chalcones, dihydrochalcones The term 4-oxo-flavonoids includes a group of complex polyphenols that share a common structure of diphenylpropanes (C6-C3-C6) characterized by two aromatic rings and an oxygenated heterocycle, Fig. (3). The three rings are referred to as the A, B, and C (or pyrane) rings. J'
.<^\
1 A
11 B 1 ^?\,/^\e^^
% / ^
c II II 0
Fig. (3). Basic structure of 4-oxo-flavonoids
Their biosynthesis derives from the condensation of three acetyl units and of a derivative of hydroxycinnamic acid leading to the formation of a conmion intermediate, tetrahydroxychalcone. This chalcone is precursor of several compounds, the most important being the 4-oxo-flavonoids [19]. The 4-oxo-flavonoids can be distinguished based upon the modifications of the nucleus, including the pyronic cycle saturation, the number and the position of hydroxyl groups and the degree of methylation and glycosylation. In plants,flavonoidsoccasionally occur as aglycones, the most commonly forms being 0-glycoside derivatives. Flavones may also occur as C-glycosides. The bond between the aglycone and sugar moieties is generally located at 7- (flavone, flavanone), 3(flavonols), or 4'-position. The sugars are mono-, di-, tri- and even tetrasaccharides, being D-glucose, L-rhamnose, glucorhamnose, galactose, and arabinose the most frequent [19,20]. Depending on their aglycone structure, at least five different groups of 4-oxo-flavonoids are distinguishable: flavonols, flavones, flavanones, isoflavones and chalcones.
264
Dietary occurrence The concentration of 4-oxo-flavonoids depends on the plant, environmental conditions, the part of the plant consumed, the degree of ripeness as well as on the food processing. Flavonoids are preferentially located in the epidermis: solubilized in the vacuolar sap (especially flavones and flavonols glycosides) or in the epicuticular zone [20]. More detailed information about the occurrence of the different compounds is given in each subchapter. 3.1
Flavonols
Flavonols (about 380 aglycones) are characterized by the presence of a hydroxyl group at position 3, Fig. (4). About 90% of the flavonols have additional hydroxyl at positions 5 and 7.
r^'^4/^
II B 1
l < ^ \
/^^/^'^^K
1 M1 ^ 1 % / ^ S " " ^OH 1 0II Fig. (4). Basic structure of Flavonols
Flavonols occur mainly as O-glycosides and the diversity of the glycoside moiety in this group is noteworthy with about 200 different quercetin and kaempferol glycosides described to date [19]. Table 6 reports the most common flavonols.
265
Table 6.
Structure of the most connnon flavonols Trivialname Aglycones Fisetin Galangin Kaempferol Morin Myricetin Quercetin Rhamnetin Glycosides Quercltrin Rutin
MW (Da)
Position of OH groups
286.24 270.24 286.24 302.24 318.24 302.24 316.27 448.38 610.53
Substituents
Position of tiie substituents
3,7,3',4' 3,5,7 3,5,7,4' 3,5,7,2\4' 3,5,7,3\4\5' 3,5,7,3',4' 3,5,3',4'
OCHj
7
5,7,3\4' 5,7,3',4'
0-Rh 0-Ru
3 3
Rh = rhamnose = 6-dcoxy-L-mannose (C6H12O5); Ru = nitinose == 6-0-D-glucose
Dietary occurrence Flavonols are present in plant foods mainly in the leaves and in the outer parts of plants with quercetin and kaempferol the most common ones. Quercetin and its glycoside are ubiquitous in fruits and vegetables. Conversely, kaempferol and myricetin are less distributed (Table 7) [2123]. Specific quercetin glycosides have been detected in onions (quercetin4'-glucoside and quercetin-3,4'-diglucoside), broccoli (quercetin-3-0sophoroside, kaempferol-3-(9-sophoroside), green beans (quercetin-3-0glucuronide) and tomatoes (rutin = quercetin-3-O-rhamnosyl-glucoside) [24,25], red wine (rutin) and tea (rutin, quercetin-3-(9-glucoside and quercetin-3-O-galactoside) [26]. Preparation of fruits and vegetables for consumption (for example peeling, skinning and cooking) can decrease quercetin and kaempferol content significantly. For example, boiling, microwave cooking and frying of onions or tomatoes involves a decrease in the content of flavonols by 30 to 80% [2].
266
Table 7.
Content of flavonois (expressed in mg/kg or mg/1) in foods determined by HPLC metliods after liydrolysis of tlieir glycosides [21-23]
Food Apple Apricot Bean, French French processed Green Broccoli Currrant, black
Red
Quercetin 20-36
25 39 17 16 30-37
Myricetin
-
-
<12 <3.8
60-72
37
1
8-13 <1.3 15-37 2-12
-
Endive Grape, black White Grape juice 4.4 4.4 Grape fruit juice (fresh) 110-120 Kale Leek 14-79 Lettuce 340-347 Onion 5.7 Orange juice 6-8.6 Strawberry 14-17 Tea, black 2-14 Tomato 63 Cherry tomato i 8.3 Wine, red
3.2
Kaempferol
46
210-470
30
5-12 14-16
-
4.5 4.5 6.2
3.0
7.9
Flavones
Flavones differ from flavonois since the hyciroxyl group at position 3 of the C-ring is missing, Fig. (5).
Fig. (5). Basic structure of flavones
267
About 300 different aglycones have been identified, and the most frequently are luteolin, apigenin (especially in parsley) and diosmetin (in Citrusfruits).Among glycosides, the 7-0- and C-forais are very conunon, and are characterized by a carbon-carbon bond between the anomeric carbon of a sugar molecule and the Ce or Cg carbon of the flavone nucleus. Table 8 describes the most common flavones [19]. Table 8.
Structure of the most common flavones
Trivialname Aglycones Apigenin Chrysin Diosmetin Luteolin Pinocembrin Glycosides Isoorientin Isovitexin Orientin Vitexin
MW (Da)
Position of OH groups
270.24 254.24 300.27 286.24 256.24
5,7,4'
448.36 432.36 448.36 432.36
Substituents
Position of the substituents
OCH3
4'
Glue Glue Glue Glue
6 6 8 8
5,7 5,7,3* 5,7,3\4'
5,7 5,7,3',4' 5,7,4' 5,7,3\4' 5.7,4'
Glue = glueosc
Flavones contribute to plant tissue color provided that they occur in high concentrations or are complexed with metal ions. Some flavones participate in taste; for example, the highly methoxylated aglycones nobiletin, sinensetin and tangeretin are responsible for the bitter taste of citrus peel. On the other hand, some glycosylated flavones (for instance neodiosmin and rhoifolin) reduce the bittemess of some substances (limonin, naringin, caffeine, quinine) [2]. Dietary occurrence Flavones are found mainly in grains and herbs and not frequently in fruits. Apigenin and chrysoeriol have been detected in parsley, while cereal grains and herbs contain apigenin and its glycosides as well as luteolin [21,27].
268
Table 9.
Content offlavones(expressed in mg/kg or mg/1) in foods determined by HPLC metliods after hydrolysis of tiieir glycosides [21,28] Food Celery leaf Stalk Sweet peper, red
3.3
Luteolin 200 5-20 5-11
Apigenin 750 16-61
-
Isoflavones
Isoflavones are a distinct class of flavonoids which stracturally differ from the commonflavonoidsin B-ring orientation, Fig.(6).
Fig. (6). Basic structure of isoflavones
The isoflavones have a chemical stracture similar to that of mammalian oestrogen 17P-estradiol [28]: the phenoUc ring and a pair of hydroxyl groups separated by a distance comparable to that occurring in mammalian oestrogens are key structural elements of most compounds that bind to oestrogen receptors [29,30]. Thus, isoflavones have recently become best known for their oestrogenic activity, hence the name "phytoestrogens". However, small differences in structures of the individual phytoestrogens can dramatically alter their activity. For instance, daidzein and genistein share identical structures except for an additional hydroxyl group on the A-ring of genistein, but this favours up to five- or six-fold oestrogenic activity of genistein in some assay systems [31].
269
Table 10. Structure of the most common isoflavones Trivialname Aglycones Daidzein Genistein Glycosides Acetyldaidzein Acetylgeiiistin
MW (Da)
Position of OH groups
Substituents
Position of the substituents
254.23 270.23
7,4' 5,7,4'
/ /
/ /
430.23 446.23
7,4' 5,7,4'
Acetyl-gluc Acetyl-gluc
6" 6"
Acetyl-gluc = Acetyl-glucoside
Dietary occurrence Isoflavones are present in plant foods either as aglycones (genistein or daidzein) or - predominantly - as different highly polar and water-soluble glycosides, including acetyl and malonyl glucosides and p-glucosides of daidzein and genistein [32]. Legumes are the main source of isoflavones. Soybeans are particularly rich in daidzein and genistein. Table 11 shows the wide range in total isoflavone concentrations in soya products and other legumes [32,33]. The concentration of isoflavones in soy foods is in the range of 0.1-3.0 mg/g. However, theseflavonoidshave been detected also in black beans, green split peas and clover [34]. Otherflavonoidsof this group, including biochanin A and formononetin, have been found in chick peas, green beans, and sunflower seeds. Table 11.
Content of isoflavones (expressed in ^g/g) in foods [3233]
Food Soya bean Tofu Soy flour Textured soya protein Soya milic Miso Soy clieese Tofu yoghurt Soy sauce Green split peas
3.4
Isoflavones (total) (fig/g)
|xg per average portion size (g)
579-3812 79-674 833-1778 701-1184 34-175 256-890 34-47 151 13-75 73
34740-228720 (60) 10270-87620(130) 16660-35560(20) 28040-47360 (40) 3400-17500(100) 4608-16020(18) 1360-1880(40) 18120(120) 65-375 (5) 2920 (40)
Flavanones, chalcones, dihydrochalcones
270
Flavanones arise from flavones after reduction of the double bond in the heterocycle (position C2/C3), Fig. (7).
Fig. (7). Basic structure of flavanones
Among aglycones, the best known are naringenin and hesperidin. Their glycosylated forms occur commonly as O- or C-glycosides, usually as rutinosides (6-0-a-L-rhamnosyl-D-glucosides) and neohesperidosides (20-a-L-rhamnosyl-D-glucosides) attached at position 7. Flavanones contribute to the flavour of citrus [19]. Table 12 reports the structures of some common flavanones. Table 12.
Structure of some common flavanones
Trivialname Aglycones Eriodictyol Hesperetin Naringenin
MW (Da)
Position of OH groups
288.26 302.28 272.26
Glycosides Hesperidin Naringin
610.57 580.54
Substituents
Position of the substituents
5,7,3*,4' 5,7,3' 5,7,4'
OCH3
4'
5,3' 5,4'
Rli-Gluc; OCH3 O-Rh-Gluc
7;4'
7
Rh = rhamnose = 6-dcoxy-L-mannosc (CeHnOs); Glue = glucose
Flavanones can be easily converted to isomeric chalcones in alkali (or vice versa in acidic media) provided that there is a hydroxyl substituent at position 2' (or 6') of the chalcone. Chalcones are unsaturated and, along with dihydrochalcones, contain an open pyronic cycle and a carbon skeleton numbered in a way different from other flavonoids, Fig. (8, 9). Native chalcone glycosides tend to transform into flavanone glycosides during extraction procedures. Chalcones per se are therefore of restricted occurence in foods [35].
271
OH
rf^^^y
, ^ ^ ^ 4 ^
OH
^==^, OH Fig. (8). Isosaiipiupurin (Chalcone)
OH Fig, (9). Phloretin (Dihydrochalconc)
Dietary occurrence Flavanones are found in a small number of foods. Chick peas (with the flavanone garbanzol), cimiin, pepperaiint (both with hesperidin), hawthom berry, licorice, rowanberry and citrus fruits are among those fews containing molecules of this group. Naringenin and narirutin glycosides are present in hawthomberry and rowanberry; liquoritigenin in licorice roots. Flavanones neohesperidose (such as naringin) are found in grapefruit and are usually bitter; the tasteless flavanone rutinosides (such as hesperidin) are present in oranges [35,36]. Flavanone glucosides are comparatively rare in species but are found for instance in different herbs [37]. Hesperidin and the aglycones naringenin, eriodictyol and hesperitin have been reported in the herbal tea (Honeybush tea) prepared from the legume Cyclopia intermedia [38]. Naringenin and eriodictyol have been reported in potato [5]. Citrusfiruitsand associated products (fiuit juices, peeled freshfruit)are a major dietary source of flavanones (Table 13) [35]. However, the distribution is quite scattered, and much higher concentrations are found in the solid tissues compared to the juice. For example, an individual drinking orange juice (250 ml) will have a daily flavone intake (as aglycones) in the range of 25-60 mg; eating the flesh of a whole orange (200 g) will provide about 125-375 mg. Chalcones are comparatively rare in foods. Naringenin chalcone is present in tomato skin and may be present in juice, paste and ketchup. Acid hydrolysis, commonly applied prior to HPLC, converts the chalcone to the corresponding flavanone (naringenin), which is naturally present only in trace amounts (2-15 mg/kg) in the tomato [23]. Dihydrochalcones (DHCs) are characteristic of apples and derived products (apple juice, cider, pomace etc.), and their content depends on
272
the cultivar. Phloretin 2'-glucoside (phloridzin), phloretin 2'(2"-xylosylglucoside) and 3-hydroxyphloridzin have been identified unequivocally and some investigators have reported phloretin 2' (2"-xylosylgalactoside). They are present in the skin, pulp and especially in tiie seeds, where they account for up to 60% of the total phenols (compared with less than 3% in the epidermis and parenchyma zones). When eating an apple, the seeds and core of the fhiit are usually discarded and thus some of the apple dihydrochalcones are not mgested. Whole apple fruits are processed industrially to produce juices and ciders, and therefore the contribution of these processed products to the intake of DHCs can be higher than that of fresh apples (250 ml of apple juice or cider supply about 1-5 mg phloretin. By contrast, a dessert apple of about 100 g supplies less than 1 mg [39,40]. The dihydrochalcones aspalathin and nothofagin have been identified as the main flavonoids in the South-Afiican Rooibos tea {Aspalathus linearis) [41]. Table 13.
Flavanones characteristic of common citrus fruits
Flavanones Eriocitrin Narirutin Hesperidin Naringin Neohesperidin
Sweet orange (Citrus sinensis) + +++ -
Sour orange (Citrus aurantium) -H^
++
Lemon (Citrus limon) ++ +++ -
Grapefruit (Citrus paradisi) •H-
Trace 4-H-
Trace
Mandarin Lime (Citrus (Citrus aurantifolia) reticulata) ++ +++ +++ -
4. Flavanols (FIavan-3-ols) Flavanols have a C-ring structure similar to that of 4-oxo flavonoids, but they are characterized by the lack of the double bond at the 2-3 position and of the 4-oxo-group, Fig. (10) [19].
Fig. (10). Basic structure of flavanols
273
The flavan-3-ols most occurring in nature are (-f-)-catechin and (-)epicatechin (EC), although gallocatechin and epigallocatechin have also been identified [42], Proanthocyanidins (or condensed tannins) include oligo- and polymeric forms of the monomeric flavanols and will be examined later. Polymerization of monomeric flavanols can occur as a result of auto-oxidation, but more often it is catalyzed by polyphenoloxidase (PPO), an enzyme that is present in most plant tissues [43].
4.1
Catechins - Dietary occurence
Catechins are widely distributed in plants; however, they are rich only in tea leaves, where catechins may constitute up to 25% of dry leaf weight. Catechins of green tea include the flavanols epicatechin, epigallocatechin, and their gallate esters (Table 14). Table 14.
Structure of the most common catechins
MW
Position of OH groups
(+).Catechin (C)
290.3
3(^-0H)
(-)-Epicatechin (EC) (cis form)
290.3
3 (
Epigallocatechin (EGC)
306.4
Epicatechin-gallate (ECg)
442.4
Trivialname
Substituents
Position of the substituents
^»M^»NOH\
3 ( VWSAAVOH)^ 5 »
3 OH
X Epigallocatechin-gallate (EGCg)
458.4
5'
3 OH
The most abundant monomeric flavanols of black tea are (-)epicatechin gallate (ECg) and (-)-epigallocatechin (EGC) and (-)epigallocatechin gallate (EGCg) [44]. Indeed, quantitative analyses of black tea reports levels of 31-79 mg/1 for EC, 5-91 mg/1 for EGC, 18-229 mg/1 for EGCg and 8-110 mg/1 for ECg; for green tea levels from 10-94
274
mg/1 for EC, 20-287 mg/1 for EGC and 60-408 mg/1 for EGCg are found [45]. During fermentation in the preparation of black tea, oxidative polymerization of flavanols occurs with the formation of theaflavin, theaflavingallates, thearubigins, and epitheaflavic acid [44]. Flavanols have been determined in apples, apricots, pears, cherries, peaches and plums [47,48]. The contents of (+)-catechin and (-)-epicatechin in red wine are relatively high (up to 208 mg/1 for catechin and 90 mg/1 for EC) [49]. Low levels of (+)-catechin (-5 mg/1) and (-)-epicatechin (-1 mg/1) have been reported for lager beers [50]. Data on grapes are limited; qualitative studies show the presence of (+)-catechin, (-)-epicatechin and ECg in black and white grape seeds and skins [51]. Recently, chocolate and cocoa have gained interest because of their contents of catechins and related polymers (procyanidin oligomers) [52]. Fruit juices processing may seriously affect flavanol content. For example, the preparation of commercial apple juice decreased the flavanol content in a stepwise manner. In particular, crushing and pressing, storage of the concentrated juice at room temperature and decolorization by treatment with activated carbon destroy theflavanolsalmost entirely [46]. 5. Anthocyanidins, anthocyanins, proanthocyanidins, tannins 5.1
Anthocyanidins and anthocyanins
The fundamental nucleus in anthocyanidins (aglycones) is flavylium chloride. Most of the anthocyanidins are derivatives of 3,5,7trihydroxyflavylium chloride. Thus, the hydroxylation pattems in the natural anthocyanidins fall into the three basic groups of pelargonidin, cyanidin and delphinidin. Anthocyanidins are rarely found in fresh plant material because of their instability [19]. On the other hand, anthocyanins, i.e. the glycosylated anthocyanidins, are an important group of water-soluble pigments occuring in 27 families of food plants (mainly red fruits and vegetables). Fig. (11) [53]. Table 15 shows the most common anthocyanidins and anthocyanins.
275
Fig. (11) Basic structure of anthocyanidins and anthocyanins
Table 15.
Structure of the most common anthocyanidins and anthocyanins MW
Trivialname Anthocyanidins Pelargonidin Cyanidin Delphinidin Malvidin Anthocyanins Pelargonidin-3-glucoside Cyanidin-3-glucoside DeIphinidin-3-glucoside Malvidin-3-glucoside
JM
Position of OH
Substituents
Position of the substituents
271.70 287.70 303.70 331.75
3,5,7,4' 3,5,7,3*,4' 3,5,7,3\4',5' 3,5,7,4*
OCH3
3*,5'
432.70 448.70 499.70 492.70
5,7,4' 5,7,3\4' 5,7,3',4',5' 5,7,4'
OGluc OGluc OGluc OGluc; OCH3,
3 3 3 3;3',5'
The natural anthocyanins vary in: • the basic anthocyanidin skeleton, i.e. the number and position of hydroxyl and methoxyl substituents; • the identity, number and positions(s) at which sugars are attached to the skeleton; the most common sugars are glucose, galactose, rhamnose and arabinose ( as 3-glycosides or 3,5diglycosides). • the extent of sugar acylation and the identity of the acylating agent(s); the most common acylating agents include ciimamic acids (caffeic, p-coumaric, ferulic and sinapic), which may themselves be glycosylated, and a range of aliphatic (for example acetic, malic, malonic, oxalic and succinic acid) and aromatic acids. The anthocyanins occur in the vacuole as an equilibrium of four molecular species: the coloured basic flavylium cation and three
276
secondary structures (the quinoidal base, the carinol pseuodobase and the chalcone pseudobase) [54]. The pH changes the colour intensity of the anthocyanins (for example, by the addition of vinegar or other acids while cooking or processing). Commonly, anthocyanins are red in acid, violet in neutral, and blue in alkaline solution. In fact, when cooking a food that is red, such as red cabbage, it may be helpful to add an acidic substance such as vinegar (or tomato juice or lemon juice) to prevent the food from tuming purple. Anthocyanins contribute significantly to the red purple, and blue color of flowers, many fruits of higher plants, vegetables and associated products, beverages and preserves. Anthocyanins and polymeric pigments derived from anthocyanins by condensation with other flavonoids, are responsible for the color of red wine. It has been recognized that anthocyanin-rich extracts might be used as food additives. Many factors influence the stability of anthocyanins. Heat and light can destroy sensitive anthocyanins during processing of fruits and vegetables. In particular, anthocyanins are rapidly destroyed in the presence of a high sugar concentration; thus processed foods containing large amounts of sugar or syrup would not have the same amount of anthocyanins as their improcessed counterparts [55], Dietary occurrence Anthocyanins are widespread in food plants, with an estimated worldwide consumption of 10000 tonnes from black grapes alone [53]. The anthocyanin content of many fruits and vegetables has been estimated by various methods (Table 16) [56-58]. The main sources of these plant pigments are fresh fruits such as cherries, plums, strawberries, raspberries, blackberries, grapes, red currants and black currants.
277
Table 16.
Content of anthocyanins in foods (expressed in mg/l or mg/lcg) [56-58]
Food Blackberry Blueberry Cherry Cliolceberry Cranberry Currant (blacic) Grape (red) Orange, Blood O'uice) Raspberry, black Raspberry, red Strawberry Cabbage, red Onion Wines, red Wines, Porto + Marsala
5.2
Anthocyanins 1150 825-4200 20-4500 5060-10000 600-2000 1300-4000 300-7500 2000 1700-4200 100-600 150-350
250 up to 250 240-350 140-1100
Proanthocyanidins
Proanthocyanidins (PAs, syn condensed tannins) are polymeric flavan-3ols whose elementary units are linked by C-C and occasionally C-O-C bonds (polymerization degree between 3 and 11), Fig. (12) [19]. Oxidative condensation occurs usually between carbon C4 of the heterocycle and carbons Ce or Cg [59]. A characteristic of PAs is that they yield anthocyanins upon heating in acidic media, hence their name, they yield anthocyanidins (hence their name). Two main types of PAs can be distinguished according to the substitution pattern of their B-ring: • Procyanidins: main constituents are catechin and epicatechin, and are characterized by the presence of two hydroxyl groups (3\ 4') in the B-ring. • Prodelphinidins: main constituten is epigallocatechin, which has three hydroxyl groups (3', A\ 5') in the B-ring.
278
r
^<^°" YY^^K'^^^^OH ./'x OH
HO^
^«^
.0^
^1*
^^5-^
" \ ^ - \ / ° ^ ^ v ^ V , OH
OH
.1
»°\,^'-v^?\^'v^' %
4^
^OH
Dimers: Procyanidin B3: R3' = H
Trimer.
Prodelphinidin B3: RS' = OH
Fig. (12). Basic structure of proanthocyanidins
Dietary occurence Common sources of PAs are fruits, such as apple, strawberry, pear and grape, beverages such as red wme and tea, and chocolate (Table 17) [59]. PAs complex protems, and are responsible for the astringency of foods and beverages (e.g. grape skin and seeds, cider, wine) [19].
279
Table 17.
Content of proanthocyanidins in foods (expressed in mg/1 or mg/lOOg) [59]
Food Apple Apple juice Barley Beer Blackberry Cacao bean Cherry Grape Grape juice Lentil Pear Pear juice Raspberry, red Strawberry Wines, red
5.3
Proanthocyanidins 17-50 nd-298 64-126 3.5-19,5 9-11 260-1200 10-23 1-160 3.546 316-1040 0.7-12 11-74 2-48 2-50 nd-500
Tannins
Tannins are compounds of intermediate to high molecular weight that distinguish them from the groups of low molecular weight plant phenolics. Tannins with a molecular weight up to 30000 DA have been found in certain Leguminosae. One of their main characteristics of tannins is the formation of insoluble complexes with proteins leading to the astringency of taimin-rich foods (for instance tea) because of the precipitation of salivary proteins [59]. Plant tannins are subdivided into two major groups (Table 18) [60]: 1. Hydrolyzable tannins: they consist of a central glucose molecule linked to molecules of gallic acid (gallotannins) or hexahydroxydiphenic acid (ellagitannins), Fig (13). They are readily hydrolyzed, hence their name. The most common hydrolyzable tannin is tannic acid, Fig. (14), which is a gallotannin formed by a pentagalloyl glucose molecule esterified by five gallic acid units.
280
OH HO.
OH
O
COOH
OH
Fig. (13). Structures of gallic (a) and ellagic (b) acid
OH
HO.
X.
/
.OH
CO
OH2C
OH
oc—o
HO"
^
I oc
"OH
OH Fig. (14). Structure of tannic acid
Condensed tannins (= proanthocyanidins): unlike hydrolysable tannins, condensed tannins are polymeric flavans that are not readily hydrolysable. They often consist of molecules of catechin and epicatechin joined by carbon-carbon bonds. Hence catechin and epicatechin are referred to as monomers; oligomers containing 2-4 (epi)catechin units are referred to as oligomeric procyanidins (OPC).
281
Table 18.
Classification of tannins Type of tannin Hydrolyzable tannins: • Gallotannins • EUagitannins and metabolites • Ellagitannin oligomers 2. Condensed tannins: • Proanthocyanidlns: Procyanidlns Prodelphinidins • Galloylated proanthocyanidlns
Example
1.
Pentagalloylglucose Geranin, Corilagin Agrimoniin Epicatechin oligomers Epigallocatechin oligomers
ABSORPTION AND METABOLISM OF SELECTED PLANT POLYPHENOLS IN HUMANS Gut absorption The absorption and the metabolism of dietary polyphenols arte determined primarily by their chemical structure, with glycosylation playing an important role. In fact, glycosylation influences the bioavailability of the polyphenols. It is generally stated that flavonoid glycosides are hydrolyzed before being absorbed [61]. Therefore, the first step of metabolism should involve the removal of the sugar moiety by enzymes-(glycosidases). Glycosidases activity can occur in the food itself (endogenous or added during process) or in the cells of the gastrointestinal mucosa or can be secreted by the colon microflora. Nonenzymatic deglycosylation in the hiraian body, such as in the acid conditions of the stomach, does not occur [62]. The absorption of polyphenols should therefore be controlled by enzyme specificity and distribution. Polyphenols with attached glucose are potential substrates for endogenous human enzymes, while attached rhamnose is not a substrate for human p-glucosidases and so is only cleaved by colon microflora arhamnosidases [63]. Deconjugation and reconjugation reactions in metabolism After the hydrolysis of a polyphenol glycoside to the free aglycone, polyphenols are conjugated by methylation, sulfation, glucuronidation or a combination. However, there are exceptions to this sequence, as
282
supported by different studies [64-68] reporting the absorption of intact polyphenols glycosides. This is a critical point, since the formation of conjugates dramatically alters the biological properties of the circulating metabolites. Furthemiore, it should be reminded that significant differences between the administration of drugs (usually in himdreds of milligrams in one concentrated dose) and the consumption of dietary polyphenols (usually <100 mg in a diluted dose) exist. These differences imply that drugs can readily saturate the metabolic pathways that rely on the supply of cofactors such as UDP-glucuronic acid. Hence, unconjugated drugs are often found in the blood. On the other hand, polyphenols found in food are not expected to saturate the metabolic pathways, therefore being in circulation in the conjugated forms [63]. Metabolism by the gut flora Polyphenols that are not absorbed in the stomach or small bowel will be carried to the colon. Polyphenols that are absorbed, metabolized in the liver and secreted with bile back to the small intestine will also reach the colon. Here, microorganisms degrade both unabsorbed and absorbed flavonoids. Indeed, colonic bacteria produce glycosidases, glucuronidases, sulfatases that can strip flavonoid conjugates of their sugar moieties, glucuronic acids and sulfates [61]. Human intestinal bacteria are able to hydrolyse 6>-glycosides [69] as well as C-glycosides [70]. In addition, the degradation involves the splitting of the heterocyclic oxygen-containing C-ring. Degradation products can be absorbed [71], and subsequently metabolized by enzymes present mainly in the liver, where 3'-0-methylation by catechol-0-methyltransferase, dehydroxylation, p-oxidation, and conjugation with glucuronic acid, sulfate, and glycine occurs [72]. These metabolites are considered to contribute to the biological effects of dietary flavonoids (antioxidants). In general, the metabolism of dietary flavonoids may be sununarized as shown in Fig. (15).
283
INTESTINE
Flavoaoid (agUcones and glycosilated fonns) and products of the microbic metabolism
FECES
Fig. (15). Metabolism of dietary flavonoids. GlcA = glucuronic acid; UGT = uridine 5'diphospoglucuronosyl transferase; Met = methyl; Sulf = sulfate; COMT = catechol-O-methyl transferase; PST = phenol sulfo transferase
1. Flavonols Absorption of flavonols from the diet was long considered to be negligible, because flavonols are present in plants bound to sugars as >8glycosides. Only aglycones were supposed to be absorbable, whereas glycosides were thought to be non- or only marginally absorbable [61]. One of the main flavonols studied is quercetin. Indirect evidence for the presence of quercetin conjugates in humans was obtained by Manach et al [73], who found the presence of quercetin in plasma after the consumption of a complex meal rich in plant products only after pglucuronidase and sulfatase treatment. Furthermore, the authors reported
284
the presence of a methylated derivative of quercetin, isorhamnetin, in three out of the ten subjects. These results are in good accordance with data obtained by Conquer et al [74], where the concentration of quercetin in fasting plasma of a quercetin-supplemented group was 23-fold higher than that of the placebo-group. Urinary excretion of quercetin increased significantly with dose and time after the consimiption of fruit juice (blackcurrant and apple juice in a 1:1 mixture) in humans [75]. Ranges from 0.29-0.47% of ingested quercetin were found in the urine. The presence of quercetin in urine shows that it was absorbed by the gut, but the urinary content does not necessarily reflect absolute absorptive efficiency because absorbed quercetin may be metabolized (conjugated), stored and excreted through other routes such as the biliary tract. However, since quercetin is present in a variety of fruit and vegetables, plasma concentrations or urinary excretion of quercetin may potentially be useful as biomarkers of habitual intake of these foods. The absorption of intact quercetin glycosides has been demonstrated by some authors [64,65,76]. Holhnann demonstrated in ileostomy subjects (who lack colon with the bacterial flora, thus circumventing the problem of microbial degradation), that the quercetin glycosides from regular foods (onions, tea) were far better absorbed than pure aglycone (52%vs24%). LC-ESI-MS analyses allowed the detection of intact flavonol glucosides (rutin) in plasma of healthy volunteers after the consumption of tomato extract [65]. Glycosides of flavonols from onions, such as quercetin-4'-0-glucoside and quercetin-3'-0-methyl-4'-0-glucoside, have been found in the plasma of volunteers with a peak of absorption of 0.5 - 4 h [64]. On the other hand, a diet-controlled cross-over-study [77] with supplementation of quercetin and its glycoside rutin showed that rutin occurred only as conjugated form (with glucuronic acid and/or sulfate groups) in plasma suggesting that this glycoside was not absorbed in its original form. Conversely, quercetin was detected in conjugated as well as unconjugated (aglycone) forms. Interindividual differences in the pharmacokinetics of both compounds were considerable. Sesnik et al [78] found also no intact quercetin glucosides and only traces of aglycone in human plasma after the administration of quercetin-3-glucoside or quercetin-4'-glucoside as an oral solution, while quercetin glucuronides were the major metabolites in plasma.
285
Interestingly, different studies demonstrated that the sugar moiety of quercetin glycosides is an important detemiinant of their absorption and bioavailibility [79-82]. Quercetin-3-rutinoside and quercetin-4'-glucoside are important forms of quercetin in foods. The first one accounts for about 40% of quercetin in black tea [83] and the second one for about 45% of quercetin in onions. [84]. Although the intake of quercetin-3-rutinoside is twice that of quercetin-4'-glucoside, the absorption of quercetin-3rutinoside is only 17% of ingested dose, whereas the absorption of quercetin-4'-glucoside is 52% of ingested dose [76]. Furthermore the bioavailibilty of quercetin-3-rutinoside is only 20% of that of quercetin4'-glucoside [81]. Olthof et al [82] have tested the bioavailibilty of quercetin-3-glucoside in comparison to that of quercetin-4'-glucoside and concluded that both quercetin glucosides are rapidly absorbed in humans, irrespective of the position of the glucose moiety. In addition, this study provided information on the metabohsm of quercetin into isorhamnetin (3'methoxyquercetin). Of the ingested quercetin glucosides, --50% is absorbed in the small intestine and subsequently converted into isorhanmetin, in the liver and in other organs. The 50% of ingested quercetin that is not absorbed in the small intestine is metabolized by the colonic microflora into quercetin aglycone and phenolic acids, which might be absorbed from the colon [73]. The bioavailibility of quercetin-glycosides from onions, containing mainly quercetin-p-glucosides, was superior to that of various quercetin glycosides from apples (containing a mixture of quercetin-p-galactosides and p-xylosides) and of pure quercetin-3-rutinoside (major species in tea). The possible matrix effect of the foods remains unclear. It seems that overall percentage of absorption, determined by measuring plasma levels of flavonols after enzymatic hydrolysis, does not exceed 2-3% of the ingested dose. It is also likely that, as with other micronutrients, the existence of a steady-state concentration of these compounds could result in diminished absorption. Thus, it is conceivable that the major parts of these flavonoids are either degraded to phenolic acids in the large intestine or excreted in the faeces [72]. 2. Flavones Data on the absorption of flavones (namely luteolin) are limited. Shimoi
286
et al [85] investigated the intestinal absorption of iuteolin and iuteolin-70-P-glucoside in rats and humans. The absorption analysis using the rateverted small intestine demonstrated that Iuteolin was converted to glucuronides during passing through the intestinal mucosa and that luteolin-7-O-P-glucoside was absorbed after hydrolysis to Iuteolin. In plasma, either free Iuteolin as well as its monoglucuronide and methylated conjugates were present, while luteolin-7-O-p-glucoside was not detected. This indicates that glucosides may be first hydrolyzed to Iuteolin by the microbacteria. The same authors reported that in humans free Iuteolin and its monoglucuronide have been detected in plasma after oral administration of Iuteolin. 3. Isoflavones Intestinal microflora plays a key role in the metabolism and bioavailibility of isoflavones [86]. After ingestion, soybean isoflavones are hydrolyzed by intestinal glucosidases, which release the aglycones, daidzein and genistein, Fig. (16).
intestinal glucosidases
malonylglucosides acetylglucosides p-glucosides
demethylation dehydroxylation reduction ring cleavage equol dihydrodaidzein 0-desmethylangolensin p-ethylphenol
daidzein genistein
absorption
hepatic conjugation-enterohepatic cycling urinary excretion
Fig. (16). Metabolic fate of soybean isoflavones in humans (Setchell 1999)
287
These aglycones may be absorbed or further metabolized to different metaboUtes, including equol, 0-
288
4. Flavanones, chalcones, dihydrochalcones The flavanones have received less attention in comparison to flavonols and isoflavones, although their intake from the diet can be high and they exhibit promising biological activity. Little infomiation is available about the absorption or the kinetic behavior of the flavanones naringenm, hesperetin and their glycosylated fomis naringin, hesperidin, and narirutin. Studies conceming urinary excretion of these compounds have confimied their bioavailibility from fruits and that they are excreted, at least to some extent, into the urine. The renal excretion of naringin, naringenin and its glucuronides after the consumption of grapefruit juice (20 ml/kg body weight) was investigated by Fuhr et al [96]. Only naringenin and its glucuronides appeared in urine after an average lagtime of 2 h. Neither naringin nor its glucuronides were found. The data suggest that cleavage of the sugar moiety, presumably by intestinal bacteria, is the first step of naringin metabolism. These data were confirmed by Lee et al [97] who detected naringenin glucuronide in urine samples after the administration of grapefruit juice (containing about 214 mg naringin). However, a recent study reported that the glycoside naringin was recovered (0.02% of the administered dose) in urine as unchanged molecule, hence confirming those glycosides are absorbable [66]. The influence of glycosylation on the metabolism of naringenin-7glucoside and its aglycone in the conscious rat model was examined by Choudhury et al [98]. It resulted that via oral route the glycoside group is cleaved by an intestinal enzyme and then the aglycone is glucuronated within the epithelium. By contrast, after intravenous dosing the majority was detected as native glucoside in the urine. Bioavailibility and kinetics of naringenin and hesperetin from orange and grapefiiiit juices was also investigated by Erlimd et al [99]. Both flavonoids were absorbed from the juices with great interindividual variations. The authors hypothesized that these variations were caused by differences in gastrointestinal microflora. Peak plasma concentrations of naringenin and hesperetin were reached between 4.8 and 5.5 h, indicating that an absorption takes place in the distal parts of the small intestine or the colon (where enzymes capable of cleaving theflavonoidglycosides in question are present).
289
5. Flavanols - Catechins Early studies (in the 1970s) on the pharmacokinetics of (+)-catechin revealed that tiiis flavanol is absorbed from the gastromtestinal tract following administration to healthy volunteers (4.2 g in the form of gelatin capsules) [100]. (•f)-Catechin was excreted in the urine together with several imidentified metabolites, and the amount excreted within 24 h was about 7.5% of the administered dose. Other authors suggested that catechins are converted to glucuronyl derivatives in the intestinal mucosa and are further metabolized by methylation, sulfation and conjugation with glucuronic acid, sulfate and glycine [101]. Indeed, Lee et al [102] determined flavanol conjugates in human plasma after the ingestion of green tea. EGCg was mainly present as a sulfate conjugate (65%), followed by the free form (20%) and the glucuronide (15%). EGC on the other hand was mainly present as glucuronide (60%), followed by sulfate (30%). About 10% was detected as imconjugated EGC aglycone. More recently. Da Silva et al [103] detected the presence of glucuronides, O-methylglucuronide sulfate, and glucuronide sulfate of epicatechin (EC) in plasma of rats fed epicatechin. Absorption of (-)-epicatechin from chocolate has been studied by different authors [104-106]. Baba et al [104] found maximum levels of total EC metabolites in plasma after 2 h of chocolate or cocoa intake. Sulfate, glucuronide and sulfoglucuronide conjugates of non-methylated EC were the main metabolites present rather than methylated forms. In urine samples, excretion of total EC metabolites within 24 h was about 30% of total EC intake after chocolate and 25 % after cocoa consumption. A 12-fold increase in plasma epicatechin concentration from 22 to 257 nmol/L was reported by Rein et al [105] after consmnption of 80 g semisweet (procyanidin rich) chocolate within 2 h after ingestion. The total antioxidant capacity of plasma increases of 31% within the same time, and plasma 2-thiobarbituric acid reactive substances decreased up to 40%. These data support that consimiption of chocolate increases plasma epicatechin concentrations and decreases plasma baseline oxidation products. These results have been confirmed in another study by Wang et al [106]. The bioavailability and metabolism of catechins was studied in humans after consumption of black tea containing 15.48 mg of EGC, 36.54 mg of
290
EC, 16.74 mg of EGCg and 31.1 mg of ECg [107]. Plasma concentrations of EGC, EC and EGCg increased significantly reaching peak plateau between 5 and 8 h (peak levels of 145,174 and 20.1 nmol/L respectively). ECg on the other hand increased linearly over the 24h-period, peaking at 50.6 nmol/L. Urinary excretion of EGC and EC paralleled the rise in plasma levels. EGC, EC and ECg peaked at 5 h whereas EGCg at Ih. Fecal catechin excretion varied widely from subject to subject, but it was significantly different from baseline for all catechins. Furthermore the authors calculated the percentage of ingested catechins. Only 1.68% of the total catechins consumed (400 mg) was found in the plasma, urine and feces, providing evidence that catechins undergo considerable metabolism and/or degradation either in the gastrointestinal tract or in the body after absorption. After ingestion of green tea infiision (400 mg of total catechins), epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) were detected in human plasma with an significant increase of these two free catechins after enzymatic hydrolysis with glucuronidase/sulfatase, indicating their presence in plasma mainly in the conjugated form [108]. At the same time, detectable amounts of final 60 mg catechin metabolites were found in plasma and urine, including 4-hydroxybenzoic acid, 3,4dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3methoxy-4-hydroxybenzoic acid. LC/ESI-MS analyses were applied to determine urinary glucuronidated and sulfated tea catechins after the administration of green tea to humans, mouse and rats [109]. The major conjugates were identified as monoglucuronides and monosulfates of EGC and EC. Besides these metabolites, also 0-methyl-EGC-O-glucuronides, 0-sulfates and Omethyl-EC-0-sulfates in human urine were detected. Furthermore, the ring-fission metabolites of EGC and (-)-epicatechin, 5-(3',4',5'trihydroxyphenyl)-x-valerolactone and 5-(3 ',4'-dihydroxyphenyl)-yvalerolactone respectively, have been detected in the monoglucuronide and monosulfate forms. It is not known whether tea catechin conjugates possess any biological activities. In a study by Manach et al [110], the glucuronic/sulfate conjugates were shown to have the same electrochemical behaviour as the parent drug. Considering that the oxidation potential of chemicals may represent their antioxidant capacity, the electrochemical behaviour of the conjugates suggests that they are effective antioxidants. Nevertheless, because the glucuronic acid/sulfate conjugates are generally more
291
hydrophilic than the parent compound, the tissue distributions of these metabolites are likely to be more limited than those of the parent catechins [111]. 6. Hydroxycinnamates, hydroxybenzoic acids Non-ruminants possess several intestinal Na'^^-dependent saturable transport systems. These include the well-known sodium-glucose cotransporter (SGLTl), responsible for the active uptake of glucose, and it appears to be specific for cinnamic and ferulic acid and possibly for other hydroxy-cinammic acids [112]. Healthy volunteers have shown to excrete caffeic, p-coumaric and ferulic acid in the urine after the consumption of various fruits [113]. The excretion of free ferulic acid in urine peaked after 7 h at a concentration of-'T jiM after the consumption of tomatoes (36-73 g containing 21-44 mg ferulic acid). The concentration of free ferulic acid plus glucuronide (and possibly sulfate) conjugates exceeded 20 |aM and accounted for some 11-25% of the dose [114]. Simonetti et al [115] studied the plasma levels of caffeic acid after consumption of 100,200, 300 ml of red wine (caffeic acid content of 9.01 mg/L) that provided about 0.9, 1.8, and 2.7 mg of caffeic acid, respectively. The highest plasma levels of caffeic acid was reached 60 min after ingestion and decreased to basal values within 180 min (for 100 and 200 ml) and within 240 min (for 300 ml). Both, the absence of caffeic acid in plasma before the trial and its significant, dose-dependent increment after red wine ingestion suggest that it may be a possible marker of consimiption of beverages containing this acid. Furthermore, 200 and 300 ml red wine intake produced a significant increase in plasma total antioxidant capacity (TRAP). Conceming HBAs, their metabolism involves conjugation with sulfate, glucuronate and glycine. Methylation may also occur, as may demethylation, dehydroxylation and decarboxylation (this only if there is a 4-hydroxyl) [3].
292
7. Anthocyanins, proanthocyanidins a.
Anthocyanins
Dietary anthocyanins have gained much attention based on the recognition of the "French paradox'* which led to the suggestion that some components of red wine (in particular anthocyanins) may protect against coronary heart disease. Limited evidence on the absorption of intact anthocyanins exist until today. There are reports, based upon spectral properties from DAD-HPLC of anthocyanin-like substances in plasma [116] and urine after acidification [117]. However, the ^max observed for the components in urine was at 430 nm. Anthocyanins should not have an absorption peak around this wavelength. Thus, the detected compounds appeared to be anthocyanin metabolites [68]. These spectroscopic data do not provide conclusive evidence for the absorption of intact anthocyanins. Conversely, anthocyanins from elderberry were detected in human plasma using a more selective approach based on HPLC coupled to UV detection (512 nm) [67]. These findings have been confirmed by a recent study on the detection of anthocyanins in their unchanged glycosylated form in urine and plasma after ingestion of 720 mg anthocyanins [68]. b.
Proanthocyanidins (PA)
The complexity and the lack of commercial pure standards of PA make their analysis difficult. Their absorption depends on their degree of polymerization. In some in vitro experiments, only PA dimers and trimers, but not polymers with an average polymerization degree of 7, were absorbed through an intestinal epithelium cell monolayer [118]. Experiments in chicken and sheep showed that polymeric PAs were not absorbed through gut barrier [119,120]. Evidence occurred that polymeric proanthocyanidins could be degraded by the colonic microflora into low-molecular-weight compounds, which would be subsequently absorbed. The group of Deprez [118] investigated their metabolism by human colonic microflora incubated in vitro in anoxic conditions using non-labeled and ^"^C-labeled
293
purified proanthocyanidin polymers. Degradation occurred almost totally after 48 h of incubation; metabolites identified were low-molecularweight phenolic acids: phenylacetic, phenylpropionic and phenylvaleric acids, monohydroxylated mainly in meta or para-position. It is supposed that, once fermentation products have crossed the intestinal barrier, they reach the liver through the portal vein, where they are further metabolized by dehydroxylation, methylation or conjugation to sulfate esters or glucuronides as it has been shown for other flavonoids [59]. POLYPHENOLS: BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES Polyphenols are endowed with different biological activities, including: 1. 2. 3. 4. 5. 6. 7. 8. 9.
Antioxidant/anti-radical activity and chelation of metal ions Modulation of some enzymes activity Anticarcinogenic activity Antiatherosclerotic activity Anti-inflammatory activity Inhibition of histamine release and spasmolytic activity Hepatoprotective activity Antiviral and antimicrobial activity Oestrogenic activity
1. Antioxidant and anti-radical activity, chelation of metal ion Polyphenols can act as antioxidants by a number of potential pathways. The most important is likely to be by free radical scavenging, in which the polyphenol can break the radical chain reaction. Polyphenols are effective antioxidants in a wide range of chemical oxidation systems, being capable of scavenging peroxyl radicals, alkyl peroxyl radicals, superoxide, hydroxyl radicals, nitric oxide and peroxynitrate in aqueous and organic environments [121]. This activity is due to the ability of donating an H atom from an aromatic hydroxyl group to a free radical, and the major ability of an aromatic structure to support an unpaired electron by delocalization around the 7i-electron system. Phenolic acids
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and flavonoids may be good antioxidants, particularly those possessing the catechol-type structure [122-125]. Phenolic acids and flavonoids (PP-H) can also act as free radicalchain (ROO*) reaction terminator, as follow: ROO* + PP-H -> ROO-H -f PP* The PP* radical is relatively stable and could react in another reaction as terminator, as follow: ROOVPP*->ROO-PP The interaction between flavonoids and phenolic acids with other physiologic antioxidants, such as ascorbate or tocopherol, is another possible antioxidant pathway for these compounds [72,126,127]. Nevertheless, like must other antioxidants, flavonoids may also act as prooxidant in particular circumstances [128,129]. Phenolic acids and flavonoids can also act as chelating agents, complexing transition metals that are responsible of the initiation of peroxidative processes (Fenton and Haber-Weiss reactions). This property is much stronger in phenolics having a catechol, pyrogallol, or 3-hydroxy4-carbonyl group [130]. 2. Modulation of some enzymes activity Interaction of low molecular weight molecules, such as phenolic acids andflavonoids,with macromolecules can modify their chemical-physical properties. Different studies confirm the ability of phenolics in modulating some enzymes, such as hydrolases, transferases, kinases, oxidases, hydroxylases, glutathione S-transferase, nitric-oxide synthase, cytochrome P450 systems, ATPases, lipases, phospholipases, adenylate cyclase, RNA and DNA polymerase, human DNA ligase I, ribonuclease, reverse transcriptase, topoisomerase, aromatase, hyaluronidase, elastase, HIV-1 proteinase and integrase, aldose reductase [131,132]. Specifically, enzymes such as xantine-oxidase, nitric oxide synthase, phospholipase, cyclooxygenase and lipoxygenase, involved in the production of free radicals in biologic systems, are also inhibited, in vitro, from different polyphenols [132,133].
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Overall, these activities may explain the correlation between polyphenols and their anticancer, antithrombotic, anti-atherogenetic and anti-inflammatory effects. However, more research is needed to determine which of these activities can realistically be translated into clinical effects. 3. Anticarcinogenic activity The antioxidant effect, the modulation of some enzymes and induction of apoptosis are at the root of the anticancer mechanism. Carcinogenesis is a multi stage process of genetic change that may be initiated by increased and persistent damage to DNA causing permanent alterations in the genetic message when the cell replicates its DNA and divides. ROS (Reactive Oxygen Species) are potential carcinogens because they can induce structural damage to DNA by oxidation, methylation, depurination and deamination reactions. The ability of some polyphenols to reduce or inhibit the oxidative damage to DNA is well documented. For example chlorogenic acid inhibits DNA damage in vitro caused by peroxynitrite [134], coumaric acids are good free radical scavengers [135], caffeic and dihydroxybenzoic acids are able to inhibit iron induced DNA damage [135,136]. Chlorogenic acid can inhibit DNA damage caused by monochloramine [137]. Many kind of flavonoids are able to scavenge free radicals protecting DNA from oxidation, including the isoflavone genistein [138,139], theflavonesluteolin and apigenin [140], the flavanol epigallocatechin gallate [141], the flavonols myricetin and kaempferol [140], quercetin [140-142], and its glycosylated derivatives, quercetin-3glycoside, quercitrin and rutin [140]. DNA damage may be also reduced by metal binding properties of flavonoids, as demonstrated for rutin and quercetin [143]. Another mechanism in reducing carcinogenesis by polyphenols is the modulation of the enzymatic system that is in charge of the metabolization of carcinogenic molecules. The cytochrome P450 family of enzymes metabolizes a large number of procarcinogens to reactive intermediates, which bind covalently to DNA and can induce mutation. Severalflavonoidsare able to inhibit the activity of this family enzymes, as reported by different authors [144-149]. The inhibition of this activity byflavonoidsis directly correlated to their antimutagenic properties. The glutathione transferases (GST), together with the tripeptide glutathione (GSH), conjugate the highly reactive and potentially
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carcinogenic substances, making such molecules more polar, thereby facilitating their excretion. Flavanones, flavones, flavonols increase hepatic GST levels and activity in rats [148,150]. Furthermore, by modification of gene expression, some polyphenols may prevent or reverse carcinogenesis, inducing apoptosis or inhibiting neoplastic transformation. Gallic acid induces selective cell death in cancer cell [151], and hamster fed caffeic acid with the diet were shown to be less susceptible to the effect of methylazoxymethanol, an initiator of colon carcinogenesis [152]. Caffeic acid phenethyl ester from propolis, given to mice bearing a germline mutation in the Ape gene and spontaneously developing numerous intestinal adenomas by 15 weeks of age, decreased tumor formation by 63% at a dietary level of 0.15% and the examination of intestinal tissue from treated animals showed that tumor prevention was associated with increased enterocytes apoptosis [153]. Protocatechuic acid from Hibiscus sahdariffa induced apoptosis in human leukemia cells [154]. Green tea polyphenols and epigallocatechin gallate increase fragmentation in several human and rodent carcinoma cells, but not in normal epidermal keratinocytes [155]. Epigallocatechin gallate also induces apoptosis in transformed fibroblast [156] and in human histiolytic lymphoma U937 cells [157]; theasinensin D, theaflavin, theaflavin digallate induced apoptosis in the same lymphoma cells [157]. Quercetin, rutin, morin, gallic acid and tannic acid inhibited the growth of human prostate cancer cell (LNCaP) at different concentrations, and induced apoptosis [158]. 4. Antiatherosclerotic activity Cardiovascular heart diseases (CHD) are considered as the clinical expression of advanced atherosclerosis. One of the initial steps in atherogenesis is the oxidative modification of LDL and the uptake of the modified lipoprotein particles by macrophages, which in tum become lipid laden cholesterol-rich cells, so-called foam cells [159]. An accumulation of foam cells in the arterial wall is the fu-st visible sign of atherosclerosis and is termed fatty streak, the precursor to the development of the occlusive plaque [160]. It is well known that oxidation of LDL can be initiated in vitro by incubating isolated LDL particles with cells (macrophages, lymphocytes, smooth muscle cells, or endothelial cells), metal ions (copper or iron), enzymes, oxygen radicals, or UV-light. However less is known about the mechanisms by which
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LDL becomes oxidized in vivo. There is evidence that LDL is protected against oxidation in plasma by water-soluble antioxidative substances, such as ascorbic acid, uric acid, or bilirubin. Thus, it is likely that the majority of oxidative modification of LDL occurs in the artery wall, where LDL is largely isolated from the plasmatic antioxidants. Recent evidence suggests that metal ions (copper or iron) and the enzymes myeloperoxidase and lipoxygenase play major parts in the modification of LDL [161]. In in vitro studies the oxidation of LDL by endotheUal cells, macrophage and Cu"*^ can be inhibited by a wide range of polyphenols and polyphenol-rich extracts [162-164]. Such effects may be due to polyphenols by direct scavenging of the oxidizing species, by regeneration of a-tocopherol in LDL [165], by their ability in binding metal ion and LDL protein [166]. In addition to in vitro studies, several animal models and trials with human subjects indicate that ingestion of polyphenols increases the resistance of LDL oxidation ex vivo [167,168]. Some polyphenols inhibit platelet aggregation reducing the risk of thrombosis [171-173]. This effect may be due to a series of interaction of flavonoids in different biochemical pathways, such as by inhibition of cyclooxygenase and lipoxygenase, that are involved in the arachidonic acid metabolism in the platelets, or by inhibition of the formation of tromboxane and of the receptor function of the same [173-176]. Regular consumption of wine, tea and chocolate has been associated to the reduction of platelet aggregation, cardio-vascular diseases and thrombosis [171,177-179]. 5. Anti-inflammatory activity Inflammation is a highly complex biochemical protective response to cellular injury. It is important in the maintenance of homeostasis when the organism is challenged by noxious agents or by tissue mechanical injury. Inflammation is associated with a drastic rise in the number of polymorphonuclear leukocytes and monocytes in the affected tissue and with the release of inflanunatory mediators such as prostaglandins and cytokines. Under normal conditions, inflammation results in the complete recovery of the integrity of the affected tissue, but if the response to the triggering stimulus is not subjected to tight regulation.
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cellular and extracellular components of the organism adjacent to the inflammation site can be injured, inducing a condition known as chronic inflammatory disease. A chronic inflammatory like environment characterizes the pathogenesis of various diseases such as atherosclerosis, arthritis, and Crohn's disease, and it is thought to be among the causative factors of more than 30% of human cancers. For these reasons it is very important to localize and reduce the inflammatory response. In this scenario, polyphenols are involved as immunomodulatory and anti-inflammatory agents, scavenging ROS and modulating the activity of key enzymes of the inflammatory response [172,180482]. 6. Inhibition of histamine release and spasmolytic activity The flavonoids quercetin, hyperoside and isoquercetin, present in the ethanolic extract of Drosera madagascariensis, are inducers of spasmolytic and anti-inflammatory effects in guinea-pig ileum by affecting cholinergic M3 and histamine HI receptors [183]. In an in vitro study by Yamada et al [184], triphenols, such as pyrogallol and gallic acid, and among flavonols, myricetin, inhibited histamine release from rat peritonei cells. Pyrogallol and gallic acid, and also o- and /?- diphenols, such as catechol and hydroquinone and all flavonols tested, strongly suppressed leukotriene B4 release in the same cells. Another in vitro study on rat basophilc leukemia cells demonstrated that, among tea polyphenols, (-)-epigallocatechin gallate (EGCg), (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECg) have different inhibitory effects on histamine release induced by a calcium ionophore, with the following magnitude: EGCg>ECg>EGC [185]. In the same study was also demonstrated that pyrogallol and gallic acid exert inhibitory activity and a mixture of these two compounds inhibited histamine release as strongly as EGCg. 7. Hepatoprotective activity Polyphenols are also endowed to have hepatoprotective effects. For example, quercetin reduces liver oxidative damage, ductural proliferation and fibrosis in biliary-ostructed rats, suggesting that it may
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be a useful liver protective agent in patients with biliary obstruction [186]. 8. Antiviral and antimicrobial activity Polyphenols may act as antimicrobial and antiviral agents as demonstrated by several studies in vitro. Polyphenol-rich extracts from various plants, such as Betula pubescens^ Epilobium angustifolium, Perillafrutescens, Pinus sylvestris, Rubus chamaemorus, Rubus idaeus. Solarium tuberosum, propolis and pure compounds, were tested to evaluate their antimicrobial activity against different bacteria and yeasts species, such as Bacillus subtilis, Escherichia coli, Mycobacterium tuberculosis H37Rv, Pseudomonas aeruginosa. Salmonella spp, Staphylococcus aureus. Streptococcus piogenes, Aspergillus niger, Candida albicans, Saccharomyces cerevisiae and showed growth inhibitory and bactericidal effect at different concentrations [187-192]. Naturally occurring flavonoids with antiviral activity have been recognized since the 1940s [193]. Quercetin, morin, rutin, taxifolin, dihydrofisetin, leucocyanidin, pelargonidin chloride, apigenin, catechin, hesperidin, and naringin have been reported to possess antiviral activity against some of 11 types of viruses [193]. (-)-Epigallocatechin gallate and theaflavin digallate inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney cells in vitro [194]. 9. Oestrogenic activity Plant-derived oestrogens may exert both oestrogenic and antioestrogenic effects, depending on several factors, including their concentration, the concentrations of endogenous oestrogens, and individual characteristics, such as gender and menopausal status [195,196]. The anti-oestrogenic activity of phytoestrogens may be partially explained by their competition with endogenous 17p-estradiol for oestrogen receptors [197]. Many of the potential health benefits of phytoestrogens may be attributable to features that do not involve oestrogen receptors, such as their influence on enzymes, protein synthesis, cell proliferation, angiogenesis, calcium transport, Na"^/K"*" adenosine triphosphatase, growth factor action, vascular smooth muscle cells, lipid oxidation, and cell differentiation. Phytoestrogens may have
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favorable effects on the risk of cardiovascular disease and are thought to be hypocholesterolemic, anticarcinogenic, antiproliferative, antiosteoporotic, and hormone altering [195,196,198,199]. Finally, flavonoids can bind to structural proteins and this feature could explain their ability to enhance the integrity of connective tissue. EPIDEMIOLOGIC EVIDENCE HEALTH BENEFITS
OF PLANT POLYPHENOL
1. Risk of CHD diseases Several epidemiological studies have reported inverse relation between intakes of flavonols and flavones and cardiovascular heart diseases (CHD). In a prospective study of 3454 men and women (age 55 years and older), a significant inverse association between the intake of catechinrich tea and radiographically quantified aortic atherosclerosis was found [200]. Similarly, inverse association between the consumption of red wine and CHD mortality (French paradox) have been suggested [201]. This beneficial effect of red wine may be due to the antioxidant ability of the wine phenolics to inhibit the oxidation of LDL to an atherogenic form [202], In the Zupthen Elderly Study [203] flavonol and flavone intake at baseline in 1985 of approximately 800 men (aged 65-85 years) was determined using the cross-check dietary history method. Men were divided into tertiles of flavonol and flavone intake. After five years of follow-up 43 men died from heart disease in this period. Flavonol and flavone intake, expressed as tertiles, was inversely associated with mortality from coronary heart disease and to a lesser extent with the incidence of first myocardial infarction. Furthermore, the association between long-term flavonol and flavone intake and risk of stroke in a cohort of 552 middle-aged Dutch men free fi"om history of stroke at baseline was also investigated within this study. Men were divided into quartiles of flavonol and flavone intake, and followed for 15 years. During this period 42 men had a first stroke event. Flavonol and flavone intake was strongly inversely associated with stroke risk. In both studies, the men in the highest category of flavonol and flavone intake (>30mg/day) had about one-third the risk of getting the disease compared
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with men in the lowest category. The major sources of dietary quercetin and other flavonols were revealed as tea and onions (fruits and vegetables had minor importance). The same authors [204] confirmed these results in the Seven Country Study. The contribution of flavonols and flavones in explaining the variance in coronary heart disease mortaUty rates across 16 cohorts from seven countries was studied. Flavonol and flavone intake was inversely correlated with mortality from coronary heart disease. Thesefindingare in line with the results of a cohort study in Finnland [205], where a significant inverse gradient was observed between dietary intake of flavonoids and total and coronary mortality. A modest but not significant inverse correlation between the intake of flavonols and flavones and subsequent mortality rates was found in a prospective cohort study of US Health Professionals by Rimm et al [206]. The authors do not exclude thatflavonoidshave a protective effect in men with established coronary heart disease although strong evidence was missing. Also other studies failed to demonstrate a significant statistical association between the intake of polyphenols and CHD. In Great Britain for instance coronary and total mortality even rose with the intake of the majorflavonolsource, tea [207]. The most likely explanation for the latter observation is that in this study tea consumption merely acted as a marker for a lifestyle that favours the development of cardiovascular disease. Indeed, men with the highest intake of tea and flavonols tended to be manual workers, and they smoked more and ate more fat [208]. 2. Risk of cancer The epidemiological evidence for a beneficial support of polyphenols in cancer disease is contradictory and less clear than its role in CHD. The Zupthen Elderly Study found a weak inverse association between flavonoid intake from fruit and vegetables sources and cancer of the alimentary and respiratory tracts combined [209]. The same authors observed no independent association with mortality from other causes between flavonoid intake and cancer mortality in the Seven Country Study [204]. ICnekt et al [210] studied the relation between the intake of flavonoids and subsequent cancer among 9959 finnish men and women during a follow-up in 1967-1991, An inverse association was observed between the intake offlavonoidsand incidence of all sites of cancer combined. Of
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the major flavonoid sources, the consumption of apples showed an inverse association with lung cancer incidence. The cancer protective effects of black and green tea consiraiption, important sources of flavonol in specific countries, have been investigated mainly in case-control studies. Kohlmeier et al [211] evaluated the epidemiologic literature about tea and cancer prevention, concluding that cohort studies do not suggest a protective role for tea drinking in the total risk of cancer. Site-specific studies give a more complex picture. For example, a protective effect of green tea on the development of colon cancer is suggested. On the other hand, evidence for black tea is less clear, with some indication of a risk of colon or rectal cancer associated with regular use of black tea. In another cohort study of a Japanese population, researcher surveyed more than 8000 individuals over 40 years of age on their living habits, including daily consumption of green tea. Results found a negative association between green tea consumption and cancer incidence, especially among females drinking more than 10 cups per day [212]. 3, Vasoprotective effects (Hypertension) Experimental studies have shown that the administration of green teaenriched water to laboratory animals is associated with a reduction in blood pressure [213]. Different epidemiologic studies have suggested that drinking either green or black tea may lower cholesterol concentration and blood pressure [214,215]. In a epidemiological study of Japanese women, a history of stroke was less common among those who drank more green tea. There was no statistically significant reduction in blood pressure alone among those women who drank more tea [206]. 4. Oestrogenic effects Phytoestrogens represent a family of plant compounds that have been shown to have both oestrogenic and anti-oestrogenic properties. Accumulating evidence from molecular and cellular biology experiments, animal studies and, to a limited extent, human clinical trials suggests that phytoestrogens may potentially confer health benefits related to
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cardiovascular diseases, cancer, osteoporosis, and menopausal symptoms. These potential health benefits are consistent with the epidemiological evidence that the risk of heart disease, various cancers, osteoporotic fractures, and menopausal symptoms is lower among populations that consume plant-based diets, particularly among cultures with diets that are traditionally high in soy products. One study over 9 months noted a significant reduction in total cholesterol in premenopausal women when they consumed soy products with 45 mg conjugated isoflavones/day in comparison to levels during a control period when they were fed isoflavone-free soy products. The treatment group difference was significant despite the small sample size and the selection of healthy, normocholesterolemic women who had limited room for detectable improvements [216]. The pattem of soy intake and its association with blood lipid concentrations in the Hong Kong Chinese population was studied in a total of 500 men and 510 women with an age range of 24-74 years by Ho et al [217]. In men, soy intake and total plasma cholesterol were negatively correlated (r = 20.09, P = 0.04), as were soy intake and LDL cholesterol fr = 20.11, P = 0.02). The respective values in women <50 y old were r = 20.11, P = 0.04 and r = 20.11,P = 0.05. In the Framingham Offspring Study a group of 939 postmenopausal women was studied to correlate the association between dietary phytoestrogen intake and metabolic cardiovascular risk factors. Mean blood pressure, waist-hip ratio (WHR) and lipoprotein levels were determined in quartile categories of dietary phytoestrogen (isoflavones and lignans) intake. In the highest quartile of intake of isoflavones, plasma triglyceride levels were 0.16 mmol/L lower (95% CI, -0.30 to 0.02) compared with the lowest quartile of isoflavones and the mean cardiovascularriskfactor metabolic score was 0.43 points lower (95% CI, -0.70 to -0.16) than the lowest quartile [218]. Hormone-related cancers of the breast, ovary, endometrium, and prostate have been reported to vary by as much as 5 to 20-fold between populations. Migrant studies indicate that the difference is largely attributable to environmental factors rather than genetics [219,220]. The highest rates of these cancers are typically observed in populations with Westem lifestyles that include relatively high fat, meat-based, low fiber diets, whereas the lowest rates are typically observed in Asian populations with Eastem lifestyles that include plant-based diets with a high content of phytoestrogens [219,221].
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In a case-control study Ingram et al [222] reported a significant reduction in breast cancer risk among both premenopausal and postmenopausal women who consumed phytoestrogens. In a study of Asian-Americans of Chinese, Japanese, and Filipino heritage, it was reported that tofii consumption was significantly and inversely associated with breast cancer [223]. Similar findings were reported from a case-control study of women in Singapore in which soy intake was inversely, and animal products intake was positively, associated with breast cancer, although these findings were significant only among premenopausal, not postmenopausal women [224]. Soy and fiber consumptions were both associated with a decreased risk of endometrial cancer among the multiethnic population of Hawaii, a finding that was limited to women who had never used oestrogens and had never been pregnant [225]. In a study conducted in Boston and Helsinki, it was demonstrated that the lowest excretion of enterolactone and equol was found in a group of postmenopausal breast cancer patients compared to healthy omnivorous and vegetarian women [226]. The continual loss of bone mass in the elderly is a natural process of aging. Women have a higher incidence of osteoporotic fractures than men due to their lower peak bone mass, but in addition, the abrupt decrease in oestrogen secretion in postmenopausal women accelerates bone loss. Currently, osteoporosis-related fractures are lower in Asia than in most Westem communities, possibly due to the phytoestrogen-rich soybeans and vegetables consumed in large quantities in the Asian diet [227]. Investigation about rates of hip fracture in Hong Kong and the U.S. reported that for men and women 85 yr of age or more, the rates in Hong Kong were roughly one third the rates in the U.S. [228]. REFERENCES: [1] [2] [3] [4] [5] [6] [7]
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
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PROMISING PHARMACOLOGICAL ACTIONS OF CROCIN IN CROCUS SATIVUS ON THE CENTRAL NERVOUS SYSTEM SHINJI SOEDAi, TAKASHI OCHIAIi, H I R O S H I SHIMENOi, HIROSHI SArr02, KAZUO ABE2, MINORU SUGIURA^, HIROYUKI TANAKA^ FUTOSHI TAURA^ SATOSHI MORIMOTO^ a n d YUKIHIRO SHOYAMA^ * ^Facidty ofPharmaceutical Sciences, Fukuoka University, Fukuoka8140180, Japan ^Graduate School ofPharmaceutical Sciences, The University of Tokyo, Tokyo 113'0033, Japan ^Graduate School ofPharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan ABSTRACT: Information on clinically available activities in both peripheral and neuronal systems dF crocin in saffron has been accumulated. The LTP-blocldng effect erf ethanol was significantly improved by oral-, intravenous-, and intracerebroventricularadministraticm of crocin, respectively. We investigated the effects of ethanol and CTOcin on synaptic potentials mediated by N-methyl-D-aspartate (NMDA) receptors in tfie dentate gyrus of rat hippocampal slices. Crocin alone did not affect synaptic potentials mediated by non-NMDA (x NMDA recq>tors. Crocin did not affect the inhibiticm of non-NMDA response by 100 mM ethanol, but significantly blocked the inhibition of NMDA response by 10-50 mM etiianol. We perfcnmed whole-cell patch receding with primary cultured rat hippocampal neurons, and confirmed tiiat crocin blocked etfianol inhibition of inward currents evoked by the application of NMDA. We also demonstrated that crocin suppresses the effect of tumor necrosis factor (TNF)-a on neuronally differentiated PC-12 cells. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-a~induced release of cytochrome c from the mitochondoria. Crocin also blocked the cyotochrome c-induced activation of caspase-3. We found that crocin inhibited the effect of daunorubicin as well. The present paper focuses on the pharmacological actions of crocin on the central nervous system and reviews briefly the findings of such studies on the prevention of neuronal progranmaed cell death (apoptosis).
INTRODUCTION Saffron {Crocus sativus L. ; Iridaceae)findsuse in medicine as well as a flavoring and coloring agent. It has three main chemical compounds. The bright red coloring carotenoids; a bitter taste, picrocrocin; and a spicy
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aroma, safranal. The carotenoid pigments consist of oooelin (JKP4>^Jiioo^^ estei; crooetin<^B-gentiobk)fiylK^^ and crocetin-di-(P-Ddigentiobiosyl)-ester (crocin) as indicated in Fig. (1). COOH
Hocx: Crocctin
COO
OOC R1-OH2C
Ri=Glc R2=Glc
Fig.(l).
R2-OH2C
Crocin
Ri=Glc R2=H
Crocctiii-(fi-D-gentiobiose)-(B-D-glucosyl)-ester
Ri=H
Crocetin-di-(6-D-glucosyi)-estcr
R2=:H
Structures of crocetin and its glycosides
Previously, we showed that crocetin glucose esters increased from the period before bloonung and reached maximum in the full blooming period [1]. They are sensitive for the presence of o^gen, light irradiation because of the polyene structure, and to an indigenous p-glucosidase which hydrolyzes crocin to crocetin di-(p-D-glucose)-ester [1]. The artificial pathway is indicated in Fig. (2). Moreover, it is evident that storage of saffron at -20 °C promotes the constant supply of saffron with a homog^ieous pharmacological activity [1]. In order to check the quantity of saffi'on, we have already prepared a monoclonal antibody (MAb) against crocin, and established a competitive ELISA using the anti-crocin MAb [2]. In the peripheral blood system, crocetin derivatives prevent an elevation in bilirubin levels [3] and also reduce elevated levels of serum cholesterol and triglyceride [4]. Anti-tumor activity of saffron is observed in mice transplanted with several types of tumor cell lines including sarcoma 180, Ehrlich ascites carcinoma, and Dalton's lymphoma ascites [5]. Saffron shows an inhibitory effect on chemical carcinogenesis in mice [6], and the effect of crocetin on skin papilloma and Rous sarcoma has been reported [7]. Escribano et al. [8] have recently demonstrated that crocin inhibits tiie growth of HeLa cells and suggested pro-apoptotic
315
QOC HOH2C O^^^OH
COO
/^OHa' O^'
OH
Crocetin-(B-D-gcntiobiose)-(6-D-glucosyi)-cstcr
Crocetin-di-(fi-D-glucosyl)-ester Fig.(2).
Artificial pathway of crocin
properties of the compound. More recently, we have reported that orally administered ethanol extract of saffron and crocin exhibit inhibitory effects on the two-stage carcinogenesis of mouse skin papillomas [9]. These results suggested that crocetin and/or crocetin glucose esters contributed to the anti-tumor activities of saffron. The development of natural products having alleviation properties for the symptoms of learning and memory impairments has been chnically expected. In the brain, the hippocampus is a very important region in the learning and memory processes, and the long-term potentiation (LTP) induced from the brain tissue is closely related to leaming and memory [10]. We reported the effects of ethanol extract of C sativus and its purified components on the central nervous system in terms of leaming behaviors in mice and the LTP in the dentate gyrus of hippocampus in anesthetized rats and in the CAl region of rat hippocampal slices [11-13]. This review also discusses the values of folk medicines in modulating apoptotic cell death, together with our recent data of crocin's effect on neuronal cell death. Neuronal cell death is required for the development of the nervous system. However, recent studies suggest that neurons die from programmed cell death (apoptosis) in brains deprived of oxygen by stroke [14] and trauma [15], and in the brains of Alzheimer's patients [16]. Therefore, prevention of neuronal apoptosis has been considered to be a desirable therapeutic strategy for treating such neurodegenerative diseases, although the value of this approach is not yet evident. We have recently reported that crocin suppresses tumor necrosis factor (TNF)-a-
316
induced apoptosis of PC-12 cells by modulating the mRNA expression of Bcl-2 family proteins, which trigger downstream signals culminating in caspase-3 activation followed by cell death [17]. Effect of crocin on LTP We already indicated that intravenous injection of ethanol blocked the LTP induced by tetanic stimulation [18]. However, when saflBron crude extracts were injected intracerebroventricularly, the blocking effect of ethanol on the LTP decreased dose-dependently [19]. Moreover, crocin prevents the ethanol-induced impairment of memory acquisition in ST and SD tests [20]. From these results it is easily suggested that crocin antagonized the blocking effect of ethanol on the induction of LTP. Crocin of 50 mg/kg ameliorated the blocking effect of ethanol on the LTP at approximately 84% compared to the control as indicated in Fig. (3). 3.00D
Control
EtOH alone
10 50 Crocin
50 100 Ctocctin gentioblose glucose ester
50 100 (mg/kg) Crocctin di-glucose ester
Fig.(3). Effects of crocin and its analogues on the LTP-blocking effect of ethanol. The vehicle or drug was intraceiebroventricularly injected 20 min before tetanus, and saline or 30% ethanol was intravenously injected at a volumn of 2 ml/kg 15 min before tetanus. The AUC from 5 to 60 min after appUcation of tetanus was calculated and defined as an index of magnitude of LTP in each group. The data are represented as the means ± SEM of the number of observations shown in parentheses. ••p<0.01 vs. control group. -Hp<0.05, -HpO.Ol vs. ethanol group in Duncan's multiple range test.
317
Effects of crocin on the induction of LTP in the CAl region of rat hippocampal slices In the control experiments, strong tetanic stimulation induced robust LTP. Eth^ol (30%; 10-15 ml/kg) did not show any significant effect on the baseline synaptic responses, but suppressed the induction of LTP following strong tetanic stimulation in a concentration-dependent manner (Fig. (4)). The effect of crocin on the LTP-suppressing effect of ethanol was investigated (Fig. (4)). The potentiation induced by strong tetanic stimulation in the presence of 20 mg/kg crocin and 30% of ethanol (15 ml/kg) was significantly larger than that in the presence of 30% of ethanol (15 ml/kg) alone, indicating that crocin clearly attenuates the action of ethanol [21]. Fig.(4). Effects of ethanol and crocin on LTP induced by strong tetanic stimulation in the CAl region of rat hippocampal slices. The inset in A is a representative evoked potential recorded from the CAl pyramidal cell layer. Calibration hards: vertical 2 mV, horizontal 10 msec. The population spike an^litude was defmed as an average of the ampUtude from the first positive peak 1 to the succeeding negative peak 2 and the ampHtude Time(iiiln) from the negative peak 2 to the second positive peak 3. A Time-course of potentiation induced by strong tetanic (23) stimulation in the control stices (O, n=23) and in the slices treated with 30% of ethanol si (15 ml/kg) ( • , n=27 ) and in the shces (7) treated with 30% of ethanol (15 ml/kg) and (16) # 20 mg/kg crocin ( A , n=7). (1) and (2) OLJE (27) indicated 30% ethanol of 15 ml/kg and 10 (7) ml/kg, respective^. Ethanol or crocin was added in the perfusing ACSF from 15 or 20 Co«t ( 1 ) ( 2 ) 10 20 30 min, respectively, before tetanic stimulation. Crocin (mg/kg) The ordinate indicates the population spike + 30% EtOH(15 ml/kg) an^Utude expressed as a percentage of the baseline values immediately before tetanic stimulation. B: Summary of the effects of ethanol and crocin on the induction of LTP. The magnitude of LTP was evaluated with the population spike an^)litude 30 min after tetanic stimulation. The numbers of observations in each groiq) are shown in parentheses. All data are represented as the mean ± SEM *^p<0.01 vs. control, ^p<0.05 vs. 30% of ethanol (15 ml/kg) alone. Duncan's multiple range test.
1 i1
318
Effects of ethanol and crocin on non-NMDA receptor-mediated synaptic potentials in hippocampal slices The synapic potential mediated by non-NMDA receptors was recorded in normal ACSF. When ethanol (10-50 mM) was added to the perfusmg medium, no significant change in non-NMDA receptor-mediated synaptic potential was observed. However, the addition of ethanol at a mghCT concentration (100 mM) induced a small reduction m non-NMDA receptor-mediated synaptic potential (Fig.(5)A). The reduction m nonNMDA response rapidly occurred after the addition of 100 mM etiianol and reached a steady state within 10 min. After washing out the ethanol, the response gradually returned to the normal level. When 10 \iM crocin was added 10 min prior to the ethanol, the non-NMDA response was similarly reduced in the presence of 100 mM ethanol (Fig-(5)B) Crocin (10 nM) did not significantly affect the inhibitory effect of 100 mM ethanol on non-NMDA response (Fig.(5)C) [22].
Ii1g.(5).
Effects of ethanol and crocin on non-
NMDA receptor-mediated synaptic potential evoked in nonnal ACSF in rat hippocampal slices.
E, to
a. 0
lOOtnMQ ethanol I i L
5
10 15 20 25 30 35 Vme (min)
B
lOOmMethanola L...
CO
—
10 MM crocin —1 i L. J
a. 0
5
L
10 15 20 25 30 35 Tvne (min)
Ettianoi (mM)
(A) Representative experiment showing the effect of ethanol on non-NMDA reseptor-mediated response. (B) Representative experiment showing the influence of crocin on ethanol-induced inhibition of non-NMDA response. Crocin (10 fiM) was applied 10 min prior to ethanol (white bar). (C) Concentration-effect curves for ethanol inhibition of non-NMDA response in the absence (O) or presence ( • ) of 10 MM crocia
319
Effects of ethanol and crocin on NMDA-induced currents in single hippocampal neurons In order to confinn the possible interaction of ethanol and crocin on NMDA receptors, we also performed whole-cell patch recording with primary cultured hippocampal neurons and measured membrane currents induced by the application of NMDA in a voltage-clamped condition. Application of 100 fiM NMDA induced an inward current of 100.2 ± 9.8 pA (n=10) at a holding potential of -60 mV. The NMDA-induced inward current was not affected by 10 \iM CNQX (data not shown), but was completely abolished by 30 \iM APV, supporting the fact that the response was mediated by NMDA receptors. Etiianol inhibited NMDAinduced currents in a concentration-dependent manner. Crocin (10 |iM) had no effect on NMDA-induced currents by itself (data not shown), but attenuated the inhibitory effect of ethanol on NMDA-induced currents. The concentration-effect curve for ethanol was shifted to the right by the presence of crocin [22]. Effect of crocin on TNF-a-induced morphological changes and DNA fragmentation in the nucleus of PC-12 cells The effect of crocin on the TNF-a-induced cell death of PC-12 cells is shown in Fig. (6). In serum-free GIT medium conditions, PC-12 control cell morphology remained intact at 24 h (panel A). However, the cells treated for 24 h with TNF-a (500 units/ml) appeared rounded and showed the characteristics of necrotic and/or apoptotic cells (panel B). In the combination with 10 fxM crocin, PC-12 cell morphology retained intact neuronal cell morphology at 24 h (panel C). Crocin alone had no effect on the morphology of PC-12 cells (data not shown). We next analyzed DNAfragmentationin the nuclei. Treatment of PC12 cells with TNF-a (500 units/ml) for 24 h caused DNA fragmentation (lane 2 in panel D), while the nuclei in control (lane 1) and inlO fiM crocin-treated cells (lane 5) were intact. Lanes 3 and 4 show that 1 and 10 jiM crocin blocked TNF-a-induced DNA fragmentation. This data suggests that crocin prevents TNF-a-induced cell death of PC-12 cells at a concentration range of 1-10 jiM. The TNF-a-induced DNAfragmentationmay have been caused by caspase-activated deoxyribonuclease (CAD) in PC-12 cells. In nonapovtotic cells, CAD is present as an inactive complex with the inhibitor r [23,24]. During apoptosis, caspase-3 inactivates I^"^, leaving CAD free to f\mction as a nuclease [25]. Therefore, we used the fluorogenic substrate, Ac-DEVD-MCA, to determine whether the caspase-3 in PC-12 cells was activated by treatment with TNF-a and/or crocin. As shown in
320
Fig. (7), TNF-a treatment resulted in an elevation of caspase - 3 activity in the cells at 6 h (a 3.9-fold increase), and the elevation lasted for 24 h. Crocin (0.1-10 jiM) suppressed the TNF-a-induced activation of caspase3 in a concentration-dependent manner. Caspase activity in the copresence of 10 (AM crocin was near the control level, while the crocin (10 \xM) alone had no effect on caspase activity in cells imtreated with TNF-a. These results suggest that crocin can suppress the TNF-a-induced cell death of PC-12 cells by blocking the activation of caspase-3. It is possible that the crocin treatment may also suppress upstream signals for caspase3 activation.
Lane 1: cx>flliol
99 ^i !#• Lane 2: TNF adone 50003000-
Lane 3: TNF+1JJL M crocta
15001000-
Lane 4: TNF+10/zM crocin Lane 5:10/i M crocin alone
1 2
3 4 5
Fig.(6). Morphological appearance of PC-12 cells treated for 24 h with vehicle alone (panel A), TNF-a (500 units/ml) alone (panel B), or TNF-a (500 units/ml) plus 10 \»M crocin (panel C). In panel D, inlemucleosomal DNAfragmentationwas detected by agarose gel electrophoresis of DNA extracted from cells treated for 24 h.
321
control IOMM crodn
TNF +l/iM crocin
-i»~TNF.a(500 U) -•><-TNF +0.1 MM crodn -A^TNF +10/1M crocin
Time (h) Flg.(7).
Effect of crocin on TNF-a-induced activation of caspase-3 in PC-12 cells. Cells were incubated
for the indicated periods in GIT medium supplemented with vehicle alone, TNF-a alone, crocin alone, or their combination. After lysis of the cells, caspase-3 activity was assayed as described in Materials and methods. Each bar represents the mean ± S.D. of three independent experiments. M P<0.01; ###P<0.001, compared to TNF-a alone.
Crocin blocks the release of cytochrome c from mitochondria by modulating the expression of Bcl-2 family protein mRNAs Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis [26]. Anti-apoptotic proteins such as Bcl-2 and BC1-XL prevent apoptosis in response to numerous stimuli. During the apoptotic process, cytochrome c is released from mitochondria, but the release can be inhibited by the presence of Bcl-2 on the organelles [27]. The released cytochrome c forms an essential part of Sie apoptosome, which is composed of cytochrome c, Apaf-1, and procaspase-9 [28]. The complex formation results in activation of caspase-9, which leads to the stimulation of caspase-3. BC1-XL has recently been reported to bind to Apaf-1 [29]. It may inhibit the association of Apaf-1 with procaspase-9 and tiiereby prevent caspase activation. Fig. (8) shows the effects of TNF-a, crocin or their combination on Bcl-2 and BC1-XL mRNA levels in PC-12 cells. TNF-a treatment had no effect on the BC1-XL mRNA levels at 3 h but significantly decreased the mRNA expression at 18 h, compared to the control (panel A). Crocin (10 fxM) alone or its combination with TNF-a appeared to rather enhance the expression of BC1-XL mRNA at 3 h. At 18 h, the TNF-a-induced decrease
322
in BC1-XL mRNA levels was reversed by 1-10 jiM crocin in a concentration-dependent manner. On the other hand, crocin alone, TNF-a alone or their combination had little or no eflfect on Bcl-2 mRNA levels at 3 h (panel B). The Bcl-2 mRNA levels at 18 h were slightly decreased by TNF-a treatment, but significantly increased by the presence of crocin, compared to the control. These results suggest that crocin alone has the ability to increase the mRNA expression of BC1-XL, but not of Bcl-2, in PC-12 cells at an early point (3 h). Bax, Bcl-Xs and LICE are known as pro-apoptotic proteins [26]. Therefore, we next determined the effects of TNF-a and/or crocin on these mRNA expressions in PC-12 cells. Fig. (9) shows that a 3-h treatment with TNF-a increased the mRNA levels of Bax (panel A), BclXs (panel B) and LICE (panel C) in the cells 2.6-, 1.8-, and 1.6-fold, respectively, compared with the control. The Bax mRNA expression in TNF-a-treated cells at 3 h was similar to that in TNF-a plus crocintreated cells. However, TNF-a-induced expression of Bcl-Xs and LICE mRNAs at 3 h was significantly reduced by treatment with 1-10 jiM crocin. Furthermore, the TNF-a-induced enhancement of Bcl-Xs and LICE mRNAs at 18 h was reduced in a concentration-dependent manner by the presence of crocin. At 3 h, crocin selectively modulated the expression of Bcl-Xs and LICE mRNAs. Together with the increase in the anti-apoptotic BC1-XL mRNA, the crocin-induced decrease in the two proapoptotic mRNAs may greatly decrease the subsequent TNF-a death singals. Since the enhancement of caspase-3 activity in TNF-a-treated cells was observed at 6 h (Fig. (9)), the modulation of the mRNA levels by crocin at 3 h may be important in determining the activation of caspase-3 following tiie release of cytochrome c from mitochondria. However, it is not known how crocin changed the balance between the mRNA expressions of anti- and pro-apoptotic Bcl-2 family proteins. The balance between the competing activities of these proteins may greatly affect the maintenance oftiieirdownstream target, mitochondria. Measurement of cytochrome c revealed that TNF-a significantly increased the cytosolic cytochrome c levels in PC-12 cells at 6 h (Fig. (10)). The increase in cytochrome c was suppressed to near the control level by the presence of 1 or 10 |iM crocin. Therefore, our present results show the possibility that crocin has a pharmacological effect that prevents apoptosis of neuronal cells [17].
323
a control BIOMM
crodn
fife "rNF-a (500U) 0TNF+lAiM crocin OTNF+5/xM crodn
3
18
DTNF+IOMM
crocin
TlmeCh)
3
18 Time (h)
Fig.(8). RT-PCR analyses of TNF-a and/or crocin-treated PC-12 cell lysates for the mRNA expression of anti-apoptotic proteins, BC1-XL and Bcl-2. The experimental procedures were those described in Materials and methods. The bands in agarose gel were visualized and photographed under UVradiation.The i^otographs were scanned and quantified by using an NIH Imager. The expression of GAPDH mRNA was used as a control. Each bar represents the mean ± S.D. of three independent experiments. *P<0.05; *• P<0.01; ***P<0.001, compared to the control. #P<0.05; ## P<0.01; ###P<0.001, compared to TNF- a alone.
324
o control "^lO/iMcrocin •TNF-a (500U) OTNF+lMMcrodn OTNF+5MMcrodn aiNF+lO/xMcroclD
3
18 Time(h)
Fig.(9). RT-PCR analyses of TNF-a and/or crocin-treated PC-12 cell lysates for the mRNA exjwession of pro-apoptotic proteins, Bax, Bcl-Xs and LICE. The experimental procedures were those described in Materials and methods. The bands in agarose gel were visualized and photographed under UV radiation. The photographs were scanned and quantified by using an NIH Imager. The exjwession of GAPDH mRNA was used as a control. Each bar represents the mean ± S.D, of three independent experiments. • • P<0.01; ••*P<0.001, compared to the control #P<0.05; ## P<0.01; ###P<0.001, compared to TNF-a alone.
325
* *
66 l - ^ 50
r
5|20
\ 1B ^ n
10 0
i^ 1
TNF(ir) crocin(MM) 1
-
500
—
— —
500 1
* «:
"
m 500 10
Fig.(10). ELISA of cytosolic cytochrome c levels in PC-12 cells, treated for 6 h with TNF-a and/or ciocin. The experimental procedures were those described in Materials and methods. Each bar represents the mean ± S.D. of three independent experiments. ** P<0.01, compared to the control. M P<0.01, compared to TNF-a alone.
DISCUSSION It is well-known that the hippocampus is very important in the learning and memory processes. The appearance of LTP by high-frequency afferent stimulation is suggested to be closely related to the cellular basis of learning and memory [10]. The relation between LTP phenomenon and its inhibition by ethanol using rat hippocampal slices has been reported by several groups [30-32]. An oral administration of crocin had no effect on memory acquisition in normal mice but improved the ethanol-induced impairment of learning behaviors of mice in passive avoidance performance tasks. This phenomenon resembled that of crude extract of saffron as reported previously. The tendencies of the effect between CSE and crocin are similar to each other. From these results it can be easily speculated that crocin is the most important principle in crude extract of saffron. Other crocetin glucoside esters weakly antagonized the blocking effect of ethanol on the LTP compared to crocin. The LTP-suppressing action of ethanol is reduced by the presence of crocin in rat hippocampal slices in in vitro experiment. Crocin alone does not affect the basehne responses or the potentiation induced by weak tetanic stimulation. It becomes evident that crocin attenuates the action of ethanol within the hippocampus supporting in vivo experiments as
326
already documented. Although impairment of ethanol for brain functions including learning and memory is well known, the mechanism is still obscure. Several groups reported the LTP-blocking effect of ethanol using rat hippocampal slices [30-32]. We confirmed that crocin prevents the LTP-suppressing effect of ethanol in the CAl region of rat hippocampal slices [21]. We demonstrated for the first time that crocin selectively antagonizes the inhibitory effect of ethanol on NMDA-receptor-mediated responses in hippocampal neurons. This action of crocin may underlie the antagonism against ethanol-induced memory impairment. Crocin should be useful as a new pharmacological tool for studying the mechanism of ethanol inhibition of NMDA receptor fimctions. We have also demonstrated that crocin prevents TNF-a-induced apoptotic morphological changes and DNA fragmentation of PC-12cells. Moreover, crocin suppressed the TNF-a-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of BC1-XL mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-a-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To confirm how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. The anthracycline antibiotic, daunorubicin, has been proven to have a therapeutic benefit in the treatment of a variety of neoplasia. Recently, daunorubicin was shown to induce apoptosis in cells by the activation of ceramide synthase [33] or of sphingomyelinase (SMase) [34]. Therefore, in contrast to TNF-a signaling through the membrane receptors, daunorubicin is capable of triggering cell death within PC-12 cells. We found that crocin inhibited the effect of daunorubicin as well (data not shown). Taken together, these results indicate that crocin can block PC-12 cell death induced by both outer and inner apoptotic stimuli. Our recent data confirm that crocin has the ability to reduce both TNF-a- and daunorubicin-induced increase in intracellular ceramide levels of PC-12 cells (unpublished data). Daunorubicin can also stimulate the generation of reactive oxygen species (ROS) [35]. In PC-12 cells, the generation of ROS activates neutral SMase to generate ceramide, which, in tum, induces cell death [36]. Glutathione directly inhibits the activation of the SMase [37]. Crocin is reported to have antioxidant properties [39], and it is conceivable that the antioxidant action of crocin contributes to the blocking of PC-12 cell death, induced by TNF-a and/or daimorubicin. In the final process of apoptosis, caspase-3 is activated to induce the activation of deoxyribonuclease. Of the crocetin derivatives (crocin, aooet]bdi(P-I>^uoo6yl)«tei;aood^ tested, crocin was the most potent inhibitor for tike TNF-a-induced
327
activation of caspase-3 in PC-12 cells (unpublished data). Many medicinal plants and their purified components modulate programmed cell death in vitro and in vivo. However, the ability of medicinal plants to induce apoptotic cell death in cancerous cells, but not to prevent apoptosis in normal cells, has been the topic of much research. Our recent studies showed that crocin also had the abilities to inhibit skin tumor promotion in mice [9] and to prevent TNF-a-induced apoptosis in PC-12 cells [17]. The pharmacological action of crocin to prevent both cell proliferation and apoptosis suggests that the two biological processes share a common patitiway at some point, where crocin may act. Apoptotic signaling by TNF-a involves recruitment of specific proteins to the death domain of the p55 TNF-a receptor (TNFRl) including TRADD, TNFKX receptor-associated factors, receptor-interacting protein, leoqAr-iitecactirg ptotanas80ciatedIch-l/CED-3 homologous protein with a death domain, and Fas-associated death domain. The formation of this TNFRl complex leads to tiie activation of a number of signahng intermediates such as cJun N-terminal kinases (JNK), phospholipase A2, sphingomyelinase, NFKB, caspases. Reports have implicated these pathways in both promotion and suppression of apoptosis by TNF-a as well as by other agents including anti-cancer drugs, radiation, and Fas. The magnitude and types of signaling events activated as well as their coordinate interaction with other pro-/anti-apoptotic pathways determine the response of a cell to TNF-a and other inducers. Therefore, the sum of these signals and their cross-regulation to each other determine survival and death outcomes. Phosphatidylinositol 3-kinase (PI3K) is reported to be a critical signaling molecule involved in regulating cell survival and proliferation pa&ways. PI3K can regulate cellular ceramide levels induced by TNF-a, suggesting the existence of cross-talk between the cell survival and death patiiways. It is of particular interest to note that crocin suppresses ceramide generation followed by TNF-a-induced activation of SMase in PC-12 cells. In this review, we confirmed the structure-activity relationship between crocin and other crocetin glucose esters. The elimination of glucose moiety from the crocin molecule greatly decreased its pharmacologic^ activity, while neither gentiobiose nor glucose moiety alone mimicked the effect of intact crocin (data not shown). Therefore, these results clearly suggest that the two gentiobiose moieties attached to crocetin play an important role in the exhibition of the biological activities. This tendency was observed in the studies on the inhibitory effect of crocetin derivatives on two-stage carcinogenesis of mouse skin papillomas [9].
328
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
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SYNTHESIS AND MODIFICATION OF MARCFORTINE AND PARAHERQUAMIDE CLASS OF ANTHELMINTICS B YUNG H. LEE
Preclinical Development, Pharmacia Animal Health, 7000 Portage Road, Kalamazoo, MI 49001, USA ABSTRACT: Three distinct chemical classes for the control of gastrointestinal nematodes are available: benzimidazoles, imidazothiazoles, and macrocyclic lactones. The relentless development of drug resistance has severely limited the usefulness of such drugs and the search for a new class of compounds - preferably with a different mode of action - is an important endeavor. Marcfortine A (1), a metabolite of Penicillium roqueforti, is structurally related to paraherquamide A (2), originally isolated from Penicillium paraherqueL Chemically the two compounds differ only in one ring; in marcfortine A, ring G is six-membered and carries no substituents, while in paraherquamide A, ring G is five-membered with methyl and hydroxyl substituents at C14. Paraherquamide A (2) is superior to marcfortine A as a nematocide. 2-Desoxoparaherquamide A (PNU-141962, 53) has excellent nematocidal activity, a superior safely profile, and is the first semi-synthetic member of this totally new class of nematocides that is a legitimate candidate for development. In this review, total synthesis of paraherquamides was also described. INTRODUCTION
Helminths, especially parasitic nematodes, cause severe health problems in humans and domestic animals. Although several classes of anthelmintics are commercially available, their suboptimal therapeutic spectrum and diminished activity due to the development of drug resistant
332
Strains of parasites [la,b] necessitates the unending search for compounds, particularly for veterinary medicine.
0-4—CH^
MarcfortineA (1)
Paraherquamide A (2)
Fig. (1). Structures of Marcfortine A and Paraherquamide A
The discovery of a new class of anthelmintic having a broad activity spectrum and acceptable safety profile is a rare event. Despite intensive efforts, only three distinct classes of anthelmintics have been commercialized over the past 40 years: the benzimidazoles, the imidazolides and the macrocyclic lactones (MCL) [2]. Pharmacia Animal Health is committed to the discovery of a new class of anthelmintics with a unique mode of action. Marcfortine A (MFA), isolated at Pharmacia from a fermentation broth in the early 90's, was shown to be active against the free-living nematode C elegans in a relatively high throughput screen. MFA (1) is a fungal metabolite of Penicillium roqueforti first reported by Polonsky et al. [3]. Paraherquamide A (2, PHA), a metabolite of Penicillium paraherquei [4], is structurally similar to MFA, the only difference being in ring G, which in PHA is 5-membered with a methyl and a hydroxyl substituent at Cu, while in MFA ring G is six membered and has no substituents. The antiparasitic activity of both MFA and PHA has been previously described by workers at Merck [5]. MODIFICATION OF MFA AND PHA Synthesis of Paraherquamides from MFA PHA is a more potent nematocide than MFA, against some species, based on a literature review. Our attempts to improve the potency of
333
MFA by simple chemical modifications, such as N-1 derivatization, oxidation, reduction etc., were not successful.
Br~A \
ON \ HgC CHo
)
VN
(70-98 %;
QH3 I f^^^^'^ CH3
-NH
CH, 1
(MFA)
1. PhSe-SePh, NaBH4 (70-90%) 2. Nal04, Heat (80%)
.
CH3
CN
H3C CH3 ,.^V°-y^^3^J;jaOH_ >
CH, Y "^
\^'\^^'f\}^^'
(50%
I.OSO4/NMO 2. Nal04 3. NaBH4
^ (50%)
6
(PHB)
Less active than MFA Fig. (2). Conversion of MFA to PHB
As mentioned earlier, the only structural difference between PHA and MFA resides in ring G, and therefore our analog program centered on this ring. An added reason was that the envisaged synthetic pathway could also provide PHA, a compound not in our possession at the time, and essential for comparative assays. Removal of the methyl and hydroxyl groups at C14 in PHA would yield paraherquamide B (PHB), a compound identical to MFA except for the size of ring G (PHB five versus MFA six). The planned synthetic sequence required the opening of ring G and an
334
oxidative removal of one carbon atom, to be followed by ring closure as shown in Fig. (2). Treatment of MFA (1) with cyanogen bromide [6] opened ring G to yield the bromo derivative 3 [7]. Attempts to dehydrobrominate 3 in one step via a base-catalyzed elimination with DBU/CH3CN, KOH/MeOH, or r^rr-BuOK/DMSO were unsuccessful. However, the required methylene entity could be introduced by converting 3 first to a selenide, then oxidation with periodate, followed by thermolysis in benzene to provide compound 4. Hydrolysis of the cyano group with NaOH in ethylene glycol [8] produced 5 (50% yield). Osmium catalyzed oxidation of 5 in the presence of 4-methylmorpholine A^-oxide (NMO) gave a diol, which was cleaved to an aldehyde upon treatment with periodate. Treatment of the aldehyde with sodium cyanoborohydride resulted in an intramolecular reductive amination to yield the desired product PHB (6). The seven step conversion of PHB to PHA is shown in Fig. (3). Oxidation of PHB (6) in THF/H2O with iodine in the presence of bicarbonate [9] gave 16-oxo-paraherquamide B (7, 40%). Treatment of 7 with LDA and phenylselenyl chloride followed by oxidation of the resulting selenide with H2O2 gave the a,p-unsaturated lactam 8. Attempted epoxidation of 8 with H202/NaOH, m-CPBA, isovaleraldehydeA^O(acac)2, or n-BuLi/H202 failed to give the epoxide 9. However, when 8 in THF was treated with r^rf-butylperoxide [10] in the presence of triton B, the epoxide 9 (58%) was obtained. It was assumed that attack on the double bond by peroxide would occur from the least hindered side to yield an a-epoxide; proof was obtained from the stereochemistry of alcohol 11 (vide infra). In any case, the stereochemistry of these chiral centers is of little consequence, since they are removed at a later stage of the synthesis. While standard methods utilizing NaBHj, BH3-THF, or superhydride failed to open the epoxide ring, samarium iodide [11] provided the required 14a-hydroxy compound 10 in a good yield (85%). Although this single electron transfer reagent has been shown to cleave epoxy-ketones, to our knowledge this is the first instance of its application to amides. Reduction of 10 with LAH/AICI3 gave 11 in a modest yield (24%). The stereochemistry of 11 was established by comparison of its ^H NMR spectrum with that reported by Blizzard et al. in their synthesis of 11 by an altemative synthetic route [5j]. Swem oxidation of 11 produced ketone 12 (71%). Reaction of 12 with methyl magnesium bromide in THF gave PHA in a 50% yield (based on recovered starting material) with the
335
formation of only a trace of the related a-methyl epimer. Semisynthetic PHA proved to be identical by TLC, ^H NMR and HRMS to the natural product kindly provided by Professor Yamazaki. We were able to then confirm that the nematocidal activity of PHA is superior to MFA, which in turn is superior to that of PHB.
1.LDA/PhSeCI
,
2. H2O2 (58%) 6
(PHB)
Y-
OH
triton B (58%)
LAH/AICI3 (24%)
Swern Oxidation
MeiVlgBr 1
•
HO
(50%)
(71%)
CH3
Hr,CCH,,^v04-^H3 2 H3C fif °
\ Q CH3'-'
Fig. (3). Conversion of PHB to PHA
(PHA)
More active than l\^FA
336
Hydroxylation of MFA at C14, C15, C16 via a Novel Cyanogen Iodide Reaction While the reaction of MFA with cyanogen bromide (BrCN) in refluxing chloroform caused ring fission to yield 3 [Fig. (4)], under the same conditions cyanogen iodide did not provide the iodo analog 13. CH^
J
14B reflux 1 h
Fig. (4). Reaction of MFA with CNI
15A
15B
Cyanogen Iodide (ICN) has been used extensively for the cyanation of alkenes and aromatic compounds [12], iodination of aromatic compounds [13], formation of disulfide bonds in peptides [14], conversion of dithioacetals to cyanothioacetals [15], formation of rran^-olefins from dialkylvinylboranes [16], lactonization of alkene esters [17], formation of guanidines [18], lactamization [19], formation of a-thioethter nitriles [20], iodocyanation of alkenes [21], conversion of alkynes to alkyl-iodo alkenes [22], cyanation/iodination of p-diketones [23], and formation of alkynyl iodides [24]. The products obtained from the reaction of ICN with MFA in refluxing chloroform were fran^-16-iodo-17-cyanomarcfortine A (14)
337
and 17-cyanomarcfoitine A (15) in 90% and 10% yields, respectively [25]. The likely mechanism of the formation of these products is shown in Fig. (5). CH3 HoC C H o x ; ^ ^ 0 4 - - C K
Fig. (5). A plausible mechanism of the reaction of MFA with CNI
The iminium ion intemiediate 16 is generated by a free radical oxidation of 1 and is in equilibrium with the enamine intermediate 17; cyanide ion addition to 16 gives a low yield of compound 15 while the favored trans addition provides the main product 14. Although generation of an iminium intermediate with chlorine dioxide [26] or bromine [27] has been reported, these reagents did not produce compounds such as 14, suggesting that equilibration with the enamine did not occur. The Polonovski-Potier reaction applied to aspidospermane generated an enamine intermediate, which upon treatment with CNBr gave a product similar to 14 in three steps [28]. The trans adduct 14 upon treatment with 45% aqueous KOH in MeOH for 3 h at room temperature gave the 16,17-dehydro derivative 18 in 90% yield. The utility of this novel cyanogen iodide reaction was further demonstrated on MFA by affording a procedure for the regiospecific introduction of a single hydroxyl group at C14, C15, and C16. These analogs were required to determine the significance of the hydroxyl group
338
on anthelmintic activity. Compound 18 thus became the common intermediate for the synthesis of 14a-hydroxymarcfortine A (23), 15ahydroxymarcfortine A (27), and 16a-hydroxymarcfortine A (29).
I.LDA/PhSeCI 2. HgOg/NaOH (65%)
More active than MFA
Fig. (6). Synthesis of 14a-hydroxymarcfortine A from 18
Preparation of 23 from 18 was achieved in five steps [Fig. (6)]. Hydrolysis of 18 with a catalytic amount of p-toluenesulfonic acid in 95% methanol at room temperature for 1 h gave 19 (90%). The C15-C16 double bond was then introduced by selenation [Lithium diisoproylamide (LDA) and phenylselenenyl chloride] at C16 followed by hydrogen peroxide oxidation and subsequent elimination of phenylselenenic acid by an aqueous alkaline workup ( I N NaOH) to give the C15-C16 dehydro analog 20 (65%). Compound 20 underwent allylic oxidation with Se02in refluxing dioxane (1.3 equiv, 1 h) to provide in a modest yield (35%) the desired a-hydroxyl derivative 21. Reduction of the double bond with lithium triethylborohydride (7 equiv/THF, 0 ""C, 0.5 h) gave 22 (86%). The regiospecific reduction of the C17 amide in 22 was achieved by treatment with BH3-DMS (10 equiv/THF) to provide 14ahydroxymarcfortine A (23, 75%). The couphng constant between the C14 hydrogen and the CI5 hydrogen is 2 Hz, indicating the hydroxyl group is axial.
339
3
1.LDA
18
Inactive
18 NMO (80%) inactive
Fig. (7). Synthesis of 15 a- and 16a-hydroxyniarcfortine A from 18
The preparation of 15a-hydroxymarcfortine A (27) and 16ahydroxymarcfortine A (29) is shown in Fig. (7). Treatment of 18 with LDA (4 equiv/THF, -78 ""C) followed by 1.5 equiv of Davis's reagent 24 [(2-benzenesulfonyl)-3-phenyloxaziridine] [29] gave the desired C15hydroxlated material 25 (y-hydroxylation, 30%) along with the 17-oxo derivative 19 (a-hydroxylation, 10%). ^H NMR of 25 indicated a single stereoisomer in which the C15 hydrogen has two pseudoaxial and two pseudoequatorial couplings. Reaction of 25 with 2.6 equiv of Se02 in aqueous ethanol at ambient temperature for 16 h furnished 26 (50%), which was reduced with LAH (2.75 equiv/THF, 0 ""C, 0.5 h) to yield 27 (25%). Treatment of 18 with a catalytic amount of OSO4 and 4.2 equiv of NMO gave 28 (80%); the carbonyl at C17 was reduced with BH3-DMS (6
340
equiv/THF, 0 ""C, 1 h) to yield 16a-hydroxy-MFA 29 (60% yield based on recovered starting material). While compounds 27 and 29 lack nematocidal activity, we found 23 to be more active than MFA. Synthesis of 14p-hydroxy-MFA (31) and 14p-methyl-14a-hydroxyMFA (32)
CH^ HoC CHq
^O-^UcHg /)
Swern Oxidation
CHg^
30
MeMgBr NaBH. CH^ HX
32
as active as PHA
^-.^04-CH3
31 (50%) + 23 (3%)
Fig. (8). Synthesis of 14P-hydroxy-MFA and 32
Because of the enhanced biological activity of 14a-hydroxy-MFA (23), the synthesis of its antipode was carried out as shown in Fig. (8). Swern oxidation (oxalyl chloride, DMSO, NEts, -78 ''C, 1 h) of 23 provided 14-oxo-MFA (30, 74%). Reduction of 30 with NaBHU (6 equiv/THF, 0 ''C, 1 h) gave M^-hydroxy-MFA 31, 50%) and 14ahydroxy-MFA (23, 3%). Compound 31 was inactive, thus demonstrating the need for correct stereochemistry of the hydroxyl group at C14. To assess the effect of the C14 methyl group on biological activity, 30 was reacted with methylmagnesium bromide to yield 32 (50% based on recovered starting material), the six membered homologue of PHA. The
341
related a-methyl epimer was present only in trace amounts. Compound 32 is the first MFA analog with nematocidal activity comparable to that of PHA. An Improved and Practical Synthesis of 14a-hydroxy-MFA (23). SPh
1.LDA 2. PhS-SPh
\
(60%)
4 V-N
33
CHo
1.,x;s^COOOMg
CI,
"COQ-
(76%)
2. heat
COOOMg f^'^o
23 (70 g)
Fig. (9). An Improved and Practical Synthesis of 14a-hydroxy-MFA
To provide additional material for clinical trials, and to eliminate the use of hazardous reagents such as ICN, PhSeCl, and Se02, an improved synthesis of 23 was devised [Fig. (9)]. Treatment of MFA 1 with sodium bicarbonate and iodine [30] in refluxing aqueous THF produced 19 in one step (76%, 350 g scale), eliminating the use of ICN and Se02. Compound 19 was reacted with LDA and phenyl disulfide to give 33 (65%). Oxidation with the magnesium salt of perphthalic acid followed by refluxing in toluene provided 34 (76%). Repeated reaction with the magnesium salt of perphthalic acid and diethylamine gave the rearranged product 22 (61%) [31]. The carbonyl group at C17 in compound 22 was reduced with BH3DMS (10 equiv/THF) to provide 14a-hydroxy-MFA 23 (70 g, 50%). The stereospecific rearrangement of 34 was previously described [32].
342
Synthesis of 15a-Methyl-14a-Hydroxy-MFA (36) and ISp-Methyl14a-Hydroxy-MFA (41) Due to the enhanced nematocidal activity observed following the introduction of a methyl group at C14 [32, Fig. (8)], its effect on C15 was next investigated.
Me2CuLI (60 %)
11 Q
BH3-DMS (50 %)
HoC--<^N—\X HO CH, 36
very active
1.LDA
35
2. MeSOgSMe (37) (57 %)
BHq-DMS (33 %)
40
Fig. (10). Synthesis of 15a-MethyM4a-Hydroxy-MFA (36) and 15p-MethyM4a-Hydroxy-MFA (41)
Conjugate addition of lithium dimethylcopper to 21 [33] gave 35 in 60% yield [Fig. (10)]. The cis addition of the methyl group is likely due to the bulky copper-oxygen intermediate [34]. Subsequent treatment of 35 in THF with the BH3-DMS complex gave compound 36 (50%) which, in our laboratory, had good activity against Haemonchus contortus and Trichostrongylus colubriformis. Compound 41 (the epimer of 36) was
343
prepared from 35 in four steps [35]. Sufenylation of 35 with methyl methanethiol sulfonate 37 and LDA gave 38 in 57% yield. Oxidation with m-CPBA in methylene chloride at -78 °C gave a sulfone intermediate which on heating under reflux in toluene/ethylene chloride yielded 39 (70%) with elimination of methanesulfonic acid. Reduction with lithium triethyl borohydride gave 40 in a 60% yield. A second reduction with the BH3-DMS complex provided the desired 41 in a 45% yield.
>/
9-BBN NaOH/HgOg
1.MsCI/NEt3 2. DBN (70%)
45
Inactive
Fig. (11). Synthesis of 45, a C14,15-fused ring analog.
While compound 36 demonstrated excellent nematocidal activity, the epimer 41 at the same concentration was totally inactive. Perhaps the conformation of the G-ring relative to the hydroxyl group is more important than the chirality of the methyl group at CI5. To further
344
investigate this hypothesis, compound 45, having a C14-C15 fused ring, was synthesized. In addition, 45 could also define the importance of hydrogen bonding at C14 [Fig. (11)]. Synthesis of 45 , a C14,15-Fused Ring Analog Compound 21 [Fig. (11)] was reacted with vinyl magnesium bromide and copper iodide in THF at 0 ^C to give the 1,4 addition product 42 in 48% yield [35]. Hydroboration with 9-BBN followed by NaOH and H2O2 workup provided a mixture of the desired alcohol 43 (20%), recovered starting material 42 (20%), and the C17 reduced alcohol 44 (5%). Further reduction of 43 with the BH3-DMS complex gave 44 in 35% yield. The two combined samples of 44 were mesylated (CH3S02Cl/NEt3) and then treated with excess DBN to give the furan-containing MFA analog 45 in 70% yield. Compound 45 is inactive, thus demonstrating that hydrogen bonding of the C14 hydroxyl group is indeed important for anthelmintic activity. The significance of the geometry of ring G on anthelmintic activity was next investigated. Fission of ring-G: The Synthesis of Analog 48 CH,
O4-CH3 // QJJ ^^" O'
C;H3
COCI2 Pyridine /toluene
U ^
J
O H^C-
bH O
CH, LiBEtgH
HaCs^aC^ H3C CH3 ^^^YO-lr-CHa
HO-4—(•-v4J> Inactive
Fig. (12). Synthesis of 48.
-NH 48
^^^^
H3C
NaBHgCN H O 80%
H,
46 40%
H H3C CH3 ^?v^o4-CH3
345
Compound 2 [Fig. (12)] in pyridine was treated with phosgene in toluene [5j] to give the carbamate 46, which on reduction with lithium triethyl borohydride gave 47 in a 40% yield. Subsequently, compound 47 was treated with formaldehyde and sodium cyanoborohydride to yield 48 (80%). Compound 48 is biologically inactive, and in our computer model (minimum energy conformation) it resembles 14p-hydroxy-MFA 31, which is also inactive. It can be concluded that the naturally occurring conformation of ring G is essential for biological activity. Economic Viability The biological profile of compounds 32 and 36 points towards their use in large food animals. The cost of these compounds, however, is prohibitive if synthesized by the procedures outlined in Fig. (8) and Fig. (9). Therefore, an alternative scheme had to be developed, as outlined in Fig. (13). The envisaged route required the selective introduction of the crucial 14-hydroxyl group into MFA via a biotransformation procedure. The resulting 23 would then be a common intermediate for the synthesis of 32 (two chemical steps) and 36 (three chemical steps).
BJotransformation Primary Fermentation
- • IVIFA
. m Q^ VN/ O
Three Chemical Steps
23 Tvw) Chemical Steps
HoC
36 Fig. (13). Economic viability
32
CH,
346
Microbial Hydroxylation (Biotransformation) at Eight Individual Carbon Atoms of MFA Many reviews and hundreds of papers have been published on the use of mono-oxygenases for the introduction of oxygen atoms onto various substrates [36]. We were especially interested in nonactivated stereospecific carbon atom hydroxylation. Therefore, a large number of biotransformation experiments were carried out utilizing cultures reported in the literature, and random samples from the Pharmacia culture collection. Screening was performed by adding a solution of 1 (10 mg) [37] in DMF (0.4 mL) to vigorously growing culture fermentations in 500 mL wide-mouth flasks. Incubation was conducted at 28 °C, and shaking was continued for 1-3 days depending on the culture. The mixture was thoroughly extracted with chloroform, centrifuged, and the solvent removed at 35 °C under reduced pressure. The residue was analyzed by TLC (three solvent systems) and HPLC. Promising samples were scaled up 10- to 100-fold and refermented in a Labraferm fermentation tank. Purification was achieved by various chromatographic techniques, and the structure determined with the aid of NMR and MS. Whenever semisynthetic samples were available, a side by side comparison was also performed.
27
CH3
l>^0-4-CH.
o I to N-Demethylatlon/^ ^ " s
Arrows indicate position of hydroxylation Fig. (14). Biotransformation products
The great majority of cultures either totally metabolized 1 or left it unchanged. However, a few cultures did provide hyroxylated products.
347
Extensive efforts were undertaken to increase the yields of biotransformation products by changing the media, temperature, time of fermentation, etc. The highest yields obtained were for compounds 23, 27 and 29, which on scale-up gave a 10-15% yield, in addition to recovered starting material (30%). In summary, utilizing biotransformation techniques, eight out of the 28 carbon atoms in MFA were successfully hydroxylated. In Fig. (14), arrows indicate the sites of hydroxylation. It is noteworthy that despite the hindered nature of C14, cultures UC 5059, UC 11141, and UC 11144 were able to introduce a hydroxyl to give 23 in a 10-15% yield. Discovery of 2-Desoxo-15a-Methyl-14a-Hydroxy-MFA (49) Scale up studies of a second potential candidate yielded the desired 36 and a small amount of a less polar compound, characterized as the 2desoxo derivative 49 [Fig. (15)]. The excellent nematocidal activity of 49 encouraged us to attempt the selective reduction of the C-ring carbonyl in MFA (1), to yield 52. Reduction of 1 with three or ten equivalents of LAH yielded 50 (carbonyl reduced in ring F) and 51 (carbonyls reduced in rings C and F) respectively, both lacking nematocidal activity; we were unable to synthesize 52. Discovery of 2-Desoxo-PHA (PNU-141962,53) To follow up the excellent nematocidal activity of 49, PHA was selectively reduced with the alane-NMe2Et complex to furnish 53, albeit in only a 5% yield [Fig. (16)]. Attempts to improve the yield of this one step procedure with various reducing agents, such as LAH, LAH/AICI3, NaBEU/Acetic acid, NaBIVCFsCOOH, Red-Al, Super-hydride, Li-9-BBN-hydride, BH3-THF, Li-tri-r-butoxyaluminum hydride and LiBHU, were unsatisfactory. The highest yield obtained was with LiBEU (10-20%). A lengthier but higher yielding synthesis of 53 was developed (4 steps 60-70%) by using our previously described process [38]. Compound 2 was reacted with 9fluorenylmethyl chloroformate (Fmoc-Cl, 1.5 equiv) in the presence of NaH (3 equiv) at 0 °C to give a quantitative yield of 54 [39]. Reduction of 54 with NaBHU in MeOH at 0 °C gave 55, which was deprotected with piperidine in THF to give the imine intermediate 56.
348
HO
;HN '/>^NH
35 (20 grams)
^"3
BH3-DMS CH3
CH3
04-CHo
O-4-CH3
oi + 49 (10 miligrams) as active as 36
36 (10 grams) CH3
04-CH, -//LAH(3or10equiv)
0
CK 52
(3 equiv)
O-UCH,
5Q
Inactive
51
Inactive
Fig. (15). Synthesis of 49,50, and 51
This was further reduced with NaBKU in MeOH at 0 °C to give 53. Compound 53 displayed excellent activity in our jird and sheep models. Indeed, in our hands, this compound was two to four times more potent in sheep than the parent compound (PHA) against the important
349
gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus colubriformis.
^
O
CH 53
PNU-141962
CHo
CH3
0-4-CHo
O-V-CH3
K)—>"
J, IN-Fmoc
H3C Jl'X ^
CHq
^'^
NaBH. ^
Overall yield 60-70%
Hqi 3
O
CH3 OH
^
CH,
56
Fig. (16). Discovery of 2-Desoxo-PHA (PNU-141962,53)
It should be noted that workers at Merck found PHA to be considerably more potent than we did [40a], a difference which may be due to the use of different vehicles. While this was an exciting development, we were concerned about the toxicity of this PHA analog, because Merck workers reported [40b] that PHA is quite toxic to mice, with an estimated LD50 of < 15 mg/kg. In dogs, the toxicity is even greater, with death seen at doses as small as 0.5 mg/kg [40b], reducing chances for commercialization. Interestingly, PHA is relatively safe in jirds, sheep and rats. Structure activity relationship studies performed by Merck and Pfizer workers did not yield analogs with lower toxicity. In contrast, compound 53 was not toxic to mice at doses up to 50 mg/kg. Furthermore, dogs treated with 53 at 20 mg/kg experienced no toxic effects besides mild and reversible
350
mydriasis. The exceptional improvement in selective biological activity due to the removal of a single oxygen atom in ring C is noteworthy indeed. In conclusion, rational drug design led to the synthesis of two highly active compounds, 32 and 36. Scale up studies of 36 yielded a minor product, the 2-desoxo-MFA derivative 49, which in turn led to the discovery of 2-desoxo-PHA (PNU-141962), a compound that is currently under development. Efficacy of MFA, PHA and PNU-141962 Drug evaluations were conducted in Mongolian gerbils (jirds) concurrently infected with the gastrointestinal nematodes H, contortus and T, colubriformiSr or monospecifically infected with O. ostertagU using established techniques [41a,b]. This model permits assessment of the activity of experimental compounds directly against target parasites in vivo, using very little drug. Treatments were given on day 10 or day 6 post-inoculation in the K contortus and T. colubriformis or O. ostertagi models, respectively. Table 1. Efficacies of MFA, PHA and PNU-141962 against gastrointestinal nematodes in experimentally infected jirds following oral dosing.
95% Effective Dose (mg/jird)
Compound H. contortus
T. colubriformis
O. ostertagi
MFA
0.33
0.11
4.0
PHA
0.33
0.11
0.5
PNU-141962
0.33
0.11
1.0
Drugs were administered orally in 0.2 ml vehicle (17% DMSO: 83% vehicle #98). Animals were examined for worm burdens on day 13 {H. contortus and T. colubriformis) or day 8 (O. ostertagi) post-inoculation (3 days post-treatment). Approximate ED95 values for MFA, PHA and PNU-141962 against 3 nematode species in jirds are shown in Table 1. Against H, contortus, the approximate ED95 for MFA, PHA and PNU-141962 was 0.33 mg/jird
351
(roughly 10 mg/kg). These 3 compounds were also approximately equipotent against T. colubriformis in the jird model, with an EDc^^ of 0.11 mg/jird. Against O. ostertagU the approximate ED95 for each compound was higher than that observed for the other nematodes. Against this species, the ED95 values for PHA and PNU-141962 were 0.5 and 1.0 mg/jird, respectively; these values were 4- to 8-fold lower than those for MFA (-4 mg/jird).
Table 2. Efficacy of Marcfortines, PHA and PNU-141962 in sheep experimentally infected with H. contortus following oral dosing of conqiounds in 60:40 propyleneglycol/glycerol formal vehicle.
95 % Effective Dose (mg/kg) H. contortus Compound
MFA
12.5
23
7
32
2
PHA
1-2
PNU-141962
0.5-1
36
4-5
,
The compounds were also tested in sheep experimentally infected with Haemonchus contortus. The treatments were given orally in propylene glycol/glycerol formal (60:40 v:v) vehicle on day 35 post-inoculation. Animals were necropsied 7 days post-treatment and examined for worm burdens. Approximate ED95 for the marcfortine compounds, PHA and PNU-141962 are shown in Table 2. Against K contortus, the approximate 95% effective dose for PNU-141962 was 0.5-1.0 mg/kg; ED95 for the marcfortines ranged from 1-12.5 mg/kg and for PHA 1-2 mg/kg. It should be noted that formulation has not been optimized for ruminants, and subsequent preliminary pharmacokinetic studies have shown that the bioavailability of PNU-141962 in sheep is low (10-15%) in the vehicle used, when dosed orally or subcutaneously (unpublished observations).
352
Mode of Action of PNU-141962, MFA and PHA Similarities in structure and anthelmintic spectra of the paraherquamides and marcfortines suggest that these compounds share an anthelmintic mechanism. This concept is supported by observations that PHA and PNU-141962 displaced [^H]marcfortine A in competition binding assays using membranes prepared from adult H, contortus; competition was also observed in binding assays using membranes prepared from the free-living nematode Panagrellus redivivus and [^H]PNU-141962 as ligand (our unpublished observations). PHA, PNU-141962, and related compounds rapidly induce flaccid paralysis of parasitic nematodes in vitro, without affecting ATP levels [42]. While the mechanism of action of these new anthelmintic agents was initially poorly understood, they appear to share a binding site with phenothiazines in membranes prepared from C elegans [43]. Recent observations in insects suggest that PHA binds to invertebrate nicotinic acetylcholine receptors (nAChR) and is an antagonist of acetylcholine (ACh) at these receptors [44]. To determine if a similar mechanism of action is found for these compounds in nematodes, we investigated the mechanism of action of this anthelmintic class using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum [45a]. Our findings can be summarized as follows. None of the compounds significantly altered A. suum muscle tension or membrane potential when given alone. However, paraherquamides blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by ACh and nicotinic agonists, including the anthelmintics levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic action of PNU-141962 and related paraherquamides and marcfortines is due to blockade of cholinergic neuromuscular transmission. To further test that concept, we examined the effects of these compounds on three subtypes of human nAChR. In these studies, a Ca^"^ flux assay was used to measure the function of nAChR expressed in cultured mammalian cells. PNU-141962 blocked nicotinic stimulation of cells expressing a3 ganglionic (IC50 = 6 \\M) and muscle-type (IC50 = 3 |LIM) receptors, but was inactive at 100 |xM vs the a7 CNS subtype. This compound also paralyzed the parasite H. contortus in culture with an IC50 value of approx. 0.1 uM, thus demonstrating the basis for host vs. parasite selectivity. It is noteworthy that PHA is more effective in blocking mammalian nAChR than is PNU141962, perhaps explaining the greater mammalian toxicity observed with the prototype drug.
353
Isotopic Labeling of PNU-141962 (53) with Deuterium [45b]
2. NaBHaCN (preferred)
(500/^
Fig. (17). Isotopic Ubeling of PNU-141962 (53) with Deuterium
Modifications of MFA and PHA led to the discovery of 2desoxoparaherquamide A (PNU-141962, 53) which is as active as PHA and has an improved safety profile. In order to do preclinical studies, we
354
wished to synthesize radio-labeled PNU-141962. In this reason, we prepared [CD3]-2-desoxoparaherquamide A (62). Although the synthesis of [C24"^H]-PHA has been reported [45c], the labile nature of position-24 with respect to acid hydrolysis rendered such labeling unsuitable for our preclinical studies. Using a deuterium labeled reagent, we have developed a synthetic strategy that is suitable for the introduction of '"C and ^H into the 14-methyl group of PNU-141962 through the appropriate choice of labeling in the reagent [Fig. (17)]. The dehydration of PNU-141962 to exo-olefin 57 through the use of DAST was readily accomplished. A method has been reported for the conversion of an exo-olefin derivative of PHA to its ketone by sequential reactions involving bromination, ozonolysis and debromination with zinc [5j]. However, these procedures were long, low yielding and, worse, the benzene ring of PHA was also brominated. In our hands, glycolation of the exo-olefin with osmium tetroxide followed by oxidation of the glycol to the ketone with sodium periodate proved more suitable to our purposes. Earlier, we reported a method for the stereospecific addition of MeMgl to 14-oxoparaherquamide B [7]. Using this methodology we successfully methylated the carbonyl at position-14 of ketones 60 and 61 in a highly stereoselective manner. Treatment of 53 with DAST [(diethylamino)sulfur trifluoride] in methylene chloride provided 57 in 50% yield. Compound 57 was treated with osmium tetroxide at 5 °C in the presence of NMO (4methylmopholine iV-oxide) for 18 h to provide 58 and 59, which were separated by silica-gel chromatography. Compounds 58 and 59 were treated with sodium periodate at 5 °C for 18 h to provide 60 and 61 respectively. Compounds 60 and 61 were treated with CDsMgl followed by NaBHsCN to give 62 in 20% and 50% yields, respectively. In conclusion, isotopic labeling of 2-desoxoparaherquamide A (PNU141962) with deuterium was achieved from PNU-141962 in four steps in anticipation of using its method of synthesis for the preparation of the corresponding ^^C and ^H labeled products.
Semi-Synthesis of 3-Epi-paraherquamide A (65) To investigate the significance of the chirality of the C3 position on anthelmintic activity, we prepared 3-epi-PHA [65, Fig. (18)].
355
CH« 0-4-CH3
H3C CH3 ^
= \=. C o^ y" 3 CHo
^ 0 bH,
O
CH,
63
56 f-BuOCI NEtg CH2CI2,0 °C
HgCf^a
rt 16h
-3 :T CH3
AcOH
o '^=\P^3 ^y
^-kteCXJ "°-. V-v^, H3C Jt-^^
65
0 CH3
CI 64
Fig. (18). Semi-Synthesis of 3-Epi-paraherquamide A (65)
Compound 56 [39] was heated under refluxing in xylene to give rearranged product 63 in good yield. Compound 63 was subjected to conditions described by Williams [46] to provide 65 in low yield. Compound 65 did not show ant anthelmintic activity . €26 Dialkyl and Spiroalkyl Analogs of MFA
HCOOH MFA
•
Fig. (19). Outlined synthesis of 67
356
To investigate the effect of changes at the C26 position of MFA on anthelmintic activity, several of C26-dialkyl and spiroalkyl analogs were prepared [47]. The analogs were synthesized in four steps starting with cathecol 66 [Fig. (19)], which was prepared from MFA in 80% yield by stirring in formic acid for 16 h.
Br
"V
\—'——^
R
K2CO3 Kl
R
66
m-CPBA
68a: R, R == cyclobutyl 68b: R, R == cyclohexyl 68c: R, R == diethyl 68d: R. R =: ethyl-methyl 68e: R, R =: dimethyl
C[
^O-^
r\
R
SnCU
•
MTPI
Fig. (20). Synthesis of 67
The general route outlined below, follows the modified method of Williams [48] used his preparation of the g^m-dimethyl dioxepin ring of PHB. By this method we were able to prepare the four dioxepin-ring anaologs in which the geminal methyl groups at C26 of MFA were replaced by a cyclobutyl 67a, cyclohexyl 67b, diethyl 67c, and ethylmethyl 67d groups [Fig. (20)].
357
The catechol 66 was coupled with the appropriate bromo reagent 68a-d in the presence of K2CO3 and KI in acetone/water to give mono-alkylated products 69a-d in 29-82% yield.Epoxidation with m-CPBA in CH2CI2 followed by workup with sodium bisulfite [49] (to remove the N-oxide) gave epoxides 70a-d in 40-100% yield. Ring closure using SnCUin THF provides alcohols 71a-d (50-85% yield) which were dehydrated with methyltriphenoxyphosphonium iodide (MTPI) in THF/DMF to provided the final products 67a-d in 20-30% yield. To verify the regiochemistry of the alkylation described above we reduced MFA with borane-methyl sulfide complex to provide 72 in 40% yield [Fig. (21)]. This compound was identical to the one prepared from the catechol 66 and 4-bromo-2-methyl-2-butene (73) using the chemistry reported in step 1 of Fig. (20), thereby conforming the assigned regiochemistry of compounds 68a-d. The biological activity of compounds 67a-d was evaluated in our standard anthelmintic assay which uses immunosuppressed Mongolian gerbils inoculated with Haemonchus contortus and Trichostrongylus colubriformis [41]. The compounds were administrated orally at a dosage rate of 0.33 mg/gerbil. Unlike MFA, none of these compounds gave the 95% clearance of helminthes we use as a criteria for determining activity, and were deemed inactive.
MFA
BH3-DMS /—\
•(
^aS. P^;
N-^K^ Ji^jJ^
I ^
^
K2CO3/KI
66
Acetone/HgO
Fig. (21). Reaction of MFA with BH3-DMS
C24 and C25 Substituted MFA Derivatives To investigate the effect on anthelmintic activity by changing the substituent pattern at C24/C25, we synthesized a number of analogs [Fig. (22)] [50].
358
Swem oxidation of 71e provided the ketone 74, which was subjected to Wittig olefination with methyltriphenylphosphonium bromide and n-BuLi to give the exocyclic methylene containing compound 75 in 50% yield. The versatile ketone 74 was also epoxidized with trimethylsulfoxium iodide and potassium r-butoxide in DMSO to give 76 in 35% yield, whereas Grignard chemistry (MeMgBr) gave a quantitative yield of 77. Treatment of 77 with DAST afforded a 35% yield of the desired analog 78.
V-OH
^
Fig. (22). C24 and C25 Substituted MFA Derivatives
We reasoned apriori that both compounds 75 and 78 would have anthelmintic activity since they contained the crucial C26 dimehtyl moiety and likewise retained the proper geometry of the A ring found in the parent compound MFA based on modeling studies. Furthermore, these structures should be less hydrolyzed under acidic conditions.
359
With the exception of compound 75 none of these compounds were active. Since the exocyclic methylene analog 75 had activity we chose to prepare a simplified analog [Fig. (23)]. Thus, the catechol 66 was reacted with 79, K2CO3 and Nal in DMF for 16 h to give the exocyclic methylene compound 80 which lacked the C26 dimethyls. Compound 80 was inactive, which further emphasizes the importance of C26 dimethyls. KgCOg/Nal CI 79
^'
Fig. (23). Synthesis of 80
STEREOCONTROLLED TOTAL SYNTHESIS OF (+)-PHB As part of on going efforts of Williams and his coworkers [46] to elucidate the biosynthesis of the core bicyclo[2.2.2] ring system of the related alkaloids the brevianamides [51], they have applied methodology originally developed for the stereocontrolled total synthesis of (-)brevianamide B [52] to complete the first stereocontrolled total synthesis of(+)-PHB. Retrosynthetic analysis As outlined in Fig. (24), a convergent synthesis of the enantiomer of the natural PHB was envisioned to contain four key carbon-carbon bondforming reactions. The first task would involve the construction of a suitably a-alkylated proline derivative [52]. The second important coupling would be the Somei/Kaetani-type alkylation [53] of the suitably protected gramine derivative 86 and the requisite piperazinedione 85. The third and most crucial C-C bond-forming reaction was a stereofacially controlled intramolecular SN2' cyclization reaction and concomitantly installs the isopropenyl group that will be utilized in the fourth C-C bond-forming reaction. Standard procedures to effect this transformation involve strong protic acids [52], and there was reason for concern about the reactivity of
360
the more highly oxygenated indole 83 as a practical synthetic precursor to 82.
^i"
84
(R)N MeOgC—'N<^^^
Y
86
85
O
Fig. (24) A conversion synthesis of (+)-PHB
Construction of the Dioxepinooxindole Ring System The known pyruvic acid 87 [Fig. (25)] [54] was oxidatively decarboxylated [55] to afford the phenylacetic acid 88, which was reductively cyclized to give the required oxindole 89 in nearly quantitative yield.
361
OH
OH
NaOH H2O2
(81-93%)
Ho Pd/C
HO OMe
NO2
O HO
(92%)
pren^ bromide^
OH
OR
(52%)
90
91
r^
NaBH.
^
BFgOEtg (46%)
f ^ T V o
OMe 89
88
f y y , ^
^-^ (99%)
^•"^"^^^^. (640/^)
R = prenyl
f
V
V
o
/ y ^ ^ HO
gj
1. protection 2. Mannich
HO
RO
93
94 R = f-BuMe2Si
Fig. (25). Construction of the Dioxepinooxindole Ring System
Oxindole 89 was cleanly demethylated upon treatment with boron tribromide. The resulting oxindole 90 was subjected to the prenylation conditions, and the desired alkylated product 91 was obtained in 52% yield. The epoxidation/Lewis acid-mediated cyclization proved to be successful on this substrate. The epoxide product was directly treated with SnCU in THF to provided the desired 92. When oxindole 92 was treated with NaBHt (1.6 equiv)and BFs'OEta (3.5 equiv) in THF, the desired 93 was obtained. The indole 93 was treated with TBDMSCl and imidazole in DMF, to provide the required 0-silylated indole, which was easily converted to the gramine 94 through the well known Mannich procedure.
362
OR'
o H
95
R = COOMe R' = f-BuPhSi
R-o
R" = f-BuMegSi
HO Reagents: (a) 94, 0.5 equiv of PBuj, CH3CN, reflux, 73%; (b) LiCl, HMPA, 100 °C, 89%; (c) Me30BF4, Na2C03. CH2CI2. 62-71%; (c) (i) BOC2O, DMAP, EtsN, CH2CI2; (ii) /1-BU4NF, THF, 85-90%; (d) NCS, Me2S, 74-86%.
Fig. (26). Synthesis of 98
Construction of the Diketopiperazines Containing a Dioxepinoindole Ring The epimers 96 (prepared in twelve steps from 95) were condensed with the gramine 94 providing the indole 97 in 73% yield as a mixture of two diatereomers. The indole 97 was converted to 98 with four steps. [Fig. (26)]
363
Construction of the Bicyclo[2.2.2] Ring System Allylic chloride 98 was reprotected with r-BuPhaSiOTf to provide 99 in 77-82% yield.
NaH, benzene
N
^i
*
BOC
^
(syn, 93%; anti, 85%) ization ^ ENDO Cyclization
f-^ RO 100
N BOC RO
99b
Fig. (27). Synthesis of 100
The stage now set to effect the SN2' reaction. [Fig. (27)] Compound 99 was refluxed in benzene with 20 equiv of NaH, resulting in a very clean and high-yielding cyclization reaction furnishing the desired product 100, and the undesired anri-diastereomer was not detected. It is generally accepted that SN2' reactions favor a syn orientation [56] (i.e., the incoming nucleophile attacks the 7C-electrons from the same face as the departing leaving group, polarizing the 7i-system in the proper orientation for a "backside" displacement on the C-Cl bond). Alternatively, a frontier molecular orbital analysis has indicated [56a] that the stabilization imparted by a HOMONuc-LUMOaiiyiic interaction is greater for the syn overlap. While the greatest level of diastereoselectivity was observed with a nonpolar aprotic solvent (benzene), a fairly significant change in the relative amounts of the syn- and anri-diastereomers can be realized by simply changing the solvent to a more polar solvent such as DMF. In the present system, additional stabilization for the endo transition
364
State may be due to the formation of a tight contact ion pair between the chlorine atom and sodium atom of the enolate species in the transition state for the formation of the C-C bond. [57] The poor ligating solvent benzene is not capable of effectively solvating the enolate cation nor the developing chloride anion in the transition state. It is reasonable that this type of association favors the rotamer that positions the allylic chloride moiety over the enolate, resulting in the desired syn stereochemistry. The Final C-C Bond-Forming Reaction on the Indole With the bicyclo[2.2.2] ring system constructed in a reliable and highyielding sequence, attention was turned to the final C-C bond-forming reaction on the indole. [Fig. (28)] A search of the literature revealed a 1982 Trost and Fortunak paper [58] wherein PdCl2 and AgBF4 were utilized to effect the Heck-type cyclialkylations of various isoquinuclidine model compounds. Compound 100 was exposed to these conditions, affording the heptacycle 101 in 6382% yields. There are several reports of methods that will selectively reduce a tertiary amide in the presence of a secondary amide[59]. The secondary lactam of 101 was protected as the lactim ether 107 and treated with diborane; however, the spectral characteristics of the major products isolated were consistent with reduction of both the tertiary amide and the lactim ether. In 1991 Martin et ai [60] successfully used alane to reduce a tertiary amide in the presence of an oxindole (secondary amide) relying on the known rate difference for reduction between these two groups [61]. However, initial experiments with this reagent gave poor results, with the secondary amide undergoing reduction along with the tertiary amide. Compound 101 [and 107, Fig. (29)] is sufficiently twisted such that the g^m-dimethyl groups effectively block the P-face of the tertiary amide, leaving the a-face relatively unencumbered. However, a modification of the alane procedure [60], proved satisfactory for this transformation. The piperazinedione 101 was pretreated with AlEts, with the expectation that this Lewis acid would form a complex with the more exposed secondary lactam [106, Fig.(29).] and leave the tertiary lactam accessible for reduction.
365
102 ; R = f-BuMegSi, R' = H, R" = BOC
105
103 ; R = f-BuMegSi, R' = Me, R" = BOC 104 ; R = f-BuMepSi. R' = H, R" = H
Reagents: (a) PdClz, AgBF4. MeCN; (b) NaBH4 (63-82% from 100); (c) 1.1 equiv of EtaAl, 5.0 equiv of AIH3-DMEA, THF, toluene; (d) 2.0 equiv of NaCNBHa. AcOH, MeOH (65% from 101); (e) 2.5 equiv of NaH, 2.0 equiv of Mel, DMF (98%); (f) 80 equiv of TEA, CH2CI2 (96%); (g) /-BuOCl, pyridine, -15 °C; (h) 90% THF, 10% H2O, pTsOH (76%); (i) MTPI, DMPU (79%).
Fig. (28). Synthesis of (+)-PHB
O
\ ^ R = f-BuMe2Si
Fig. (29). Structures of 106 and 107
Following 10 min of precomplexation with AlEts, 5 equiv of AIH3Me2NEt complex was added, followed by quenching with NaCNBHs,
366
acetic acid, and methanol to provide the desired amine 102 in 63% yield. Compound 102 was smoothly alkylated with methyl iodide, affording the N-methylated product 103 in 95-98% yield. Compound 103 was subsequently deblocked with 80 equiv of TFA in CH2CI2 to yield the penultimate heptacycle 104 in 97% yield. The stage was now set for the final transformations involving the oxidative pinacol-type rearrangement and dehydration. Thus, treatment of 105 with r-BuOCl and EtsN in CH2CI2 provided the two chloroindolenines 108 and 109 (2.25:1 ratio, respectively, Figure 30). The solvent was removed, and the crude reaction mixture was refluxed with a solution of 95% THF, 4% H2O, and 1% TFA, giving a 62% yield of oxindole products (43% of the desired 105 and 19% the epi product 110). The C-3epi-isomer (110) was easily distinguishable from the desired isomer (105) by the upfield shift of the g^m-dimethyl signals in the ^H NMR spectrum. The relative amounts of products (105 and 110) indicate that the cyclization was stereospecific under these conditions. It was thus deduced that an increase in the ratio of the desired oxindole 105 to the undesired isomer 110 could be achieved simply by finding a method that would increase the ratio of chloroindolenines (108:109).
108
Fig. (30). structures of 108,109 and 110
The a-face of 104 is considerably more hindered than the p-face, a supposition that was supported by the difficulties encountered in the reduction of 101 and 107. Increasing the steric bulk of the chlorinating agent should favor attack on the p-face of 104, thus providing a greater relative amount of chloroindolenine 108. When 104 was treated with tBuOCl in pyridine instead of triethylamine, the desired chloroindolenine
367
108 was produced in a much more stereoselective fashion. It can be speculated that r^rr-butyl hypochlorite forms a bulky complex with pyridine, delivering the chlorine more selectively to the least hindered aface of 104 (only a small amount, 5%, of the undesired isomer 109 was formed under these conditions. Employing a minor modification of the solvent system, the crude mixture of 108/109 was refluxed with a solution of 90% tetrahydrofuran, 10% H2O containing 15 equiv of p-toluenesulfonic acid to give the desired oxindole 105 in 76% yield (from 104), with only 4% of the undesired 110 being formed. The final dehydration reaction (MTPI, DMPU, 18 h) on the alcohol 105 produced (+)-PHB (81) in 79% yield. This substance proved to be identical to the natural product by comparison of the ^H and ^^C NMR spectra, mobility on TLC, IR spectra, mass spectra, and UV spectra. Comparison of the CD spectra of the natural (-)-PHB (6) and the synthetic (+)-PHB (81) confirmed the expected enantiomeric relationship between these two products. ASYMMETRIC, STEREOCONTROLLED TOTAL SYNTHESIS OFPHA[62] Williams and his coworkers have previously described as symmetric synthesis of the simplest paraherquamide derivative, PHB from (5)proline. In approaching the synthesis of PHA which contains the unusal Phydroxy-|3-methylproline residue, a new method needed to be developed to generate a suitably functionalized a-alkyl-P-hydroxyproline moiety that could be conscripted for a multistep construction of PHA. Construction of the a-Alkyl-p-Hydroxyproline Moiety As described previously [63], p-ketoester 111 [Fig. (31)] was subjected to Baker's yeast reduction to afford the optically active P-hydroxyester 112 (60-80% yield). Dianion alkylation of 112 with (E)-3-methyl-4-(0-/^rrbutyldimehtylsilyl)-2-butene afforded the desired a-alkyl product 113 in 58-70% isolated yield.
368
Boc I N>^COOEt
Q:
(60-80%)
Boc I ^N
O
Boc I cOOEt
^N
U^-^Y^OTBS
OH
113
^"
112
111
cOOEt
(86%)
^Y^NH V - - ^ v ^ ^ OTBS
(93%)
^ . . ^ - ^ ^ OTBS
c.d.e
f,g (79%)
^N
cOOEt OTBS OMOM 114
Reagents: (a) Baker's yeast; (b) LDA, THF, HMPA, (E)-ICH2CH=C(Me)CH20TBS; (c) 5.7 equiv MOMCl, (/Pr)2NEt, CH2CI2; (d) 2.7 equiv ZnBr2, CH2CI2; 0 °C (e) K2CO3. 2 equiv BrCHiCOBr, CH2CI2; (f) NH3 in MeOH (5.7 M), 25 °C; (g) 3 equiv NaH, toluene, HMPA, 25 °C; (h) 1.3 equiv n-BuU, THF, 11.1 equiv ClCOOMe, - 78 °C; then 4 equiv ClCOOMe, 5 equiv LiN(TMS)2. - 78 °C. Fig. (31). Synthesis of 116
Protection of the secondary alcohol as the corresponding methoxy methyl (MOM) ether, followed by removal of the Boc group with ZnBr2 in dichloromethane and acylation of the incipient secondary amine with bromoacetyl bromide in the presence of K2CO3 afforded the bromoacetamide 114 in 86% yield from 113. Treatment of 114 with methanolic ammonia afforded the corresponding glycinamide which was directly subjected to cyclization in the presence of NaH in toluene/HMPA to afford the bicyclic compound 115 in 79% overall yield from 114. Next, a one-pot double carbomethoxylation reaction was performed by the sequencial addition of n-BuLi in THF followed by addition of methylchloroformate, that carbomethoxylated the amide nitrogen atom. Subsequent addition of four equiv of methyl chloroformate followed by the addition of 5 equiv of LiN(TMS)2 afforded 116 as a mixture of diastereomers in 93% yield that were taken on directly without separation.
369
Construction of the Tryptophan Derivative 120 Somei-Kamatani coupling of 116 [Fig. (32)] with the gramine derivative 94 in the presence of tri(n-butyl)phosphine gave the tryptophan derivative 117 as a 3:1 mixture of diastereomers in 70% yield. TBS* n
O
br"*"*^' R' = COOMe,R" = MOM :MOM
94
(70%)
OTBS OMOM
OTBS
116 R' = COOMe
(68 -71%)
•
/--p^N V - N , ^
120
OTBS
Boc
Boc
Reagents: (a) 0.7 equiv («-Bu)3P, MeCN; (b) 5 equiv LiCl, H2O, HMPA, 105 °C, 5 h; (c) 2.5 equiv Me30BF4, CS2CO3. CH2CI2.25 °C; (d) DMAP, 3 equiv (Boc2)0, CH2CI2; 0 °C (e) 3.3 equiv TBAF, THE, 25 °C; (f) 1.1 equiv Msci coUidine, CH2CI2, 0 °C; (g) 3.3 equiv TBSOTf, 2,6-lutidine, CH2CI2,0 °C. Fig. (32). Synthesis of 120
Decarboxylation of 117 was effected by treatment of 117 with LiCl in hot, aqueous HMPA at 105 °C providing 118 as a mixture of diastereomers that were separated and carried forward individually. Protection of the secondary amide group as the corresponding methyl lactim ether was accomplished by treating 118 with trimethyloxonium tetrafluoroborate in dichloromethane that contained cesium carbonate. Next, the indole nitrogen atom was protected as the corresponding Boc derivative by treatment with dicarbonic acid bis(f^rr-butyl)ester in the presence of DMAP and the silyl ether was removed with tetrabutylammonium fluoride to provide diol 119 in 52-78% overall yield from 118. Selective conversion of the allylic alcohol to the corresponding
370
allylic chloride was accomplished by mesylation in the presence of coUidine. Silylation of the secondary alcohol with rerr-butyldimethylsilyl triflate in the presence of 2,6-lutidine afforded the key allylic chloride 120 in 68-71% yield over the two steps. Construction of the Bicyclo[2.2.2] Ring System and the Final C-C Bond-Forming Reaction on the Indole
OMOM, h (85%), i (97%) OH
Reagents: (a) 20 equiv NaH, TOP, reflux 30 h; (b) 3.1 equiv AgODp4, 4.68 equlv PdCh. MeCN, propylene oxide; then NaBH4, EtOH; (c) 0.1 M HCl, THF; (d) 2-hydroxypyridine, toluene, 120 °C, 2 h; (e) 5 equiv (iBu)2AlH, CH2CI2. 0 °C; (f) NaH, Mel, DMF, 0 °C; (g) 6 equiv B-bromocatecholborane, CH2CI2, 0 °C; (h) Sequiv l,l,l-triacetoxy-l,l-dihydro-l,2-benziodoxol-3(7f/)-one (Dess-Martin periodinane), CH2CI2, 25 °C; (i) TFA, CH2CI2, 25 °C.
Fig. (33). Synthesis of 124
The stage now set to effect the SN2' reaction. [Fig. (33)] Compound 120 was refluxed in THF with 20 equiv of NaH, resulting in a very clean
371
and high-yielding cyclization reaction furnishing the desired product 121, and the undesired anr/-diastereomer was not detected. Closure of the seventh ring was effected by treatment of 121 with 4.68 equiv of PdCl2 and 3.1 equiv of AgBF4 [58] in acetonitrile containing propylene oxide as an acid scavenger. The incipient heptacyclic apallladium adduct was worked up immediately by the addition of the ethanol and NaBH4to afford the desired indole 122. Cleavage of the lactim ether 122 was effected with 0.1 M HCl to give the corresponding ring-opened amine methyl ester that was recyclized by treatment of this material with 2-hydroxypyridine in hot toluene. Chemoselective reduction of the secondary amide was effected by treatment of of the product obtained from the previous step with excess diisobutylaluminum hydride in CH2CI2 to furnish 123 (50-72% yield) [64]. Methylation of the secondary amide 123 proceeded in 96% yield. Cleavage of the MOM ether with bromocatecholborane [65] (91% yield) followed by oxidation of the secondary alcohol with Dess-Martin periodinane [66] (85% yield) and cleavage of the Boc group and TBS ether with TFA (97% yield) gave the ketone 124. Formation of the Spiro Oxindole The final, critical oxidative spirocyclization of the 2,3-disubstituted indole to the spiro oxindole was effected by treatment of 124 with tertbutyl hypochlorite in pyridine to provide the labile 125 [Fig. (34)]. The Pinacol-type rearrangement was conducted by treating compound 125 with p-toluenesulfonic acid in THF/water. It is assumed that the chlorination of 124 proceeds from the least hindered face of the indole, to give the a-chloroindolene 125. The hydration of the imine functionality must also occur from the same a-face that is syn to the relatively large chlorine atom furnishing the ^yn-chlorohydrin 126, that subsequently rearranges stereospecifically to the desired spiro oxindole 127. The dioxepin ring was then formed by dehydration of the secondary alcohol 127 with MTPI in DMPU to afford 14-oxo-PHB 12 [7]. This material has been previously described by a Pharmacia group (obtained semi-synthetically from MFA) and comparison of the authentic and synthetic materials [^H and ^^C NMR, IR, exact mass, mobility on thinlayer chromatography (TLC)] conformed the identity of this substance. Treatment of the synthetic ketone with MeMgBr gave (-)-PHA (2) in 42% yield that was identical in all respects [^H and ^^C NMR, IR, exact mass,
372
mobility on TLC, m.p. (250 °C (dec)), [ajo = -22 (C= 0.2 MeOH)] to the natural PHA [4]. OH TsOH
f-BuOCI
O
THF/HgO
OH
CH3
^*^LNx^xk^O
• (55%) O 54 % from 125
3
CH3O 127
MeMgBr (42%)
12
Fig. (34). Formation of the spiro oxindole
CONVERSION OF MFA TO PARAHERQUAMIDES VIA A NOVEL PLATINUM-OXYGEN-MEDIATED RING CONTRACTING REACTION [33] Our earlier conversion of MFA to PHA required 13 steps [7]. By employing a ring contracting reaction utilizing platinum and oxygen (Pt/Oi) at a key point in the synthesis, we were able to directly convert marcfortine A to the intermediate 16-oxoparaherquamide B (7), thereby eliminating six steps in our earlier synthesis.
373
H3C Ch CH«
A
Pt/C dioxane/water
o 7
CH,
(14-23%)
o p \J/
H3C CH,
06^
CH^ 128
(2-3%)
129
130
(9-19%)
(14-19%)
MCPBA 130
7
(80%)
1
4
MCPBA
H
o
p^JiiU/M^c CH3
P
p<j^^H^C
CH3
CH-, Fig. (35). Reaction of MFA with oxygen
Although the Pt/02 reaction has been used for oxidation of primary and secondary alcohols [67], hydroxylation of 12a-deoxytetracyclines [68], oxygenation of cholesterol [69], and oxidation of tertiary amines [5f], this
374
reaction has not been used extensively compared to singlet oxygen reactions [70]. When marcfortine A was treated with Pt on carbon (10%) in dioxane/water under an oxygen atmosphere (balloon pressure, room temperature, 2-4 days), four products (7, 128-130) were isolated [Fig. (35)]. The reaction was not accelerated by heating. Performing the reaction under oxygen at 1000 psi resulted in acceleration with very little effect on the product ratios. Yields varied depending on the activity of the platinum.
o p
^3^. p^3
%J/
Pt/Og followed by MCPBA
H
HO
23
10 (43%)
HQC
CH<3
Pt/0«
J\.
HoC
32
HOH,C
CH3 132 (10%)
o
(3%)
CHQ
>-N
°
>-N 131
HO-f-^4v> H3C
H3C CHo
V ^
o
133 (10%)
H3C CH3 ,
,,^
M:^40<J (3%)
H3C
Y\
CH^
132
Fig. (36). Reaction of 23,32 with oxygen
PHA AIHg-NMegEt
375
Dioxo compound 130 was converted to 7 in 80% yield by treatment with m-chloroperbenzoic acid (m-CPBA). According to the literature, sixmembered rings containing a 1,2-dicarbonyl moiety are converted to fivemembered ring hydroxy acids only in the presence of a strong base [71]. By contrast, our method is performed under neutral conditions and is more efficient. Subsequently, we examined several MFA derivatives as substrates for the Pt/02 reaction. When 32 was subjected to Pt/Oa chemistry, three products (132-134) were isolated in poor yield [Fig. (36)]. In the case of 23, the Pt/02 reaction mixture was treated straightaway with m-CPBA without isolation of any intermediate products, giving the desired compound 10 (43% yield). Compound 10 can be converted to PHA (2) in three steps. Compound 132 was converted to PHA (2) by treatment with alane-dimethylethyl amine complex in THF (30% yield). HqC CHo
Pt/0«
HO^
O
36 135
Fig. (37). Reaction of 36 with oxygen
(6%)
376
When 36 was subjected to Pt/02 chemistry [Fig. (37)], four products (35, 135-137) were isolated. Although the Pt/Oi chemistry performed on 36 does give 137 (the desired product) in trace amounts, it can be more readily prepared by oxidation of 136 with m-CPBA (80% yield). Subsequently, 137 was reduced with LAH/AICI3 in THF to give 138 (40% yield).
CH«
Pt/C
0-0
\
[O] B
\ Partial aiiyiiic oxidation o
CH3
C: X = H,H D: X = H,OHorO [0]
130
[H2O2]
*•
7
oo [H2O2]
[0]
128+ 129
o CH3 E: X = H,OHorO >
Fig. (38). A plausible mechanism for the Pt/O^ meddiated ring contracting reaction
377
A plausible mechanism for the Pt/Oi-mediated ring contracting reaction is shown in [Fig. (38)]. In this mechanism, A exists in equilibrium with B (one can speculate about the formation of A). Intermediate B undergoes molecular oxidation accompanied by partial allylic hydroxylation to give intermediates C and D, Each of these is further oxidized to give putative intermediate E and product 130. A final oxidative step, probably involving hydrogen peroxide generated in situ and proceeding by a mechanism paralleling that described for the m-CPBA reaction, leads to products 7, 128, and 129.
ACKNOWLEDGEMENTS We are grateful to Professor Yamazaki at Chiba University for furnishing a sample of natural paraherquamide A.
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(a) Conder, G.A.; Johnson, S.S.; Guimond, P.M.; Cox, D.L.; Lee, B.L. J. Parasitol 1991, 77, 621; (b) Conder, G.A., Johnson, S.S. J. Parasitol 1996, 82, 100. Thompson, D.P.; Klein, R.D.; Geary, T.G. Parasitology 1996, 775, S217. Schaeffer, J. M.; BHzzard, T.A.; Ondeyka, J.; Goegelman, R.; Sinclair, P. J.; Mrozik, H. Biochem. Pharmacol 1992,43, 679-684. Nauen, R.; Ebbinghaus, U.; Tietjen, K. Pestic. Sci. 1999,55,608-610. (a) Zinser, E.W; Wolfe, M.L.; Alexander-Bowman, S.J.; Thomas, E.M.; Groppi, V.E.; Davis, J.P.; Thompson, D.P.; Geary, T.G. J. Vet. Pharmacol Ther., submitted, (b) Lee, B. H. / Labelled Compd Radiopharm 2002, 45, 0000. (c) Blizzard TA, Rosegay A, Mrozik H, Fisher MH. / Labelled Compd Radiopharm 1990,2S, 461-464. (a) Gushing, T. D.; Sanz-Cervera, J. F.; Williams, R. M. J. Am. Chem. Soc. 1996, 77S, 557. (b) Gushing, T. D.; Sanz-Cervera, J. F.; Williams, R. M. J. Am. Chem. Soc. 1993, 775, 9323. Lee, B. H.; Clothier, M. F. Bioorg. Med Chem. Lett, 1997, 7, 1261. Gushing, T. D.; Williams, R. M. Tetrahedron Lett. 1990, 31, 6325. McWhorter, W. W.; Gleave, D. M.; Savall, B. M. Syn. Comm. 1997,27, 2425. Lee, B. H.; Clothier, M. F. Bioorg. Med Chem. Lett, 1998, 5, 3415. Sanz-Cervera, J. F.; Glinka, T.; Williams, R. M. J. Am. Chem. Soc. 1993, 775, 347. Williams, R. M.; Glinka, T.; Kwast, E.; Coffman, H.; Stille, J. K. J. Am. Chem. Soc. 1990, 772, 808. (a) Somei, M.; Karasawa, Y.; Kaneko, C. Heterocycles 1981, 16, 941. (b) Kametani, T.; Kanaya, N.; Ihara, M. / Am. Chem. Soc. 1980, 702, 3974. (a) Beer, R. J. S.; Clarke, K.; Davenport, H. F.; Robertson, A. /. Chem. Soc. 1951, 2029. (b) Bennington, F.; Morin, R. D.; Clark, L. C , Jr. J. Org. Chem. 1959,2^,917. Kosuge, T.; Ishida, H.; Inabe, A.; Nukaya, H. Chem. Pharm. Bull 1985, 33, 1414. (a) Magid, R. M.; Fruchey, O. S.; Johnson, W. L.; Allen, T. G. J. Org. Chem. 1979,44, 359.(b) Magid, R. A. Tetrahedron 1980,36, 1901. The idea that the stereochemical outcome of an intramolecular enolate alkylation is determined by chelation in the transition state was recently demonstrated by Denmark and Henke, who observed a marked preference for a "closed" transition state (coordination of the cationic counterion to an enolate and the developing alcohol) resulting in a syn product. For example, the highest syn.anti ratio (89:11) was obtained in toluene and the lowest syn:anti ratio (2:98) was obtained with a crown ether. These observations parallel the facial selectivities described herein and in ref 11 on the intramolecular SN2'reaction; see: (a) Denmark, S. A.; Henke, B. R. / Am. Chem. Soc. 1991, 775, 2177. (b) Denmark, S. A.; Henke, B. R. /. Am. Chem. Soc. 1989, 777, 8022. Trost, B. M.; Fortunak, J. M. D. Organometallics 1982, 7, 7. In a recently reported synthesis of gelsemine, a tertiary lactam was reduced in the presence of a secondary lactam with DIB AH. However, this reagent failed on substrates 101; see: Dutton, J. K.; Steel, R. W.; Tasker, A. S.; Popsavin, V.; Johnson, A. P. J. Chem. Soc, Chem. Commun. 1994, 765.
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Martin, S. F.; Benage, B.; Geraci, L. S.; Hunter, J. E.; Mortimore, M. J. Am. Chem.Soc.l991J 13, 6161. (a) Yoon, N. M.; Brown, H. C. J. Am. Chem. Soc. 1968, 90, 2927. (b) Marlett, E. M.; Park, W. S.; /. Org. Chem. 1990, 55, 2968. (c) Jorgenson, M. J. Tetrahedron Lett. 1962, 559. (d) Another very recent synthesis of gelsemine reported the reduction of the same gelsemine precursor (as in ref 59) with AIH3. Newcombe, N. J.; Fang, Y.; Vijn, R. J.; Hiemstra, H.; Speckamp, W. N. /. Chem. Soc, Chem. Commun. 1994, 767 Williams, R. IVl.; CaO, J.; Tsujishima, H. Angew. Chem. Int. Ed. 2000,39, 2540. (a) Williams, R. M.; CaO, J. Tetrahedron Utt. 1996, 37, 5441. (b) Cooper, J.; Gallgher, P. T. ; Knight, D. W. J. Chem. Soc, Perkin Trans 1 1993, 1313. Fukuyama, T.; Liu, G. Pure Appl Chem. 1997,69,501. Boeckman, R. K.; Potenza, J. C. Tetrahedron Utt. 1985,26,1411. (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155; (b) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc 1991,113, 7277. Sneeden, R. P. A.; Turner, R. B. J. Am. Chem. Soc 1955, 77, 190. Muxfeldt, H.; Buhr, G.; Bangert, R. Angew. Chem., Int. Ed. Engl. 1962,1, 157. Sakamaki, H.; Take, M.; Akihisa, T.; Matsumoto, T.; Ichinohe, Y. Bull. Chem. Soc Jpn. 1988,61,3023. Wasserman, H. H.; Ives, J. L. Tetrahedron 1981, 37, 1825. (a) Bhatt, M. V. Tetrahedron 1964, 20, 803. (b) Aoyage, P.; Moriyama, Y.; Tsuyuki, T.; Takahashi, T. Bull. Chem. Soc Jpn. 1973,46, 569.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
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ACARICIDES OF NATURAL ORIGIN, PERSONAL EXPERIENCES AND REVIEW OF LITERATURE (1990-2001)* GUIDO FLAMINI Dipartimento di Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy ABSTRACT: Acari are responsible for millions of dollars worth of damages each year as a result of infestations in animals, plants and man. They affect our health directly and prosperity as animal and plant parasites, vectors of disease, and producers of allergens. The indiscriminate use of pesticides has destroyed many of their natural enemies of hitherto harmless species and quickly induced resistance in many parasites. At present, the control of acarid parasitic diseases in agriculture, human and veterinary medicine is mainly based on the use of drugs; for this reason the lack of effective drugs often prevents the control of some parasitic diseases, making them more serious and important. The use of commercial drugs involves many problems; besides the drugresistance shown by the most important parasites, the environmental damage and the toxicity of many synthetic drugs, represent the main problems that strongly limit their use. In addition, drug residues in plant and animal food products are important reasons forfiirthereconomic losses for farmers and must be regarded as potentially hazardous to man and envh-onment. Plant-derived compounds are generally more easily degradable and could show a smaller negative environmental impact with respect to synthetic drugs. For these reasons, the evaluation of the antiacarid activity of plant extracts is increasingly being investigated in order to obtain new leads, as demonstrated by recent studies that have evaluated and confirmed the effectiveness of many plant compounds on bacteria,fimgi,protozoa, hehnints and arthropods. This review will be limited to the class Arachnida, sub-class Acaridi, particularly to their control in agriculture, veterinary and human medicine with natural methods.
INTRODUCTION Mites and ticks, collectively known as the Acari, constitute the second most diverse group of animals on the planet today and are of interest to humans for a variety of reasons. They affect our health and weel being directly as plant, animal and human parasites, vectors of disease, and producers of allergens. They are responsible for millions of dollars worth
Dedicated to the memory of Prof Serena Catalano
382
of economic losses each year as a result of infestations of animals, man and plants. The acari are ubiquitous, being members of every major ecosystem on earth, and their specific habitats run the gamut from the familiar to the truly bizarre. From our backyards to the geothermal springs of the Yellowstone caldera and from the subcutaneous tissue of turtles to our very own hair follicles, mites carry out a nearly invisible existence. While awareness of the acari dates back to ancient Egypt (1550 BC) and was further demonstrated through the writings of the major Greek scholars, the science of acarology originated in 18th century in Europe. Linnaeus described the first mite species, Acarus siro, in 1758, thus laying the groundwork for the field of systematic acarology. Today, over 48000 described species of mites and ticks have been described worldwide, while current estimates place total diversity at over a halfmillion species. The Class Arachnida, to which the order Acari belongs, together with the Class Insecta, the Class Crustacea and others, constitute the Phylum Arthropoda. All the classes contain species useful to man, but also many pests that can cause economic losses and/or diseases. This review will be limited to the order Acari, particularly to their control with natural methods in agriculture, veterinary and human medicine. The indiscriminate use of inorganic pesticides, destroyed many harmless species including natural enemies of these mites and ticks [1]. From 1940 onwards, organochlorine and organophosphate pesticides were introduced, but tresistance was quickly acquired in many arthropod parasites, including acari; fortunately many useful predatory mites became resistant too. The emergence of resistance to parasiticides is one of the most serious challenges faced by modem farmers. Perhaps it is the simplicity of treating parasite attacks with very effective drugs or pesticides on a routine basis and the proven cost-effective gains in productivity that will accrue in the short term, that has led to the predominance of synthetic pesticides [2]. Broadly speaking, resistance is the ability of the parasites to survive doses of drugs that would normally kill parasites of the same species and stage. It is inherited and selected for because the survivors of the pesticide treatment pass genes for resistance on to their offspring. These genes are initially rare in the population or arise as rare mutations in genes but as selection continues, the proportion of resistance genes in the population increases and so does the proportion
383
of resistant parasites. Drug susceptibility is a resource that needs to be preserved using appropriate techniques of parasite management. In many cases susceptibility to certain pesticides in some parasites has been lost forever. One approach to this problem is to develop new susceptibilities by using novel drugs. However, the application of synthetic chemical substances is still the common method to control or eradicate parasites of plant and animals. Unfortunately, many acaricides have non-specific properties, affecting other organisms (crops, non-vertebrates and vertebrates) to varying degrees. The damage to these non-target organisms may be reflected by different effects, like direct mortality, long term population reductions, bioaccumulation within the food web. The indiscriminate dispersion of these substances must be regarded as potentially hazardous to man and environment. Plants are the richest source of organic compounds on Earth. Many of these natural chemicals are endowed with pesticide properties. It is well known that some pesticides of plant origin have been in use before the time of the Romans (i.e. pyrethrum, obtained from the flower heads of Chrysanthemum cinerarifolium). In 1959, the term Integrated Pest Management (IPM) was coined [3]. IPM consist in the application of the best mix of control tactics for a given pest problem in comparison with the yield, profit and safety of the alternative mixes [4]; these control methods ought to be environmentally acceptable and sustainable [5]. However, many of these studies have little bearing on what is actually happening in the field. In veterinary medicine, the control of ectoparasites is of great importance due to their effects on livestock profitability and the health status of animals. Infestations on livestock can cause intense irritation leading to poor condition, weight loss, reduced milk yield, and hide or fleece damage. Furthermore, many species of acari are responsible for transmission of diseases to the host animals themselves or act as vectors of a number of diseases to humans [6]. APICULTURE During the 90s, several cases of resistance to common acaricides employed in beekeeping by Varroa mites (Acari: Varroidae) were reported from different countries [7-10]. In Italy, the consequences of the resistance were disastrous colony losses. Available statistics show that in
384
certain districts, losses often exceeded 70% and in some locations even reached 90% [11]. Varroa mites originated in southeast Asia, where they parasitized the Asian honeybee. Gradually they have spread and now^ afflict most of the European honeybees. Though the Asian honeybee has been able to tolerate the mite: it was a pest, but not a fatal one, European honeybees have almost no resistance, and the mite has proved fatal to millions of the colonies in Europe and America. Varroa mites suck the body fluids from adults and brood, prefering the latter, especially drone brood. The problem of developing suitable treatments was difficult in the case of the Varroa mites because most substances effective against the parasites have unacceptable side-effects on bees. Towards the end of the 80s products based on pyrethroids were used. These substances were very effective on mites, and without any appreciable effects on bees. Pyrethroids are synthetic analogs of pyrethrins, secondary metabolites obtained from the flower heads of Chrysanthemum cinerarifolium. Synthetic pyrethroids replaced the natural ones because they apparently exhibited no negative effect on plants, animals and humans. Unfortunately, the limits of this "magic bullet" soon became evident, with serious impact on beekeeping because of the resistance developed by Varroa. Since the creation of EU Varroa experts' group, several lines of research in alternative control measures have been explored: apicultural techniques for reducing the number of mites, increasing bee resistance, and searching for acaricidal products that are generally recognized as safe for humans, such as some natural derivatives [12]. Among these compounds many simple carboxylic acids have been used, such as formic, oxalic and lactic acids. The first report on the use of formic acid as miticide was published in 1980 [13]. Five years later Wachendorfer et al. [14] developed and carried out field trials on the "lUertisser Milbenplatte" (=mite plate), an adsorbent cardboard soaked in formic acid on which the bees alighted before entering in the hive. Other methods of treatment included dispensation of formic acid by means of evaporators, wood shavings, or use of liquid reservoirs with wicks [15-27]. The mortality rate of Varroa mites was however quite variable, with percentages ranging from 12% [24] to 98.8% [21], with a prevalence of intermediate values (47%-56%) [18,21,26]. A strong dependence upon the administration method was observed: in western Switzerland the percentage of mites killed was 80% when formic acid was applied on the plates, but 90%
385
when applied on viscose cloth [16]. Many of the above techniques involve removal of one or more frames from the hive, and most involve handling of liquid acid by the beekeeper, with the risks associated with dispersion and the corrosive nature of this substance. To resolve these problems, recently a gel formulation of formic acid has been developed. Kochansky and Shimanuki [28] experimented with various gelling agents, obtaining the desired rheological characteristics and releasing behavior of the active principle, with fumed silicas and Carbopols 934 and 941. The fumed silicas gave the best results, with optimum evaporation speed for application to bee hives (2-3 weeks). A gel formulation tested in Argentina under autumnal climatic conditions showed a 92% mortality in Varroa jacohsoni with a low variability, indicating that this kind of formulation could be the best one [29]. Formic acid was evaluated for its effect on the honey bee colony; Westcott and Winston [30] examined various parameters such as colony weight gain, brood survival, sealedbrood area, emerged-bee weight, number of retumed foragers, pollen-load weight and worker longevity in treated and control colonies. Only sealedbrood area was lower in formic acid treated colonies; furthermore, also the honey production was lower in treated colonies, but this difference from control was not statistically significant. Another study confirmed that the use of formic acid can be considered safe, in fact the average length of life of the treated bees was 24.4 days compared with 23.8 days of controls [31]. Also during heavy Varroa Jacobsoni infestations and hives weakened by the dry summer conditions, it was demonstrated that 8 consecutive treatments with formic acid during the month of August gave satisfactory mite control without harming or irritating the bees [15]. However, these conditions are not sufficient for a proper use of formic acid in apiculture, but it is necessary to evaluate whether this substance can accumulate in honey, wax and propolis. Various HPLC methods have been developed [32,33], but only one [34] compared treated honey samples and controls, conclusively showing that the former contained higher levels of formic acid (25-51 mg/kg) than the latter (20 mg/kg). Recently, the influence of formic acid on honey taste has been evaluated by means of an expert test panel [35]. The acid was artificially added to different honeys and the testing persons remarked an increased acid taste in the honeys adulterated with acid quantities above the taste threshold, that is the lowest correctly distinguishable concentration of an additive in
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honey. The taste threshold for acacia honey was about half of the one for honeydew honey, because the latter has a much stronger aroma and can whitstand more acid than acacia honey, which has only a weak aroma. The taste thresholds of formic acid were 150-300 mg/kg in acacia honey and 300-600 mg/kg in honeydew honey; in pure water it was 10 mg/kg, about 20 to 50 times below that in honey. Similar thresholds were also found in Italy [36]; Italian authors also noted that after application in autumn at the end of the bee season, the formic acid content increases significantly and may exceed the natural content. Later on, the formic acid content decreases slowly due to evaporation, to reach the original level in the following spring. Therefore, formic acid treatments should be performed in autumn to avoid adverse consequences to the taste of honey, harvested the following spring. Further to formic acid treatment, oxalic acid [21,21,37-40] and lactic acid [17,18,20,21,41-46] have been evaluated for their activity against Varroa jacobsoni and V. destructor. The application of oxalic acid has been studied both in different periods and by means of different methods. It seems that the best period is autumn-winter, the broodless period (average efficacy 99.44% vs. 52.25% in the presence of brood) [40]. These data have been confirmed in Italy, where a spray treatment of oxalic acid in water was compared with a topical application of the acid in a sucrose-water solution; the best result was obtained using the former method. In general, for both treatments, the highest mite mortality coincided with the treatment applied with no sealed brood [38]. With regard to the toxicity of oxalic acid, two contrasting studies are present in literature. The first one claims that bee mortality in treated hives was not significantly different from the bee mortality in controls [37], while the other paper suggests negative long-term effects, with a statistically significant negative effect on brood development and the death of some queens [39]. On the presence of oxalic acid in honey, it seems that its content in samples taken from the nest after treatment did not differ significantly between treated and control ones [37,38]. If the application is performed in autumn, the honey content of oxalic acid is comparable to its natural amount, that is below its taste threshold, determined in 300400 mg/kg for acacia honey and 700-900 mg/kg for honeydew honey [35]. Lactic acid was less active than the previous organic acids [18,21] and again it showed a greater effectiveness during late summer-winter
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treatments [18]. When its activity was compared with some commercial acaricides, results show it is less effective than coumaphos, a synthetic coumarin (umbelliferone) derivative, but also less toxic [42,45]. Rademacher [47] prepared a new formulation containing a synthetic acaricide, cymiazole hydrochloride, and lactic acid as active ingredients; furthermore, the content of cymiazole was 50% less than in a commercial preparation. The mixture resulted highly toxic to Varroajacobsoni, with the tolerance of honey bees a 100 times greater than that of the mites; field tests have shown that the mixture killed 83% and over 90% of mites in colonies with and without brood, respectively. Some authors reported an increased bee mortality after treatments with lactic acid [18,41]. Immediately after applications in autumn, the lactic acid content of honey increased up to 1500 mg/kg, but four weeks later it decreased to about 500 mg/kg: this was below the taste threshold of 800-1600 mg/kg in rape honey [35]. Formic acid was also found effective against another bee parasite, Tropilaelaps clareae (Acari: Laelapidae). This mite develops in brood cells and causes disturbances in metamorphosis, resulting in abnormal, incompletely developed individuals; in the case of a severe attack, bee larvae are killed. It caused substantial losses of Apis mellifera colonies in Pakistan, but a single application of formic acid (20 ml) to infested colonies gave complete control of the mite population, however, it also destroyed all the brood and some adult bees. Two treatments of 15 ml also gave complete control of the mite population, but caused only minimal destruction of brood [48]. The same findings were obtained in another study [49]. A following study confirmed these results and also demonstrated the effectiveness of sulfur (450 mg/frame) for T, clareae control [50]. Furthermore, sulfur was not toxic for bees [31]. Tracheal mites were also killed by formic acid [51]. This parasite, Acarapis woodi (Acari: Tarsonemidae), lives in the tracheal tubes of adult honey bees. The bees die because of the disruption to respiration caused by the mites clogging the tracheae. In summary, although these organic acids are natural honey constituents, the international food legislation prohibits honey additives adulterating its taste. Therefore, the residues of these substances in honey have to remain below their taste threshold [35]. Another natural substance tested against bee mites was the oil obtained
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from neem tree, Azadirachta indica, kernels. Its first use in beekeeping as acaricide dates back to 1995, when it was tested against the tracheal mite Acarapis woodi [52]. A commercial preparation of neem oil containing 3000 ppm was used to prepare a syrup containing 3 or 6 ml/1 of the drug. The colonies of Apis mellifera were treated in mid-May, and in August no mites were found in the colonies treated with 3 ml/1, while they were still high in controls. Furthermore, the treatment not only killed adult mites but also reduced the number of mite eggs. Treated colonies collected more pollen and produced more honey. Topical applications of neem oil in laboratory to infested bee were highly effective against both Varroa jacobsoni dead Acarapis woodi [53]. Approximately 50-90% V.jacobsoni mortality was observed 48 h after treatment with neem oil, with associated bee mortality lower than 10%. Although topically applied neem oil did not result in direct A. woodi mortality, it offered significant protection of bees from reinfestation. Significantly, azadirachtin-rich extract was ineffective at controlling both the mites, suggesting that azadirachtin. Fig. (1), one of the main insecticide compounds of neem tree, has no significant miticidal properties; in fact, honey bees were also deterred from feeding on a sucrose syrup containing more than 0.01 mg/ml of azadirachtin-rich extract. MeOO£. 1 ^OH
MeOOC MeOOC
^i
o
Fig. (1). Structure of azadirachtin
The same research group evaluated neem oil in the field [54]. They sprayed a 5% solution of the oil on infested honey bee colonies, killing about 90% Varroa mites but obtaining only a slight but not statistically significant decrease in tracheal mite infestation levels. Unfortunately this treatment caused 50% queen loss and the treated colonies showed onethird as many adult bees and one-sixth as much brood as untreated
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colonies. The authors hope that these negative effects on bees could be remedied by changes in the formulation, the application technology, and the season of application. In another trial the same researchers compared the spray applications of neem oil with feeding oil or azadirachtin-rich extract in a sucrose syrup [55]. They concluded that spray application was more effective than the other methods only on Varroa. They also reported that spray treatments had no effect on adult honey bee populations, however treatments reduced the amount of sealed brood in colonies by 50% and caused queen loss at higher doses. Besides neem oil, Majeed also tested Azadirachta indica dust obtaining an effective level of control and increased honey production with both the formulations [56]. Also pure azadirachtin, both commercially formulated and purified, was tested for its effects on Varroa mites, infested bees and healthy bees [57]. Azadirachtin was administered in 50% sucrose syrup at various concentrations, ranging from 6 to 162 |i
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Perhaps essential oils are the most studied natural substances as alternative acaricides in apiculture. In fact these substances are already present in hive products, i.e. Lejeune et al. asserted that propolis contains 5 to 10% of essential oils [61]. Furthermore, essential oils are well-known parasiticides: they are being used as fungicides, bactericides and insecticides for many years. Furthermore, the acaricidal properties of essential oil constituents have already been proved since the beginning of last century [62]. More than 150 essential oils and components of essential oils have been evaluated as miticidal substances in laboratory screening tests. However, very few of them proved successful when tested in field trials. Thymol and thymol blended with essential oils or essential oil components offer a promising exception [63]. For the twelve year period employed in this review, many papers have appeared in literature. In 1990 Colin tested the essential oils of two Labiatae species. Thymus vulgaris L. and Salvia officinalis L., against Varroa jacobsoni [64]. He also developed a formula to calculate the efficiency of the applications. He treated bee colonies with aerosols from aqueous emulsions ( 1 % for thyme and 0.5% for sage) and compared the results with those obtained from a positive control treated with amitraz. The compared treatments showed similar efficiencies. However, a major drawback is that essential oils must be used in the absence of brood or at least with a small brood area. Consequently, the most suitable time for treatment is not winter, but after a large summer honey flow, which reduces queen egg laying. In this case the great majority of female mites are exposed to the aerosol action. Concerning the honey, sensory tests did not give any evidence of thyme or sage after essential oil application. However, traces of some essential oil components were revealed by GC. The miticidal properties of menthol against the tracheal mite Acarapis woodi were first reportede in 1968 [65]. Duff and Furgala [66] determined the effectiveness and proper timing for menthol application as a control for A. woodi infestations in Minnesota. They found that menthol is somewhat difficult to deliver equally in a field study, depending on outdoor temperature, bee population, size of the hive and amount of direct sunshine on the colony that can affect menthol vaporization. However, they concluded that, in Minnesota, spring application was the best treatment, as it reduced the percentage of honey bees infested and precluded winter mortality. Later, in 1993, the same authors substantially
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confirmed their previous results [67]. In 1992, the same authors again evaluated the effects of menthol on healthy honey bees. They stated that the use of menthol in spring reduced brood production and confined the bees spatially away from the menthol bag; however, menthol did not affect honey production [68]. A similar study about the effect of Thymus vulgaris and Salvia officinalis essential oils (2% water emulsions) on mite-free honey bees was conducted by Marceau [22]. The colonies, treated with these essential oils, both by microdiffusion and evaporation, were not negatively affected. The same results were obtained in a following study as well [23]. A more accurate study on the effect of essential oils as miticides was performed by Kevan et al. [69], which determined the following LD50 of some essential oils and pure constituents administered to bees as feeding solutions: cinnamon oil 150 ppm, clove oil 200 ppm, wintergreen oil 500 ppm, pinene 1500 ppm, thymol 100 ppm. In another paper the same researchers reported that menthol was ineffective at causing mortality of mites with the highest doses adminesterable [70]. Thymol seems to be a very promising natural acaricide in apiculture, but has an inconsistent efficacy against Varroa [17,64,71-75]. During the 90s, some commercial preparations against Varroa appeared. They contained natural products, mainly thymol. The first one was Apilife VAR® (Chemicals LAIF, Italy), a vermiculite tablet impregnated with a 20 g mixture of thymol (76%), 1,8-cineole (16.4%), menthol (3.8%) and camphor (3.8%). Various research groups tested this preparation both in laboratory and field conditions [76-79]. All the authors reported high efficacy (74-95%), particularly if employed under optimal temperatures (>13°C). A formulation without camphor showed the same positive results as well [78]. Apilife VAR was not toxic for honey bees and the residues (only thymol) found in honey and wax were innocuous to humans. Another registered product, Thymovar®, consists of a sponge cloth that functions as a vehicle for the drug thymol (15 g). The effects of this preparation were similar to that of Apilife VAR [80]. Thymol was also employed as "Frakno thymol frame". It consists of thymol crystals placed into an evaporation box built into a brood frame and hung next to the brood nest, to be replaced about 2-3 times a year. However, its efficacy was judged insufficient for long-term treatment [81]. Finally, the latest thymol-based acaricide is Apiguard®; a gel formulation designed for
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a more controlled release. It was tested both against Varroajacobsoni and Acarapis woodi [82]. Varroa mortality in treated groups was about 4 times higher than natural mortality, whereas in treated Acarapis infested groups it was 6-8 times higher. Mattila et al. found this preparation not toxic for adult bees or for 4-5 days old larvae, but it was quite toxic for younger larvae [83]. Honey production during treatment was significantly reduced in colonies treated with Apiguard, though the yield of the entire season was not significantly different from controls [84]. A patent application has been presented for Varroa control by means of acyclic and cyclic terpenes, mainly linalool, linalyl acetate, eugenol and anethole; total control of the mite was observed when honey bees were fed 50% sugar syrup containing 1% linalool [85]. Besides thymol, other terpenes have been tested for their toxicity against Varroajacobsoni. Imdorf et al. determined in vitro the effective miticidal air concentrations, but with minimal effects on the bees as follows: 5-15 |ig/litre air for thymol, 50-150 jig/litre for camphor and 2060 |iig/litre for menthol; 1,8-cineole was too toxic for honey bees [86]. Another interesting paper considered the efficacy of different isomers of menthol on Acarapis woodi [87]. The natural crystals obtained from the plant, synthetic crystals and the L-form gave more than 96% mite mortality, while the D-form crystals only a 37% mortality. Colin et al. proposed a different method for characterizing the biological activity of essential oils on Varroa mites [12]. Starting from the supposition that all the field or laboratory experiments were based only on counting the dead mites after therapeutic administration, they affirmed that to describe a more real pattem of the biological activity of these compounds, lethality tests had to be complemented by behavioural tests. Authors used four essential oils: Thymus vulgaris (containing 30% of thymol). Salvia officinalis (with thujanols as major components), Chenopodium spp. (with ascaridole as main constituent), and Anona spp. (with geraniol and linalool among the principal components). They first determined the lethal dose, in order to choose the proper doses for the following behavioural tests. These consisted in repellency test with choice and without choice. They concluded that the essential oils of Thymus, Salvia and Chenopodium not only exhibited an acute toxicity in direct contact tests, but they prevented also the treated bee pupae from being parasitized: the repellent effect was so strong that a majority of
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mites do not move close to the treated pupae and probably died from starvation [12]. A recent survey about essential oils and their pure constituents used to control Varroajacobsoni, contained three interesting tables that reported the toxicity of essential oils for V. jacobsoni and Apis mellifera after 24, 48 and 72 hours in a topical application and in an evaporation test, and the effects of essential oils on behavior and reproduction of V. jacobsoni and on the bee brood [63]. The most interesting oils were those of cinnamon and clove, with 100% mite mortality after 24 h and no significant toxicity on honey bees. Furthermore, clove essential oil produced small brood mortality, and it was an inhibitor of mite reproduction. Other effective oils were anise, fennel, lavender, rosemary and wintergreen, which killed 100% mites after 48-72 hours. On the contrary, the oils obtained from garlic, onion, oregano and thyme, were found to be very toxic for honey bees. Among pure constituents, camphor, linalool, linalyl acetate and pinene resulted small brood mortality and inhibited mite reproduction. The variable responses observed are probably the main drawback for the practical use of essential oil as miticides. It must be pointed out that the same plant species often produces essential oils with variable composition because of environmental and/or genetic factors; many species have varieties, the so called chemotypes; for instance at least seven chemotypes are known for Thymus vulgaris [88,89]. Also the extraction process influences the composition of the essential oils. For these reasons, it is advisable that authors report the composition of the essential oils used in the biological investigations. Unfortunately, only one paper reported this important information [64]. Summarizing, the use of natural products as miticides in apiculture, with the exception of some substances, is not widespread. In extensive laboratory tests many compounds showed significant acaricidal properties. However, very few of them have proven to be effective when applied in field trials. Considerable variations in local environmental and colony conditions can affect efficacy. In case of mixtures, such as essential oils, the difficulty in obtaining standardized compounds also affects treatment predictability. Nevertheless, identifying new acaricide compounds with low toxicity to honey bees is fundamental for providing candidate compounds for field trials. Furthermore, the development of
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effective delivery systems could greatly contribute to the effectiveness of some promising molecules infieldconditions. VETERINARY AND HUMAN MEDICINE a) Ticks In many areas of the world, particularly the tropics, arthropod-borne diseases are among the major limiting factors to the efficient production of livestock and poultry. These diseases result in weakening, lameness, blindness, wasting, congenital defects, abortions, sterility, and death of the infested animals. Some exotic arthropod-borne diseases of livestock are zoonotic and affect humans as well as animals. Some of the most devastating of all animal diseases caused by arthropod-borne blood protozoa, include babesiosis of cattle, sheep, goats, horses, and swine; theileriosis, the East Coast fever syndrome, and Mediterranean fever; the trypanosomiases causing illness in cattle, sheep and goats, camels, pigs, dogs, and many wild game species; as well as several arthropod-borne protozoa that cause diseases of birds. The most prominent groups of arthropods that transmit etiological agents pathogenic to livestock are those that are blood-feeding (hematophagous), such as ticks. They are the most versatile vectors, for they parasitize all vertebrate groups, except fishes. The tick-borne diseases they transmit are among the most significant animal health deterrents to efficient livestock production. Ticks are obligate ectoparasites of vertebrates. The family Ixodidae comprises approximately 80% of all tick species, with the most economically important ixodid ticks attacking livestock in tropical regions belonging to the genera Amblyomma, Boophilus, Rhipicephalus and Hyalomma, Amblyomma sp. (Acari: Ixodidae) is a three-host hard tick, commonly found in cattle, sheep and goats in Asia and Africa. The African species are of the greatest economic significance because they transmit the rickettsial pathogens Cowdria ruminantium, which causes Heart-Water (Nairobi sheep disease) and Coxiella burnetii in cattle and sheep. Furthermore, it is associated with streptothricosis, the actinomycete infection of the skin of cattle, caused by Dermatophilus congolensis [90,91]. It also causes theileriosis in cattle [92]. Infected manraials bite the sites of infection or rub their bodies against hard objects damaging their skin. As a result, they do not fetch a good prices in livestock
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markets and their skin can be worthless for use in the manufacture of leather goods. Furthermore, because of the blood sucking ticks, the animals become weak and anemic, resulting in reduced milk and meat production; severe infestations usually leads to premature death. Many synthetic acaricides have been used to control this tick, including organochlorine derivatives, organo-phosphorous compounds and carbamates. However, besides resistance problems, these compounds are expensive, especially for third world countries, and sometimes they cause toxicity problems in animals and farmers [93,94]. Ndumu et al. evaluated the effectiveness of Azadirachta indica seed oil against the larvae of this parasite [95]. They administered the oil as hydroalcoholic solutions ranging 4.2-100% and computed the mortality within 60 hours. Authors observed that the mortality of larvae was concentration and time dependent; 100% mortality was observed with 100% pure neem oil after 48 h. The LD50 of different concentrations were 33.3% (56 h) and 66.7% (48 h). Author also observed little or no adverse effects on treated animals. Furthermore, they stated that the open wound caused by tick bites and therefore exposed to potential fungal and bacterial attacks, could be protected by the microbicidal properties of the neem oil. Previously, the effectiveness of neem oil was also observed by Williams and Mansingh against another tick species of the same genus. A, cajennense, another cattle tick [96]. Malonza et al. observed that the leaves of a Capparidaceous plant, Gynandropsis gynandra, exhibited repellent properties against all stages of Amblyomma variegatum [97]. In field conditions the ticks were not found up to 2-5 m from the plant in areas where this species was predominant, while, in the laboratory, ticks which were continuously exposed to its leaves, died. The effectiveness of the plant was most pronounced on juvenile stages and least pronounced on adults. Another plant, snakeweed {Gutierrezia sarothrae and G. microcephala, Asteraceae) was found useful against A. americanum [98]. Domestic rabbits that had received diets containing 5% or 10% of this plant leaves showed 39% and 33% reduction of tick attachment, respectively. Dichloromethane extracts of the leaves, topically administered on sheep skin, significantly reduced tick attachment with respect to control. A third species, Margaritaria discoidea (Euphorbiaceae), showed acaricidal properties against A, variegatum [99]. Its water extract was effective on
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nymphs, but not on adults. The hexane extract at 6.25% was found more effective, causing 100% mortality of adults. Its application at a 50% concentration on the ears of rabbits prevented the attachment of adult ticks for at least 4 days, while its direct application on engorging ticks induced mortality of 70% adults on rabbits and 50% adults on cattle in the field. Ivermectin is a macrolide antibiotic produced from a fungus first isolated from a soil sample in Japan, Streptomyces avermitilis. In the mid80s, ivermectin was introduced as probably the most broad-spectrum anti-parasite medication ever. It is effective against most common intestinal worms (except tapeworms), most mites, and some lice. It is also effective against larval heartworms (the "microfilariae" that circulate in the blood), but not against adult heartworms (that live in the heart and pulmonary arteries). Avermectins, to which ivermectin belongs, are agonist for the neurotransmitter y-aminobutyric acid (GABA). GABA is a major inhibitory neurotransmitter. In mammals, GABA-containing neurons and receptors are found in the Central Nervous System; while in arthropods and nematodes GABA is found primarily in the Peripheral Nervous System (neuromuscular junction). This difference in the localization of the GABA receptors, could be the reason for the large margin of safety of avermectins-containing products in mammals. The binding of avermectins to a neuronal membrane increases the release of GABA. GABA binds to the GABA receptor-chloride channel complex of postsynaptic neuronal membranes, causing an influx of chloride ions. This influx hyperpolarizes the neuronal membranes, making them less excitatory and decreasing nerve transmission. The hyperpolarization of neuronal membranes mediate a flaccid paralysis in arthropods and nematodes [100-101]. Ivermectin has been used in different way of administration for the control of Amblyomma. Soil et al. experimented an intraruminal bolus against natural infestations of five different African tick species on cattle, among which Amhlyomma hebraeum (the other species were Boophilus decoloratus, Hyalomma spp., Rhipicephalus appendiculatus ^ndi Rhipicephalus evertsi evertsi) [102]. Unfortunately, the observed reduction in the number of engorged female ticks was not statistically significant. An experiment aimed at controlling A. americanum in free-living white-tailed deer, Pound et al. prepared whole kernel com treated with 10 mg of ivermectin per 0.45 kg corn to use as
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bait [103]. Monitoring of the free tick populations through two years of study, showed 83.4% fewer adults, 92.4% fewer nymphs, and 100.0% fewer larval masses in the treatment versus control. In 1997, Miller et al. compared the treatment of pasture cattle with ivermectin (administered orally at 200 |ig/kg or by injection at 40 |ig/kg) against A. americanum [104]. They observed no significant difference in the number of unengorged, small, and medium sized female ticks to the untreated control compared with those treated orally. However, the number of large female ticks was reduced. Significantly, fewer small, medium, and large female ticks were found on the injection treated cattle, compared with the untreated controls. There were also significantly more unengorged females on the animals treated by injection than on those treated orally or left untreated. Wilson et al. evaluated the effects of ivermectin on the volume of blood ingested by A, americanum [105]. Adult females were collected from Bos taurus hosts, treated and untreated with ivermectin, and they observed that the mites feeding on treated animals contained smaller quantities of blood. Recently, a bioabsorbable injectable microsphere formulation has been developed to provide long-lasting delivery of the drug [106]. Some researchers noticed that some chemicals attract adult of Amblyomma [107,108]. Among these substances many were of natural origin, such as nonanoic acid, methyl salicylate, benzyl alcohol, benzaldehyde, heptadecane and squalene. Other authors exploited this feature to prepare some drugs in which the acaricides were associated to the pheromone-like chemicals to control Amblyomma [109,110]. Another "natural" way to control Amblyomma is to use its hyperparasitic fungi, such as Beauveria bassiana and Metarhizium anisopliae [111,112]. In adult of ^. variegatum, M. anisopliae induced a mortality of 37%, while B, bassiana induced no mortality. However, both fiingi induced significant reduction in engorgement weights, egg masses and egg hatchability; B, bassiana completely inhibited egg hatchability. Authors concluded that these fimgal species were capable of inducing high mortalities, decreased fecundity and egg hatchability, and they could represent a great potential for tick control. Their ability to reduce fecundity and egg hatchability is of greater importance than adult mortality, in fact a reduction of egg hatchability by 99-100% may mean a very significant reduction in the next generation of ticks, and has a greater
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impact on tick population than the direct mortaUty on engorging females, which may destroy only a few dozen ticks. Boophilus sp. are one-host ticks, which occur in all tropical and subtropical regions of the world, where they feed preferably on cattle. They are the main vectors of Babesia species, B, bovis and B. bigemina, causing babesiosis in cattle. Boophilus ticks, together with many other tick species, also transmit Anaplasma marginale, the rickettsia that causes anaplasmosis of cattle on all continents. Many natural remedies have been tested against this tick. Among them we can find essential oils and their purified constituents. Brazilian researchers evaluated the effectiveness of the essential oil and of its components a- and p-pinene, obtained from the grass Melinis minutiflora (Poaceae) [113]. All the chemicals showed lethal effect on Boophilus microplus larvae. Later, the same authors tested 1,8-cineole and «-hexanal, from the same oil, that showed individually 100% lethal effect on cattle-tick larvae within 10 min [114]. It must be pointed out that these two papers are among the few articles that report the composition of the tested essential oil. Two further essential oils, Cymbopogon citratus and C nardus (Poaceae), have been examined by Chungsamamyart and Jiwajinda [115]. They found that the oils extracted from fresh leaves, diluted in EtOH, exhibited a higher activity against adults and larvae of J5. microplus than the oils extracted from dried leaves. Fresh C citratus volatile oil exhibited 85-100% mortality against engorged female ticks at all the dilutions tested (up to 1:4), whereas fresh C. nardus oil exhibited 85-90% mortality up to 1:3 dilutions. On larvae, fresh C citratus volatile oil showed >90% mortality up to 1:16 dilution, while fresh C. nardus oil was endowed with a similar activity up to 1:12 dilution. The same research group also evaluated the activity of the peel oils and pure limonene of some cultivars and species of Citrus (Rutaceae) against the same ectoparasite [116]. The oils from C reticulata and C. maxima cv. Thong-dee showed a good acaricidal activity against engorged female ticks at the 1:10 dilution, activity being 2 times higher than that of limonene. C sinensis and C. maxima peel oils, diluted 1:10, exhibited high larvicidal activity, while the oils of C. hystrix, C. reticulata, C. suncris and C. maxima (immature fruits) showed moderate larvicidal activity, about 1.5 times stronger than that of limonene. The essential oils extracted from berries, bark, leaves and twigs of Pimenta dioica (Myrtaceae) were compared for their effectiveness against Boophilus microplus with that of
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eugenol, isoeugenol and four commercial synthetic acaricides [300]. The berry essential oil was more effective at inhibiting oviposition (no egg laying at 3 mg/g body weight) and causing mortality of the ticks (100% at 3 mg/g) than all the other extracts, the synthetic products and methyl eugenol. Authors hypothesized that the activity of the berry essential oil could be attributed to eugenol (100% toxicity at 3 mg/g and no egg laying at 2 mg/g), which accounted for more than 65% of the whole oil. Korpraditkul et al. conducted an experiment using a vetiver {Vetiveria zizanioides, Poaceae) extract to control cattle tick [118]. Three ecotypes of vetiver grass were used, 'Si Sa Kef, 'Uthai Thani', and Thetchabun'. Two methods of essential oil extraction were employed, steam distillation and solvent extraction (using two solvents, ethanol and dichloromethane). When adjusting the oil's concentration at 10%, applied to treat dairy cow tick at larval and adult stages as well as egg-laying stage, the results indicated that the chemicals extracted from the roots of different ecotypes possessed different efficiency in controlling ticks. The extract obtained by steam distillation of the dried 'Uthai Thani' vetiver root killed ticks at both stages at the highest rates, with mortality rate of larvae and adults of 50.7 and 20.0%, respectively. In addition, the condition of the root also played a role in controlling ticks; extract from dry vetiver root was able to control larval stage ticks better than adult stage, while extract from fresh root was able to control adult stage of ticks better than larval stage. It was also found that the oil extracted from vetiver root showed no significant difference in controlling ticks when compared with citronella. The ethanol extract from dried roots of Thetchabun' ecotype showed very good results, giving 99.4% mortality of larvae and inhibiting adults from laying egg at 46.7%. The results indicated that the extract from vetiver roots was able to control the growth of ticks during larval and adult stage, and including egg-laying stage of ticks. The ethanol extract had the highest potential for controlling cow ticks. According to these authors, if the extraction method is improved, or its concentration is adjusted to the optimum level, the experiment may give better resuhs. Many non-volatile extracts have been studied for their effectiveness against Boophilus sp. Those obtained from legumes plants seem to be particularly promising. Cruz-Vazquez et al. evaluated the tropical species Stylosanthes humilis and S. hamata (Fabaceae) against the larvae of Boophilus microplus in plots experimentally infested [119]. They
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observed that after four weeks the percentage larval survival was 5.1% for S, humilis, 7.5% for S. hamata, while in control plots it was 18.9%. Khudrathulla and Jagannath used the methanolic extract of another species of Stylosanthes, S. scabra, on different life stages of three tick species, B, microplus, Haemaphysalis intermedia and Rhipicephalus sanguineus [120]. In vitro trials, conducted by the tea bag method, showed a dose-dependent activity both on larval and nymphal mortality, with the exception of H. intermedia nymphs, which were not killed by the extract. Finally, authors declared that, in preliminary assays, the aqueous extract showed better acaricidal properties. Regassa reported the results of a questionnaire survey about the traditional tick control methods used in three provinces of Western Ethiopia [121]. The most frequently employed drugs were the latexes of Euphorbia obovalifolia (Euphorbiaceae) and Ficus brachypoda (Moraceae), the juice obtained from the leaves of Phytolacca dodecandra (Phytolaccaceae) and Vernonia amygdalina (Asteraceae), the fruit juice of Solanum incanum (Solanaceae), the seeds of Lepidium sativum (Brassicaceae) mixed with fresh cattle faeces, the juice of crushed leaves and bark of Calpurnea aurea (Papilionaceae), and the commercially available spice of Capsicum spp. (Solanaceae) mixed with butter. The same author tested in vitro the activity of these preparations of Capsicum spp., E. obovalifolia, S. incanum and F. brachypoda in vitro against Boophilus decoloratus, obtaining 30-100% killing effects. Following in vivo treatments with the same extracts ofE. obovalifolia and F. brachypoda on naturally infested indigenous cattle, reduced infestation up to 70%. In another survey on the activity on Boophilus microplus of several crude EtOH extracts of Jamaican plants, the authors evaluated the ability of the drugs to kill engorged ticks and inhibit the oviposition or embryogenesis [122]. They associated an acaricidal index, ranging from 0 to 100, on the basis of the effectiveness verified. The most active species were Quassia simarouba (100) (Simaroubaceae), Symphytum officinale (99) (Boraginaceae), Nicotiana tabacum (95) (Solanaceae), Hibiscus rosa-sinensis (93) (Malvaceae), Ricinus communis (82) (Euphorbiaceae), Salvia serotina (80) (Lamiaceae), Stachytarpheta jamaicensis (79) (Verbenaceae), Ocimum micranthum (76) (Lamiaceae), and Spigelia anthelmia (75) (Loganiaceae). Recently, Chungsamarnyart and Jansawan tested the extracts in water or in 10% EtOH of the mature Tamarindus indicus
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(Fabaceae) fruits, from which the seeds had been removed, on engorged female of Boophilus microplus using the dipping method [123]. The pure organic acids contained in the fruits, oxalic, malic, succinic, citric and tartaric acids, were bioassayed at 0.5% and 1% concentrations. Among the extracts, the most effective were the crude 1:2 ones. Among the organic acids, 0.5% and 1% oxalic acid exhibited the highest acute acaricidal activity, while 1% tartaric acid showed the highest delayed activity. All the compounds caused patchy haemorrhagic swelling lesion on the parasite skin, as documented by photos. The EtOH extracts of five marine algae were assayed against Boophilus microplus [124]. The author evaluated the effects of topical applications on mortality, oviposition and embryogenesis in the ticks. The most toxic extracts were those of Laurencia obtusa (Rhodomelaceae) and Liagora elongata (Liagoraceae), whereas Liagora farinosa, Padina vickerisiae (Dictyotaceae) and Stypopodium lobatum (Dictyotaceae) resulted barely effective. On embryogenesis the results were different, with L obtusa, L farinosa and S. lobatum as the most effective extracts. Different preparations obtained from neem tree, Azadirachta indica, were tested on Boophilus sp. Williams investigated the adverse effect of EtOH extracts of neem (and of Artocarpus altilis, Moraceae) on egg laying and hatching in B, microplus [125]. Egg laying was inhibited by 50% at an extract concentration of 0.54 |ig/tick; the same dose also caused a 65% hatching failure; the extracts of ^. altilis were more effective (0.46 |Lig/tick and 80%, respectively). The activity of the extracts has been reported to be due to the inhibition of protein and lipid sequestration by ovaries and oocytes. Kalakumar et al. compared the activities in vitro and in vivo of neem oil with those of Annona squamosa (Annonaceae) seed oil and pyrethrins against B. microplus [126]. Neem oil was the least effective substance: it was only 60-75% effective in infested cattle and buffaloes and it was unable to inhibit oviposition, while the other two extracts were 100% effective both against infestation and oviposition. Neem oil was also tested in association with Eucalyptus (Myrtaceae) and Pongamia (Fabaceae) oils on Boophilus microplus infested cattle and goats [127]. The most effective mixture was neem/eucalyptus oils. Authors evidenced a significant reduction of total proteins and total lipids in the treated ticks. The herbal formulation AV/EPP/14, containing extracts of A cor us calamus (Araceae), Azadirachta indica (Meliaceae), Pongamia pinnata
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(Fabaceae), Cedrus deodara (Pinaceae) and Eucalyptus globulus (Myrtaceae), and Pestoban, an Indian herbal solution containing extracts of Cedrus deodara, Azadirachta indica and Embelia ribes (Myrsinaceae), were tested by various researchers. AV/EPP/14 was 100% effective against larvae and nymphs of Boophilus microplus within 24-48 h from application; furthermore, it reduced 95% egg laying and hatchability [128,129]. Pestoban was found effective in a single application in light infestations in Buffaloes and cattle, while a second application was required for heavy infestations [130]. Similar results were obtained by Srivastava and Sinha, which observed that the larval and nymphal stages of ticks were more susceptible than the adults [131]. On calves, this herbal preparation did not induce inflammatory or other tissue changes, contrary to synthetic products which caused severe tissue reactions [132,133]. Many avermectin derivatives (see above) have been tested on Boophilus spp. Maske et al. observed tick elimination within 72 hours and prevention of reinfestation for 30-32 days with ivermectin [134]. Better results were obtained when ivermectin was administered as an intraruminal sustained-release bolus: treated animals showed significant less re-infestations of Boophilus annulatus for 90 days [102]. These results were recently confirmed by Miller et al. [135]. In Brazil a subcutaneous injection of 1% ivermectin (200 |lg/kg) showed a very good efficacy for about one month against B. microplus [136]. Also doramectin was highly effective in removing tick populations and in controlling reinfestations under conditions of continuous field challenge [137-140]. Many purified natural compounds were assayed for acaricidal activity against Boophilus sp. From the EtOH extract of the aerial parts oiBontia daphnoides L. (Myoporaceae), Williams et al. isolated the sesquiterpene furan epingaione. Fig. (2), that showed growth regulatory activities on gravid adult female of Boophilus microplus [141]. It inhibited 50%) egg hatching at 0.4 mg/g tick body weight. The activity was due to the inhibition of the sequestration of protein into eggs; histological examination of ovarian sections from the treated ticks revealed significant degeneration of funicle cells and reduction in yolk content. The dichloromethane extract of Hyptis verticillata (Lamiaceae) yielded the sesquiterpene cadina-4,10(15)-dien-3-one. Fig. (2) [142]. Besides its insecticidal activity against Cylas formicarius, this chemical inhibited the
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metabolism of lipids during embryogenesis of 5. microplus eggs in a doserelated manner. Thus, 69.78% lipid was metabolized in the control eggs, compared to 59.61% and 35.93% in the eggs produced by ticks treated with 0.9 and 1.8 mg/g of the cadinene derivative, respectively. Authors speculated that the inhibition of oviposition could be due to the effect of this substance on neuromuscular binding sites, since egg laying in Acarina is a neuromuscular process.From the EtOH extract of the roots of the herbaceous tropical plant Petiveria alliacea L. (Phytolaccaceae), dibenzyltrisulfide. Fig. (2) was isolated [143]. This compound showed a LD50 value of 0.92 |Lig per adult tick in topical treatments; the same values for synthetic acaricides were 5-10 times higher. Dibenzyltrisulfide was also effective as inhibitor of oviposition, with a IOD50 of 0.221 |lg/tick (the doses of commercial acaricides were 1.3-29 times higher). The compound was capable in reducing the hatching success of eggs oviposited by the treated ticks.
Epingaione Cadina-4,10(15)-dien-3-one
Dibenzyltrisulfide Fig. (2). Structures of epingaione, cadina-4,10(15)-dien-3-one, and dibenzyltrisulfide
Williams et al. tested the acaricidal activity of five pure natural phenylpropanoid derivatives, the compounds that accounts for 80-85% of the essential oil of Pimenta dioica [144]. Eugenol was the most effective compound in killing the adult ticks and in reducing egg laying and hatching at 3.2 mg/kg body weight. For mortality, the effectiveness was 100% for eugenol, 50% for isoeugenol, 40% for safrole, 30% for methyleugenol, while benzo-l,3-dioxole was ineffective; famesynic acid, a
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commercial insect growth regulator, was comparable to methyleugenol. Similar results were observed for oviposition and egg hatchability. Authors gave some consideration to the structure/activity relationships: with respect to eugenol, which was the most effective compound. Benzo1,3-dioxole, which lacks the propenyl substitution, was not active; substituting the hydroxyl by methoxyl reduced the activity, while changing the olefin center from 1-2' to 2 - 3 ' in isoeugenol reduced the killing efficacy by 50%. The methylene bridge in safrole was more effective than the methoxy groups in methyleugenol. The aqueous solutions of the monoterpenes piquerol A and B, purified from Piqueria trinervia (Asteraceae), showed an acaricidal potential on larvae of B. microplus, but neither compound prevented oviposition. Piquerol A, Fig. (3), was also toxic for gravid female ticks [145]. Spinosyns are natural metabolites produced under fermentation conditions by the actinomycete Saccharopolyspora spinosa. One such product, with the proposed common name of spinosad, a mixture of spinosyn A and spinosyn D, Fig. (3), has been developed by DowElanco and evaluated against B. microplus [146]. The results of this study demonstrated that a single whole-body spray treatment with spinosad, applied to cattle infested with all parasitic life stages of the tick, provided 85-90% control and almost complete protection against larval reinfestation for 1-2 weeks. Recently, many hyperparasites of the tick have been evaluated as control methods, in particular the entomogenous fungi Metarhizium anisopliae [147-149], Beauveria bassiana [112], Verticillium lecanii [150] and the bacteria Cedecea lapagei 117 and Bacillus thuringiensis var. kurstaki [151]. Other important tropical ixodid ticks species belong to the Rhipicephalus and Hyalomma genera. They can be vectors of many pathogens, such as the medically important Coxiella burnetii and Rickettsia conorii and the canine ones Anaplasma, Babesia and Ehrlichia spp. These two ticks have been also found on humans who venture into tick-infested caves and burrows [152,153]. In India, the two parasite species infesting goats, have been treated with an emulsion of tobacco leaf extract, mustard oil, DDT and copper sulphate, securing their removal within 2 days and preventing reinfestation for 33 days [154]. Comparison of the acaricide effectiveness of Annona squamosa (Annonaceae), Azadirachta indica (Meliaceae) oils
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and pyrethrins against Rhipicephalus haemaphysaloides and Hyalomma anatolicum showed 100% efficacy for A, squamosa and pyrethrins, whereas neem oil was only 60-75% effective [126].
Piquerol A
R R=H spinosyn A R=CH3 spinosyn B Fig. (3). Structures of piquerol A and spinosad.
The Indian herbal preparation Pestoban (see above) have been evaluated by many authors, obtaining 50-100% control on adults or larval stages of the two ectoparasites [130-132,155]. Also, the herbal ectoparasiticide AV/EPP/14 (see above) showed similar results on different hosts [129,156-159]. An African ground mixture of natural products, containing dried tobacco leaves and 'Magadi Soda' (principally sodium bicarbonate), commonly sold in local markets in East, West and Central Africa, prevented the completion of all feeding phases of Rhipicephalus appendiculatus, suppressed the oviposition capacity of the engorged ticks and drastically reduced the hatchability of the eggs. Larvae and nymphs were killed within 24 h from the application of the substance, while many adults were killed within 2-3 days. This product could replace commercial acaricides among resource-poor farmers in Africa [160]. An African plant, Gynandropsis gynandra (L.) Brig.
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(Capparidaceae) showed repellent and acaricidal activities against all life stages of Rhipicephalus appendiculatus, and was particularly effective on nymphs. Field investigations indicated that ticks were not found up to 25 m from the plant in areas where the plant was predominant; this species could be introduced, as pasture plant, for tick control among resourcepoor farmers in Africa [97]. The same research group evaluated the tickrepellent potential of the essential oil and its constituents obtained from the same plant [161]. The repellency of the essential oil hydrodistilled from the fresh aerial parts against R, appendiculatus was tested at four different test concentrations (lO-'^-lO"! |LI1) using a climbing bioassay. Apart from methyl isothiocyanate, all the identified constituents were assayed and their effectiveness compared with the commercial arthropod repellent DEBT (A^,A^-diethyl-toluamide). At the higher doses (0.1 and 0.01 ]\X) the percentage repellency of the oil was comparable to DEBT, while at lower doses it was slightly less effective. The most active components were m-cymene, nonanal, a-terpineol, a-cyclocitral, pcyclocitral, nerol, geraniol, carvacrol, a-ionone, (£')-geranylacetone, nerolidol and cedrene (isomer not specified), all of which had repellencies comparable to that of DEBT at the higher treatment levels; next in hierarchy benzaldehyde, phenylacetaldehyde, p-ocimene, linalool, phenylacetonitrile and methyl salicylate were found. The essential oil obtained from the leaves of another African plant, Ocimum suave (Lamiaceae), showed repellent and acaricidal properties against all the stages of 7?. appendiculatus [162]. The LC50 of the essential oil, dissolved in liquid paraffin, was 0.024% in vitro, while a 10% solution killed all the immature forms and more than 70% of adults on rabbits, and further protected them from reinfestation for at least five days. Another plant species, Margaritaria discoidea (Euphorbiaceae) showed acaricidal activity on R, appendiculatus [99]. Its water extract was effective both on nymphs and adults. The hexane extract from dry wood was found even more effective, causing 100% mortality of nymphs and adults at 6.25%. Its application at a 50% concentration on rabbit ears prevented the attachment of adults for at least 4 days, while its application directly on engorging ticks induced mortality of 70% adults on rabbits and 50% adults on cattle in the field, thus resulting as effective as the standard concentration (0.05%) of the synthetic acaricide chlorfenvinphos. Many papers report studies about the use of abamectins (see above)
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against Rhipicephalus and Hyalomma spp. on various hosts, both as acaricides and as reinfestation preventives. All the papers reported good efficacy [102,134,155,163-165], with the exception of a case of Hyalomma dromedarii on dromedaries treated with ivermectin [166]. Finally, also for these ticks, the biological control with the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae [111,112] or different strains of the bacterium Bacillus thuringiensis [167,168], showed different degrees, of effectiveness. Other important ticks, mainly distributed in North America, are Ixodes spp. (Acari: Ixodidae); certain species are vectors for Babesia spp. or Anaplasma spp. of cattle; /. dammini is the vector for Lyme disease (Borrelia burgdorferi). These bacteria are transmitted to humans by the bite of infected ticks; individuals who live, play or work in residential areas surrounded by tick-infested woods or overgrown brush are at risk of getting Lyme disease. Rash and flu-like symptoms are present in early, localized disease, while disseminated disease includes arthritis, carditis and neurologic disorders; in the U.S. about 15000 cases are reported annually. The control of the tick is performed mainly by means of synthetic acaricides, and very few papers are present in the literature about use of natural products or derivatives. The first one reports the effectiveness of Urariapicta (Fabaceae) against /. ricinus [169], a plant used in Nigerian folk medicine for control of ectoparasites. The aerial parts were extracted with MeOH, and the residue partitioned with EtOAc and water; the water-insoluble fraction was further partitioned into alkaline-soluble and alkaline-insoluble fractions. Furthermore, another aliquot of aerial parts was extracted with water. All the total and fractionated extracts were assayed on non-engorged /. ricinus ticks. All the extracts showed acaricidal activity. Water extract was the least effective (35% mortality at 5% concentration). The MeOH extract was very effective, showing 100% mortality at 1% concentration; its waterinsoluble fraction killed 98.86% of the ticks at 0.8%. The alkalineinsoluble and the alkaline-soluble constituents of the above fraction showed 80% and 97.76% acaricidal activity at 1% and 0.5% concentrations, respectively. The effective fractions were analyzed for the presence of different classes of compounds and authors suggested that it was attributable to more than one class; in fact the phytochemical screening indicated the presence of phenolic and flavonoid derivatives in
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the alkaline-soluble fraction, while sterol and terpene derivatives were detected in the alkaline-insoluble fraction. Panella et al determined the acaricidal activity of the extracts of 13 plants on immature ticks (13-16 weeks old), using 10 different doses, ranging from 0.0001 to 2% [170]. Nine extracts resulted effective, in particular the essential oils obtained from Chamaecyparis nootkatensis, C. lawsoniana (Cupressaceae) (LC5o=0.151% and 0.487% w/v, resp.), Juniperus viriginiana and J. occidenatalis (Cupressaceae) (LC5o=0.328% and 0.633%, resp.), Calocedrus decurrens (Cupressaceae) (LC5o=0.343%), Thuja plicata (Cupressaceae) (LC5o=0.821%), Artemisia tridentata (Asteraceae) (LC5o=1.180%), Sequoia sempervirens (Taxodiaceae), (LC5o= 1.673%), Foeniculum vulgare (Apiaceae) (LC5o=0.744). Authors also assayed some pure terpenes, and four of them resulted effective, namely acedrene (LC5o=1.524%), cedryl acetate (LC5o=1.556%), thujopsene (LC5o=3.168%) and 4-terpineol (LC5o=3.860%). Another paper citing use of natural chemicals against Ixodes ticks, reports the isolation and the effectiveness against /. ricinus of two naphthoquinones from Calceolaria andina (Scrophulariaceae), Fig. (4) [171]. The nymphal stages were topically treated with 0.25 p.l of the compound in acetone, and the LD50 of the naphthoquinones resulted 120 and 50 ng, respectively. Highly significant, the acute toxicity of the two compounds on mammals was very low, 1366 and 1072 mg/kg respectively in oral tests, and >2000 mg/kg in dermal tests.
o BTG505R=H BTG504R=COCHb Fig. (4). Structures of naphthoquinones from Calceolaria andina.
Other "natural" methods to control Ixodes sp. contemplate the use of entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis [172], or flocks of free range helmeted guineafowl (Numida meleagris) feedy on infested meadows [173].
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b) Mange mites Mange is a common skin disease of animals caused by different species of mites. The most severe form of mange is caused by the mite Sarcoptes scabiei, which is also the cause of human scabies. Some forms of mange are known to all domesticated animals, no matter how well-taken care of or pampered they are. The main genera implicated are Sarcoptes, Notoedres (Acari: Sarcoptidae), Psoroptes, Chorioptes, Otodectes (Acari: Psoroptidae), Demodex (Acari: Demodicidae), and Knemidocoptes (Acari: Knemidocoptidae). In the management of livestock, both for production and breeding purposes, the mite genera mainly responsible for causing mange are Psoroptes, Chorioptes and Sarcoptes mites. Modem animal husbandry practices create favorable conditions for the multiplication of ectoparasites, such as mange mites. The subclinical form of the infestation, which reduces productivity considerably, is well recognized. Mange mites are mostly transmitted from animal to animal by contact. Since mange mites are able to survive outside the host, sheds, boxes and pasture fencing must be considered as sources of reinfestation. The increased incidence of mange and resistance to the usual forms of treatment, with increasingly frequent therapeutic failures, has prompted researchers to look for more efficient forms of acaricide therapy, which are more acceptable to human and animal patients. Although traditional topical treatments, including polysulfurous compounds, possess good antiparasitic action, they have numerous undesiderable characteristics including an unpleasant odour, require numerous applications, frequently provoke local irritation and occasionally treated human and animal patients develop signs of systemic toxicity. There are also other causes for the failure in treatment of scabies: reinfestation after the end of treatment, paucisymptomatic forms with rare mites (difficult to find), possible resistance of these mites to some antiparasitic compounds at atoxic concentrations, unmotivated human patients or with immune deficiencies that are strongly prone to parasitosis, and patients who are sensitive to the active ingredient or substances contained in commercial preparations [174]. There is no synthetic acaricide yet with which the eggs of the mange mites can be reliably destroyed. A second treatment is always necessary to kill the larvae, which have hatched from the eggs in the meantime. The mites are often concealed in the crusts and scabs.
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where they are often not directly reached by the acaricide during spraying; for this reason a mange remedy with vapour effect should be favoured. Psoroptes mites prefer hairy parts of the body; by piercing the skin and sucking lymph fluid they cause pustules that spread rapidly, burst and form typical yellowish-sticky scabs and crusts. They are particularly active in the cold season and infest sheep, cattle (especially fattening bulls), and horses. The animals suffer from intense itching, become restless, bite and rub the affected parts and the hair or wool becomes detached. In young animals growth is arrested at first, followed by severe emaciation and death if the infestation is heavy. While in the past psoroptic mange in cattle was considered rare, it is today one of the main problems in bull fattening management. Sarcopies mites, which are also the cause of human scabies, prefer hairless parts of the body, which is why mange in the pig, for example, is always sarcoptic mange. The mites burrow tunnels in the skin, suck lymph and feed on young epidermal cells. At first acute symptoms, such as skin reddening and pustules, are observed on the affected parts of the skin, causing severe itching; even a few mites can cause severe clinical symptoms. In horses and sheep Sarcoptes mites cause the so-called "head mange", in susceptible horses this can spread to the neck and shoulder region. In cattle sarcoptic mange can become a problem particularly in dairy herds, the body areas most affected being the head, neck and udder. Sarcoptes mites can also cause mange in dogs and cats. Sarcoptes mites can survive for approx. 2 weeks off the host. Chorioptes mites preferentially attack the fetlocks and the base of the tail, where they chew at the skin surface, thus causing inflammations, scaly lesions and a powdery coating. The so-called foot and rump or tail mange can occur in the horse, sheep but especially in dairy cows. Demodectic mange is caused by small lancet-shaped follicular mites, belonging to Demodex genus, whose parasitism is not always accompanied by pathological lesions. These mites are also encountered in healthy animals and man, and need additional factors in order to produce clinical demodicosis. The disease has greatest significance in dogs. In other species, such as horse, cattle, pig, sheep, goat and rabbit, host-specific Demodex species can occur, but they rarely cause disease. The genera Psoroptes and Sarcoptes have been subjected to intense
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Study, at least from the natural miticidal products point of view. Historically, in 180 BC, Cato the Censor advocated the anointing of sheep after shearing, with equal parts of olive oil dregs, water in which lupines had been steeped and the lees of good wine to control the disease. During the 19th Century, many internal cures appeared, including the feeding of sulphur, but all failed. Dipping was developed in the 19th Century, the method is still used today and it was the first to achieve any real success. William Cooper in 1843 produced the first commercial dip. Among natural products, many different materials were used, including hellebore and turpentine. Sulphur, nicotine and arsenic were the most commonly used and effective, however, they stained, damaged and devalued fleeces and caused sheep to lose weight [175]. In the literature, among the natural substances, there are more than 350 reports about the use of avermectins (see above) in mange control, too many for a complete survey (for the most recent ones, both in human and veterinary medicine, see i.e. [176-179]). Despite the large use of these microbial derivatives, no cases of resistant mites have been reported till now. Our research group has evaluated various natural substances for the control of sarcoptic mange. In 1994 we tested in vitro the essential oil of the lamiaceous plant Lavandula angustifolia Miller (composition reported in the paper) and of some of its main constituents (linalool, linalyl acetate and camphor), against the adult stage of Psoroptes cuniculi [180]. All the tested substances were placed in 6 cm airtight petri dishes and resulted toxic for the mites, that were immobilized within 15 min to 1 h. Linalyl acetate and camphor were active only at the highest doses (6 |il/dish), with 96.7% and 30.0% mortality, respectively. The essential oil showed 98.3% mortality at 0.50 |Lil (100% at 2 |il), while linalool was the most effective compound (96.7% at 0.25 ^il, 100% at 0.50 ^il). In further research on the essential oil of Z. angustifolia and its constituent linalool, we evaluated the activity of this essential oil on the same parasite using inhalation, rather than direct contact between mites and compounds [181]. The mites were placed into 6 cm petri dishes covered with filter paper, that enabled gas-exchange, and inserted then into 9 cm petri dishes containing the test substance. The essential oil caused a mortality significantly higher than the controls in the range 2.5-6.0 |il (59-100%, respectively), while linalool showed a similar action down to 0.7 jil. To verify if linalool was the only effective constituent, we prepared an
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artificial mixture, in physiological saline, composed of 27% linalool, the percentage found in the essential oil, and tested in the same way. From the dose/mortality curves, linalool was the most active compound, while the mixture was the least active. We obtained the ED50 and ED90 values reported in Table (1). Table 1. Acaricidal activity of essential oil, linalool and artificial mixture from Lavandula angustifolia on Psoroptes cuniculL
EDsodil)
ED9o(lll)
Linalool
1.633
3.505
Essential oil
2.387
3.350
Mixture
5.418
10.558
Compounds
The static headspace analysis of the essential oil, showed that linalool was the most volatile constituent, and indicated that this substance could easily reach the mites in this kind of experiment. At higher dosages, the miticidal activity of the essential oil cannot be ascribed only to linalool, in fact the oil was more active than the artificial mixture. Moreover, the computed ED50 and ED90 did not overlap, giving a clear indication that these substances really had different activities, and demonstrating that linalool was not the only active compound in the oil. We also submitted to GC analyses the solution obtained by crushing the dead mites in Et20 after the bioassays with the essential oil. This examination revealed only linalool, so it can be stated that the miticidal activity by inhalation of the essential oil of L. angustifolia is mainly due to its linalool content. Linalool was one of the most active compounds, so we have investigated it effectiveness for the topical treatment of parasitic otitis caused by P. cuniculi in the rabbit and the goat [182]. The naturally infested animals were treated with 2.5 ml of three different linalool concentrations (10%, 5% and 3%) in a mixture composed of vaseline oil (2%) and physiological saline (98%). Both rabbits and goats, treated with the solution containing 5% linalool, recovered completely; no animal presented signs related to any toxicological effect of linalool, but in some rabbits, treated with higher concentrations, a transitory erythema of the ear skin was evidenced. The therapeutic efficacy of linalool was similar to the drugs commonly
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employed for the therapy of ear mange (topical application of 2.5 ml of Acacerulen®, or systemic administration of 200 |ig/kg of Ivomec®), with the exception of a goat treated with Neguvon®, which remained positive even though the number of treatments (six) was much higher than that advised by the manufacturer (two). The same goat, treated later with 5% linalool, completely recovered. Previously, we have already observed the efficacy of linalool against rabbit psoroptic mange, but we also evaluated the presence of linalool residues in euthanized animals 24, 72 hours, 5,10 and 21 days after treatment. An analysis for residues in the skin, adipose tissue, skeletal muscles, liver, kidney, lung and in milk of pregnant females was also carried out. Significant amounts of linalool were found only in the skin and in the adipose tissue of animals sacrificied within 72 hours after the treatment. However, these residues were below the toxic dose of linalool, so it should not represent a hazard to human health if the meat of these rabbits was used as food [183]. In our opinion, a comparative study of the activity of each compound, even if it does not permit assessment of the potential synergy and antagonism among the components of an essential oil, could enable a determination of the necessary structures for their pharmacological action; this information should also allow the prediction of the biological activity of other structurally related chemicals, and the assessment of their possible modes of action. For this reason, we have also performed a study about the structure/activity relationships of some natural monoterpenes against P. cuniculi, both in direct contact and vapour diffusion assays [184]. In the former tests, all the hydrocarbons, either acyclic or cyclic (limonene, myrcene and y-terpinene), did not show any miticidal activity at all the doses tested (0.125-1% in physiological saline). Thus the double bond position and/or number seems to be unimportant for this kind of activity. In contrast, the terpene alcohols (linalool, geraniol, nerol, menthol, 4terpineol and a-terpineol) were able to kill nearly 100% of mites at the doses tested. Therefore, it appears that oxygenated functional groups potentiate the acaricidal properties among these compounds. Neither the acyclic nor the cyclic nature of the compound appeared to influence miticidal activity. Similarly, neither the site of linkage (to the ring or to a side-chain), nor the nature of the hydroxyl group (primary, secondary or tertiary), influenced the activity. The cis/trans isomerism (nerol and geraniol) also appeared unimportant. Thymol and eugenol killed nearly
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100% of the mites at all dosages used, indicating that a phenolic function can enhance the miticidal properties of terpenes. The low susceptibility of parasites to linalyl acetate, particularly at the lowest doses, could be related to the esterification of the oxygenated function. Estragole, structurally close to eugenol, but with a methylated phenolic group exhibited, at 1% concentration, an activity comparable with the same dose of eugenol, but at 0.25% this miticidal action decreased (63%) and fell to zero at 0.125%. These results indicate that the best miticidal activity, in direct contact tests, can be related to compounds with free alcoholic or phenolic groups. In vapour diffusion tests, at 6 |Lil, the results were comparable to the direct contact tests. Thus, while hydrocarbons were ineffective, alcohols and phenols maintained almost 100% toxicity. Lowering the dose to 3 |Lil, all the alcohols preserved nearly the same acaricidal activity, except nerol (83.3%) and 4-terpineol (41.7%). At 1 |Ltl geraniol, menthol and thymol maintained about 100% effectiveness, whereas the activity of linalool, eugenol, a-terpineol and nerol was diminished. Linalyl acetate and estragole, like hydrocarbons, were partially or completely ineffective at all doses tested. We have evaluated also the activity, in vitro and in vivo, against eggs, larvae, nymphs and adults of P. cuniculi of the essential oil (10, 5, 2 and 1%) and two water extracts (20% and 7.5%) oiArtemisia verlotorum (Asteraceae) [185]. The in vitro studies indicated that the essential oil was highly effective against this mite; it killed 100% of larvae, nymphs and adults at all concentrations and inhibited 100% egg hatching at concentrations of 10, 5 and 2% and 95% mortality was registerd at 1%. Both the aqueous extracts killed 100% of larvae, nymphs and adults, and the 20% concentration strongly inhibited (94%) egg hatching. The in vivo efficacy was evaluated for the oil diluted at 5% and the water extract at 20%. The compounds were applied directly to the infected ears (2.5 ml). The treatment with the essential oil resulted in a clinical and parasitological recovery in all the treated rabbits: neither clinical symptoms nor mites in ear cerumen were found in these rabbits seven days after the treatment. This was not the case for the aqueous extract, which completely cured only one of the treated rabbits; in the other ones clinical lesions disappeared, but eggs and mites were still present in the ears. Many other herbs or pure compounds have been tested against manges. Among them the most studied are probably the extracts of Azadirachta indica. In
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literature two contrasting studies are present: Dakshinkar et al. [186] reported a positive response against eggs, nymphs and adults of P. cuniculi, while O^Brian et al. [187] noticed a failure in the complete elimination of P. ovis on sheep. Other papers referred to positive effects, ranging from moderate to very promising, against Sarcoptes scabiei both on human and animal patients [188-190]. The same researchers evaluated also the activity of the tea tree {Melaleuca alternifolia, Myrtaceae) oil, which was found to give far better results than neem. Another promising extract against P, cuniculi seems to be the one obtained from garlic. Allium sativum (Liliaceae). Two different papers reported its effectiveness, both against adults or nymphs and eggs [186,191]. Dakshinkar et al. [186] also reported the efficacy of Annona squamosa extract. An emulsion obtained from tobacco leaves (Nicotiana tabacum, Solanaceae) and other synthetic ingredients gave protection for 29 days, besides Sarcoptes and Psoroptes, also against Demodex mites [154]. When used alone, tobacco was less effective than synthetic drugs [192]. During our research, we have investigated the effect in vivo of Thymus vulgaris essential oil (composition reported) in a group of budgerigars (Melopsittacus undulatus) with a natural infestation caused by Knemidocoptes pilae [193]. The animals were topically treated with 10% and 5% essential oil (diluted in DMSO) on beak, vent, legs, wings and aroimd the eyes. The results were compared with ivermectin and DMSOtreated controls. The clinical and parasitological recovery was observed only in the birds treated with 10% and 5% thyme essential oil and ivermectin. The two concentrations showed the same effectiveness, but at 10% the essential oil caused death in two animals, while in birds treated with 5% solution no adverse reaction was observed. No other investigation with natural compounds (apart from avermectins) against Knemidocoptes is present in literature. In India, a comparative study between the essential oil of Cedrus deodar a (Pinaceae) and benzyl benzoate have been performed [194] against sarcoptic mange in sheep. The drugs were topically applied (doses not given) and the essential oil, of unstated composition, was the most effective, producing a complete recovery of the treated animals after the fifth application, while treatment with benzyl benzoate gave only a partial recovery; the essential oil gave also better results in haematological responses. The general conditions of the animals improved after the first
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application, and the itching and rubbing disappeared after the third one. Benzyl benzoate is one of the most studied pure natural derivatives in mange control, both in man and animals, but it can be unpleasant to use because of its unpleasant smell, can cause itching, buming and stinging [195-197]. Furthermore, in literature many conflicting reports may be found about its real effectiveness. Positive results were described on humans infested by Sarcopies scabiei [190,198], against Psoroptes and Sarcopies mites in rabbits [199] and against Demodex folUculorum in man [197]. In contrast, failures or incomplete recovery were obtained against sarcoptic mites on pigs [200], Psoropies in rabbits [201] and Demodex in dogs [202]. In Rwanda, scabies is the most important problem in parasitic dermatology; in order to find new anti-scabies agents, Heyndrickx et al. [203] tested a series of 15 plants used in the Rwandese traditional medicine to treat this disease. The plants were extracted in a percolator with EtOH and assayed at 30 mg/ml. The 100% active extracts were assayed at lO-l, 10"^, 10"^ and 10'"^ mg/ml. The hexane, CHCI3, water and EtOH extracts of the active plants were also bioassayed. Out of 15 plants tested, four showed a 100% Psoropies mortality: Heieromorpha irifoliaia leaves (Apiaceae), Neorauienenia miiis roots (Fabaceae), Penias longiflora roots (Rubiaceae), and Psorospermum febrifugum roots (Guttiferae). The EtOH extracts of A^. mitis and P. longiflora showed the greatest activity, killing the mites up to 10"^ and 10"^ mg/ml, respectively. Also the hexane and CHCI3 extracts of these two species showed good acaricidal activity: both the extracts were effective up to 10'^ mg/ml in the case of A^. miiis, and up to 10"^ mg/ml in the case of P. longiflora. Several commercial herbal preparations have been tested, mainly in India, against mange mites. Among these we can found Himax (M/s Indian Herbs, Saharanpur), containing Cedrus deodara, Polyalihia longifolia and P. excessa. This preparation always showed good acaricidal action against psoroptic, sarcoptic and demodectic mites on dogs, goats, sheep, rabbits [191,204-206]. Charmil, another herbal preparation (Dabur Ayurvet Ltd., India), containing extracts of Cedrus deodara and Pongamia pinnaia, showed even better results against Sarcopies, Psoropies and Noioedris mites on buffaloes, cattle, pigs, dromedaries, dogs and rabbits [207-212]. Other very effective commercial herbal preparations are Ectozee and Pestoban (containing extracts of Cedrus deodara, Azadirachia indica, Emhelia ribes) and AV/EPP/14 {Acorus calamus, Azadirachia indica,
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Pongamia pinnata, Cedrus deodara. Eucalyptus globulus). They have been tested against various mange mites associated with different hosts (i.e. see [213-217]). In comparison, for the genera Otodectes and Chorioptes, with the exception of avermectins, no natural compound has been evaluated. c) House Dust Mites The term "house dust mites" is applied to a large number of mites found in association with dust in dwellings. Unlike some other kinds of mites, house dust mites are not parasites of living plants, animals, or humans. House dust mites primarily live on dead skin cells regularly shed by humans and their animal pets. Skin cells and squames, commonly called dandruff, are often concentrated in parlour and sitting room, mattresses, frequently used furniture and associated carpeted areas, and may harbour large numbers of these microscopic mites. For most people, house dust mites are not harmful. The medical significance of house dust mites arises because their microscopic moulted skins and faeces being a major constituent of house dust, induces allergic reactions in some individuals. For those individuals, inhaling the house dust allergen triggers rhinitis or bronchial asthma. Expert panel reports and position statements from the European Union, the US National Heart, Lung and Blood Institute (NHLBI), and the American Academy of Allergy, Asthma and Immunology (AAAAI) have recommended dust mite allergen avoidance as an integral part of asthma management [218-221]. House dust mites belong to different genera and species, the main ones are Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei (Acari: Pyroglyphidae). However, there is a great variation in the acarid fauna among the different regions of the world. The diversity of mite fauna in a given (micro)habitat is not only due to the direct influence of environmental temperature and humidity upon the mite development and survival, but ecological and evolutionary factors may also play a role in mite diversity. The term 'house dust mites' is used originally to refer to those mites belonging to Pyroglyphidae. At present, the term 'dust mites' is more widely used, and this is in reference to all pyroglyphid and nonpyroglyphidites that are implicated in dust-borne respiratory allergy. Dermatophagoides pteronyssinus (literally "skin-eating mites") is considered as the true house dust mite and has a cosmopolitan
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distribution. Together with D. farinae (=flour, also infests stored food), it accounts for 80-90% of the total mite population generally found in houses. No pesticides are currently labeled for house dust mites. However, some commercial products are available for treatment of house dust mites and their allergens. The active ingredients are benzyl benzoate and tannic acid. Benzoic acid esters, such as benzyl benzoate, are very effective acaricides in both laboratory and field evaluations. Health risks appear to be slight as benzoates are rapidly metabolized in the body to hippuric acid, which is excreted in the urine. Most of the studies evaluated the effectiveness of these compounds on house dust mites, and all the authors agree about their effectiveness and safety, both in laboratory tests and homes, i.e.: [222-226]. Other studies concerned the activity of some plant essential oils. Among these, Chang et al. investigated the antimite activity of the essential oil and their constituents obtained from the heartwood of Taiwania cryptomerioides (Cupressaceae) against Dermatophagoides pteronyssinus and D. farinae [227]. The tests were performed in resin plates, using Et20 solutions containing various concentrations of the essential oil or pure components; the mites were introduced after air drying. The results showed that, at either high (12.6 [xg/cm^) or low concentration (6.3 jiig/cm^), the activity in decreasing order was a-cadinol (100%), T-muurolol (100% and 80% on D. pteronyssinus, 83.3% and 56.7% on D, farinae), ferruginol (80% and 56.7%, 68.1% and 36.7%, respectively), and T-cadinol (70.0% and 4.7%, 20.4% and 14.1%, respectively). The mortalities due to the treatments with 12.6 [ig/cm^ of the whole essential oil were 67% and 36.7% on D, pteronyssinus and D. farinae, respectively. Authors suggested some structure-activity relationships, in particular an equatorial OH at C-9 (a-cadinol) seems to be an important factor for antimite activity. In contrast, the type of ring junction (C-5/C-10) was less important: whether in cis configuration (Tcadinol) or trans (T-muurolol) with axial C-9 OH, Fig. (5), their antimite activities were lower than a-cadinol. Yatagai et al. studied the essential oils of the leaves of six Melaleuca species (Myrtaceae), a well-known insect repellent plant [228]. The oil obtained from M hracteata exhibited the strongest activity against D. pteronyssinus, killing all mites after 24 hours at the two doses tested (0.13 and 1.28 jig/cm^). M argentea, M. dealbata and M saligna showed mild
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activities, with mortalities greater than 50% at the lower dose after three days. Although the essential oils of M symphyocarpa and M acacoides at the higher dose killed all mites after three days, the mortalities at 1/10 of that dose were only 13% and 0%, respectively. Other Japanese researchers tested the activity of the essential oils obtained from the leaves of Lauraceae trees {Cinnamomum camphora, C. japonicum, Persia thunbergii, Actinodaphne lancifolia, Neolitsea sericea and Under a umbellata) [229]. A^. sericea oil showed the greatest activity against both D. pteronyssinus and D. farinae. The major active constituents were acadinal, caryophyllene oxide and the rare furane sesquiterpene isosericenine Fig. (5). D. farinae resulted more susceptible than D. pteronyssinus.
a-cadinol
T-cadinol
COOMe T-muurolol
isosericenine
Fig. (5). Sesquiterpenes from Taiwania cryptomerioides and Neolitsea sericea
House dust mites were of interest also for our research group. In particular, we have evaluated the activity of the essential oils of four plants, Lavandula angustifolia, L stoechas, Mentha x piperita (Lamiaceae) and Eucalyptus globulus (Myrtaceae), against a mite of stored food, Tyrophagus longior (Acari: Acaridae) [230,231]. We have analyzed by GC-MS all the essential oils and applied two different methods to test the activity of these compounds: one by direct contact and the other by vapour diffusion. In the direct contact assays five different quantities of
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each undiluted substance (6, 2, 1, 0.5 |Lil) were spread on the internal surface of petri dishes. The activity by inhalation was tested using two petri dishes of different sizes: the smaller one, containing the mites, was covered with a filter-paper disk and enclosed in a bigger dish containing 6 or 2 |il of each undiluted substance. At the highest doses, the essential oils of the two lavender species and of peppermint killed 100% of the mites, both by direct contact and inhalation. Eucalyptus oil was the least active. We have also tested the activity of the main constituents of the above essential oils, specifically linalool (27.3% in L angustifolid), linalyl acetate (32.1% in L. angustifolia), camphor (10.0% in L stoechas), fenchone {A12% in L. stoechas), 1,8-cineole (76.0% in E, globulus), menthone and menthol (31.8% and 21.7%, resp., in M. piperita). Among these compounds, menthol showed the highest activity, killing 100% of the mites at the lowest dose (0.25 |il) by direct contact and at 6 |il by inhalation. Linalool, fenchone and menthone showed good acaricidal activity (100% mite deaths at 2.0, 1.0, 0.5 |LI1 in direct contact and 6.0 |Lil each by inhalation). 1,8-cineole was the least effective compound, killing 81.7% mites at 6.0 |Lil and 50% at 6.0 |LI1 in direct contact and inhalation tests, respectively. At present, we are evaluating the acaricidal activity of the essential oils and main pure components of the branches of four Pinus species against another pest of stored food, Tyrophagus putrescentiae (Acari: Acaridae) [301]. The species used in this study were P, pine a, P. halepensiSy P. pinaster and P. nigra, their essential oils being characterized by GC-MS. The acaricidal tests were performed against mites isolated from samples of seasoned Parma ham, avoiding direct contact of the substances with the mites, but evaluating only their volatile fractions. Each compound was tested at 8 and 6 |LI1. All the essential oils, with the exception of P. nigra, had a good acaricidal activity. P. pinea oil was the most effective one, killing 100% mites at 8 |LI1 and 20% at 6 |LI1. P. halepensis and P. pinaster oils killed 60% and 53% mites at 8 |Lil, respectively, while at 6 |il they were ineffective. P. nigra oil was completely ineffective, a-pinene, p-pinene, myrcene, limonene, 1,8cineole, P-caryophyllene and germacrene D, the main constituents of these oils, were assayed in the same way. 1,8-cineole was the most active compound, killing 100% mites at 8 and 6 |il, followed by limonene which killed 100% mites at 8 |il and 32% at 6 |xl. All the other compounds were completely ineffective. Thirteen terpenes were tested by Sanchez-Ramos
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and Castanera against Tyrophagus putrescentiae [232]. The authors used myrtanol, pulegone, pinene, valencene, 1,8-cineole, linalool, linalyl acetate, fenchone, menthone, a-terpinene and y-terpinene, and unspecified isomers of caryophyllene and terpineol (valencene and caryophyllene were incorrectly considered monoterpenes). The miticidal assays were performed in cylindrical plastic cages by inhalation. Seven monoterpenes showed a high acaricidal activity, in particular pulegone, 1,8-cineole, linalool, fenchone, menthone, a-terpinene and y-terpinene. Their LC50 (vapour concentration, |il/l) were 3.7, 14.9, 7.0, 9.0, 4.7, 32.3 and 33.2, respectively. Other authors have concerned that the differences in our results [230,231] may either be due to the smaller size of T. putrescentiae in relation to T. longior and/or different specificity of the components on these two species. Interestingly, larvae and males of T. putrescentiae had a significantly higher mortality, about 2-fold, compared to females when exposed to the same dose of monoterpenes. All the compounds were ineffective on eggs. Essential oils can be also used as laundry additives for killing house dust mites [224,233]. In fact, both bedding and clothing may contain high populations of these mites and their allergens. Authors noted that washing in warm water removes most accumulated allergens but has little effect on mites, and effective long-term control requires killing of the mites. Higher water temperatures cannot always be used, so miticidal additives are required. Low concentrations of five essential oils have been evaluated: citronella, eucalyptus, spearmint, tea tree and wintergreen oils. They were mixed 4:1 with Tween 20 as dispersant and tested at 0.8%. All the oils killed more than 80% of mites after 30 mins, and with the exception of citronella, they killed more than 60% of mites after 10 mins. For shorter exposure times, tea tree oil was the most effective, killing 79% mites in 10 mins. Recently, French researchers extracted the bark of Uvaria pauciovulata (Annonaceae) with EtOH and with CH2CI2 and assayed the extracts on D. pteronyssinus [226]. The alcohol extract was weakly effective, killing less than 50% mites at 1.67 g/m^, while the CH2CI2 extract, at the same dose, killed 100% mites. Bioassay-guided fractionation of the non-polar extract led to the isolation of two active compounds, benzyl benzoate and a bis-tetrahydrofuran acetogenin, squamocin, Fig. (6). The EC50 were 0.33 and 0.06 g/m^ for benzyl benzoate after 1 and 24 hours, respectively, while for squamocin they
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were 2.7 and 0.6 g/m^, respectively. From the bark of Neolitsea sericea (Lauraceae) two acaricidal lanostane triterpenes, 24Z-ethylidenelanost-8en-3-one and 24-methylenelanost-8-en-3-one, Fig. (6), were isolated and characterized [234].
Squamocin
R=CH-CI^ 24Z-ethylidenelanost-8-en-3-one R=CH2 24-methylenelanost-8-en-3-one Fig. (6). Structures of squamocin from Uvaria pauci-ovulata and lanostanes from Neolitsea sericea
When mites were exposed to 16 [ig/cm^ of 12 and 13 for 72 hours, the percentages of immobilized mites were 17.8 and 31.4%, respectively, and 26.9 and 31.4% when exposed to 32 iLig/cm^. Authors affirmed that these data suggested that triterpenes having a 24-methylene group in the side chain were more powerful miticides than compounds having the 24Zethylidene moiety. Since Nathanson demonstrated that caffeine and other methylxanthines interfere with insect feeding and reproduction [235], American researchers have investigated its acaricidal effect on D. pteronyssinus cultivated in vitro and its main allergen levels [236]. There was a significant inhibition in the growth of treated cultures and in the production of allergens. However additional studies are necessary to determine the safety of caffeine on humans and animals. Other effective substances for control of house dust mites seem to be fungicides because fungal digestion of skin scales is a prerequisite for mite utilization [237]. Finally, an alternative to organophosphorus pesticide treatment in stored grain has been found in the use of inert substances,
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susch as diatomaceous earths [238,239]. These are dusts composed of Si02 from fossihzed diatoms. They act by absorbing waxy elements of parasite cuticle, causing desiccation. Diatomaceous earths have a very low^ mammalian toxicity and leave no chemical residues. These authors demonstrated the effectiveness of three different diatomaceous earths and an amorphous precipitated silica against two storage mites, Acarus siro and Lepidoglyphus destructor. Field treatments did not exceed 5 g/kg, and nearly 99% of any dust applied to the grain was removed during normal milling processes for flour production. AGRICULTURE Plant-feeding mites play important roles as agricultural pests of timber, fruits, vegetables, forage crops, and ornamentals. In many instances, lack of information about the correct identity of mites, as well as inadequate knowledge regarding their biology and ecology, have hampered our ability to effectively combat these mite pests. Their small size and cryptic appearance make mites difficult to detect, and thus infestations are often overlooked. Once established in a new area, certain biological characteristics allow rapid escalation to pest status. These include high egg production, various modes of reproduction (parthenogenesis, paedogenesis, and sexual), short life cycles, a myriad of dispersal techniques, and adaptability to diverse ecological conditions. These traits, combined with an exponential increase in world trade, have set the stage for potentially devastating situations that may threaten the sustainability of the world's agroecosy stems. Miticidal compounds, as in veterinary and human medicine, cannot be toxic for the plant host and no harmful residues must be found in foods: Furthermore, in agriculture an additional feature is requested: they must be devoid of undesirable effects on useful non-target organisms, like pollinators and predator arthropods [240-242]. There are several different species of mites that can cause damage to a wide variety of plants. The main species are Tetranychus sp., Oligonychus sp. (Acari: Tetranychidae), Phyllocoptruta oleivora, Tegolophus australis (Acari: Eriophyidae). Among these, the twospotted spider mite, Tetranychus urticae, a polyphagous pest, is probably one of the most dangerous for crops and ornamentals, particularly in glasshouse. Its high reproductive capacity enables it to cause serious damage in a short period of time. Furthermore, this parasite
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has developed resistance to many synthetic acaricides [see i.e.: 243-246], apart from the fact that many of these substances are toxic to useful nontarget arthropods [see i.e.: 247-249]. Numerous papers about its control by mean of natural substances are present in literature. Serbian investigators prepared the 1:1 EtOH fluid extracts of the aerial parts of Taraxacum officinale (Asteraceae), flowers of Sambucus nigra (Caprifoliaceae) and leaves of Juglans regia (Juglandaceae) [250]. They assayed these extracts and their 50, 10 and 2% dilutions against Tetranychus urticae isolated from Lamium purpureum plants. Taraxacum was the most active extract, killing 100% of the mites at 50% and about 90% at 10% dilution; at 2% it killed 57% of the mites. Sambucus showed a very similar effectiveness, with 96.4% mites killed at 50% and 91% at 10%, but at 2% only 31.7% of the mites were killed. Juglans extract was less active, killing 100% of the mites only at 100% concentration, and only 73% at 10%. Hiremath et al. compared the activity of MeOH extracts obtained from 21 different African plant species against adults of T. urticae using the leaf-dipping method [251]. The most active ones were the extracts from the whole plant of Celosia trigyna (Amaranthaceae) and Combretum micranthum (Combretaceae), leaves of Combretum glutinosum, and leaves and fruits of Prosopis chilensis (Fabaceae). The insecticidal properties of Meliaceae plants have been known for a quite long time, so Ismail evaluated the relative toxicity of Melia azedarach extracts and some synthetic acaricides against newly hatched larvae of T. urticae and third-instar larvae of an useful arthropod, its predator Stethorus gilvifrons [252]. The methanol extract of the plant was the most effective among the tested extracts, followed by acetone and petroleum ether extracts, respectively. The toxicity of plant materials was far less against the predator compared with two-spotted spider mite, whereas a synthetic acaricide was equally toxic to the pest and its predator. The study of the joint action revealed a strong synergism in the mixture of bromopropylate with the methanolic extract of M azedarach; interestingly, this mixture showed no effect on the predator. Melia azedarach extracts also greatly affected the fecundity of the parasite, especially when mixed with synthetic acaricides. The author suggested that M azedarach extracts could be used in integrated pest management programs for mite control. The crude alkaloids, the EtOH extract and the oil of the bulb of the omamental plant Pancratium maritimum, a member
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of the Amaryllidaceae family with strongly scented, white, narcissus-like flowers, was active against T. urticae. Their LC50 values were 0.2, 0.36 and 1.5%, respectively [253]. The lipophilic fraction of seeds, leaves and roots of Glossostemon bruguieri (Sterculiaceae), specifically the unsaponifiable part, was tested against T, urticae [254]. The leaves were the most toxic plant part to both adult and egg stages of T. urticae, with LC50 values against eggs of 1.7 mg/ml. At 1.25 mg/ml oviposition was totally inhibited. Artemisia absinthium (Asteraceae) is a well-known insecticide, and its water extract is used worldwide against aphids; however, the same extract showed very weak acaricidal activity [255]. The same was true for the water extract of Pinus sylvestris. When mixed 1:1, a clear synergistic interaction between absinth and pine shoot extracts was detected with the leaf dipping method against T. urticae, with 77.5% and 92.3% mortality on immature and adult stages, respectively. Synergism was also evidenced by mixing synthetic acaricides with jojoba {Simmondsia chinensis, Buxaceae) seed oil [256]. Thus, it was possible to reduce the dose of acaricides used for control of the two-spotted spider mite Tetranychus arabicus. Some correlations have been postulated about the natural concentration of foliar essential oils in six strawberry (Fragaria sp., Rosaceae) cultivars and their different degree of susceptibility towards T. urticae [257]. On the basis of leaf damage, the cultivars were classified as highly susceptible, intermediate to susceptible, intermediate to resistant and resistant. It was observed that the first two classes had a lower linalool, a-terpineol and p-cyclocitral content. Resistant plants could be used to obtain effective acaricidal compounds, as demonstrated by Amer and Rasmy [258]: when larvae of T. urticae were reared on excised leaves of Conyza dioscoridis (Asteraceae) or Carina indica (Cannaceae), they did not develop to the protonymphal stage, whereas when reared on Trigonella foenum-graecum (Fabaceae) or Brassica rapa (Brassicaceae) developed to the adult stage in a significantly longer period and the resulting females laid fewer eggs compared with controls. Crude extracts of C. dioscoridis and T. foenum-graecum leaves showed a remarkable toxic effect on adults and eggs, while extracts of C. indica and B, rapa showed less intense ovicidal action. Many essential oils and their pure constituents have been tested against Tetranychus mites. Egyptian authors tested Thymus vulgaris oil
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and pure thymol against T. urticae and both compounds were found effective [259]. Thymol was more potent than thyme oil as a deterrent factor for reducing egg laying by the mite. Mortality percentage reached 100% with both materials used, however, at lower concentrations, the effect was more pronounced with thymol than thyme oil. A very interesting paper deals about the insecticidal and acaricidal activities of many monoterpenes and their possible phytotoxicity on host plants [260]. Twenty-nine compounds, belonging to different chemical classes, were assayed against T. urticae by mean of the leaf-dip method. In particular, the alcohols carveol, carvomenthenol, citronellol, geraniol, 10hydroxygeraniol, isopulegol, linalool, /-menthol, perillyl alcohol, aterpineol and verbenol, the phenols carvacrol, eugenol and thymol, the ketones t/-carvone, /-carvone, /-fenchone, menthone, pulegone, thujone and verbenone, the aldehydes citral and citronellal, the acid citronellic acid, the ether 1,8-cineole and the hydrocarbons limonene, a-terpinene and y-terpinene were used. All the compounds were tested, in water with Triton X-100 as wetting agent, at 10000 and 1000 ppm, and the activity was assessed 24, 48 and 72 hours after treatment. The toxicity differed depending on the concentrations and the exposure times. The monoterpenes tested, except for 1,8-cineole, 10-hydroxygeraniol, aterpineol, verbenol and verbenone, caused 100% mortality at the highest concentration after 24 hours. Carvacrol was the most effective at the lowest concentrations, followed by citronellol. Geraniol produced 100% mortality, whereas its analog 10-hydroxy geraniol showed 0% mortality. Longer exposure time increased acaricidal effects. The most effective monoterpenoids (carvacrol, carvomenthenol, carvone, citronellol, eugenol, geraniol, perillyl alcohol, 4-terpineol, thymol) were evaluated in more detailed tests. Of these, carvomenthenol and 4-terpineol showed greater acaricidal activity (LC5o= 59 and 96 ppm, respectively) than others. Furthermore, authors examined the phytotoxicity of some compounds to both com roots and leaves 3 and 10 days after treatments, /-carvone was the most phytotoxic compound, while pulegone was the safest. A table with detailed data is reported in the paper. Two species known for a long time as potential pesticides, particularly as insecticides and insect repellents, have also been investigated as acaricides against T. urticae [261]. The essential oils of Artemisia absinthium and Tanacetum vulgare (Asteraceae) were obtained from whole cultivated plants harvested in fiiU
427
bloom by three different methods of extraction: a microwave assisted process (MAP), distillation in water (DW) and direct steam distillation (DSD), and their relative toxicity assayed by direct contact. All the oils were tested at 1, 2, 4 and 8% as emulsions prepared in water containing 9% of denatured EtOH and 0.32% of Alkamul EL-620 as emulsifier, and mite mortality was assessed after 48 hours. All three oils of ^. absinthium were lethal to T. urticae, however there were differences in the degree of toxicity, depending on the extraction methods, as reported in a table in the paper. For example, at 4%, oil extracted by the MAP and the DW methods caused 52.7 and 51.1% mite mortality, whereas oil obtained by DSD resulted in 83.2% mortality. Consequently, the LC50 of the oil extracted by DSD was lower (0.043 mg/cm^) than those obtained by MAP (0.134 mg/cm2) and by DW (0.130 mg/cm2). The extracts of T. vulgare obtained by DW and DSD showed greater acaricidal activity than the extract prepared by MAP. At 4% concentration they showed 60.4, 75.6 and 16.7% mortality, respectively. Chemical analysis of the T. vulgare extracts indicated that p-thujone is by far the major compound of the oil (>87.6%), and probably contributes significantly to the acaricidal activity of the oil. The paper demonstrated the importance of a further factor affecting the variability in the effectiveness of extracts obtained from the same species, that is the technique used for the extraction. Once again, I would like to underline the significance of knowing the composition of the essential oils used in the tests; unfortunately, in the case of the three absinth oils, authors were not able to identify its major constituent; moreover, another sesquiterpene, present in the DSD oil and absent in the other two, that could be responsible of the greater toxicity of the former oil, was also unidentified. The toxicity of vapours of the essential oils obtained from four plant species, seeds of Cuminum cyminum and Pimpinella anisum (Apiaceae), leaves of Origanum syriacum var. bevanii (Lamiaceae) and fruits of Eucalyptus camaldulensis (Myrtaceae) against another species of the same genus, the carmine spider mite, T. cinnabarinus, was investigated in Turkey [262]. This mite is a major greenhouse pest in this country and throughout the world. It attacks a large range of 100 cultivated crops and weeds. It is a serious pest on beans, eggplant, pepper, tomatoes, cucurbits, and many other vegetables. It is also a pest of papaya, passion fruit, and many other fruits. The carmine spider mite also attacks many flowers and ornamental
428
plants such as carnation, chrysanthemum, cymbidium, gladiolus, marigold, pikake, and rose. The oils were assayed at 0.25, 0.50, 1.00 and 2.00 |il/l of air. The phytotoxicity of the vapours of these essential oils was estimated by exposing tomato, bean and cucumber seedlings to the highest dose for 96 hours. All essential oils, except that of eucalyptus, caused 100% mortality of the carmine spider mite at or below the maximum dose after 2-3 days of exposure. When the essential oils were compared on the basis of their LT50 and LT99 values, the order of toxicity was oregano>cumin>anise>eucalyptus. Phytotoxicity to seedlings was manifested as discoloration and eventual drying of the first two leaves. Cumin and anise oils were toxic to all plants tested, oregano was toxic only to tomato, whereas eucalyptus to none. Some terpenes, commonly found in essential oils, are pheromones of some arthropods species. Pheromones are volatile chemicals used for communication within individuals of the same species and, occasionally, between different species (usually the latter are known as allomones) [263]. Two examples are represented by famesol and nerolidol. Fig. (7), two highly attractive compounds to Tetranychus mites. These compounds have been added to common synthetic acaricides to improve their effectiveness against moving stages of mites [264,265].
nerolidol
CH.OH famesol Fig. (7). Nerolidol and famesol, two sesquiterpene pheromones
Also many non-volatile compounds have been found effective against Tetranychus mites. Among these chemicals, particularly interesting as a new class of acaricides, are naphthoquinones, in particular two derivatives isolated from Calceolaria andina (Scrophulariaceae), protected by patent application, and designated as BTG 505 and BTG 504, Fig, (4) [171,266,267]. These chemicals were found to exhibit high activity, even against strains of T, urticae that are most resistant to a wide range of
429
commercial acaricides. Furtheraiore, the levels of activity were low on many beneficial species, both insects and acari {Phytoseiulus persimilis and Typhlodromus piri). Naphthoquinones have long been known to inhibit mitochondrial respiration, however the primary site of action may vary, depending upon the nature of substituents. Complex III was found to be the primary site of action for the two 2-hydroxy-l,4naphthoquinones isolated from C. andina. Complex III (ubiquinol:cytochrome c oxidoreductase), is found in mitochondria, photosynthetic bacteria and other prokaryotes; the general function of the complex is electron transfer between two mobile redox carriers, ubiquinol (QH2) and cytochrome c. Electron transfer is coupled with the translocation of protons across the membrane thus generating an electrochemical proton potential that can drive ATP synthesis by ATP synthase. Since the primary loss mechanism of applied naphthoquinones from leaf surfaces was proved to be volatilization (though some degradation also occurred leading to a half life of about 20 hours), authors have shown that different types of formulations (not reported, patent pending) can increase the efficacy against parasites and reduce phytotoxicity. Concerning the commercial potential of these products, the authors observed that naphthoquinones can occur in concentrations up to 5% w/w in dried aerial parts of C andina, but this herb would be a poor source for commercial production of the natural products. However, this genus is amenable to hybridization, and from preliminary studies it seems that it could be possible to produce more vigorous and highyielding cultivars. Furthermore, these compounds can be synthesized in 2-3 steps. Other synthetic (and natural, see below) acaricides, i.e. tebufenpyrad, pyridaben, fenazaquin, have been shown to be active by inhibition of another electron transport system of the mitochondrial respiratory chain, Complex I (NADH:ubiquinone oxidoreductase). Because of their high activity against various mite species, these METI (mitochondrial electron transport inhibitors) acaricides are in widespread use, but some strains of T. urticae from different parts of the world have been reported to exhibit resistance to these substances. Very recently, a strain of T. urticae from hops in UK was confirmed to have crossresistance to all the METI acaricides, despite having only been exposed to a single compound. Naphthoquinones inhibit Complex III in the mitochondrial respiratory chain, a system distinct from the Complex I,
430
but given the unpredictability of cross-resistance patterns and the fact that metabolic resistance can confer resistance between chemical groups, it was important to evaluate whether METI resistance also protected mites against the naphthoquinones [268]. Experiments with METI acaricide resistant strains and the standard reference susceptible strain, ascertained that the activity of naphthoquinones against T. urticae remained uncompromised.
•Mil
Fig. (8). Acetogenins from Annona glabra
From the seeds of another species, Abrus precatorius (Fabaceae), other classes of compounds have shown promising effects against T. urticae. From the non-saponified fraction of a crude petroleum ether extract, coumarin, p-amyrin and a mixture of sterols were isolated and tested against females and eggs in laboratory conditions. P-amyrin was the most effective compound against both stages. Spraying females with sub-lethal doses of p-amyrin caused a significant reduction in fecundity and the viability of resulting eggs [269]. From the seeds of Annona glabra (Annonaceae), three acetogenins, squamocin. Fig. (6), desacetyluvaricin and asimicin, Fig. (8), have been extracted and their toxicity against insects and mites evaluated; they have shown good insecticidal activity, but no acaricidal effect against T. urticae [270] while, recently, squamocin was found effective against the house dust mite Dermatophagoides pteronyssinus [226]. The genus Pimpinella (Apiaceae) produces rare phenylpropanoids with an unusual substitution pattern at the phenyl ring: the (l^*)propenyl-2-hydroxy-5-methoxybenzene skeleton of these compounds
431
has been named pseudoisoeugenol [271], Fig (9). Also derivatives of (1£)propenyl-4-hydroxybenzene have been isolated in some Pimpinella species. The activity in the contact assay of 100 ppm of eight Pimpinella phenylpropanoids against the red spider mite, T. telarius, has been evaluated [272]. Epoxy-anoltiglate was the most effective compound, killing 100% of the mites, while four other substances, epoxypseudoisoeugenolisobutyrate, epoxy-pseudoisoeugenoltiglate, pseudoisoeugenolisobutyrate and isoeugenolisobutyrate showed lesser effectiveness, killing 80-90% of the mites.
X) HoCO'
H3CO' Epoxy-anoltiglate
epoxy-pseudoisoeugenolisobutyrate
isoeugenolisobutyrate
^ O H3CO"
"^^
epoxy-pseudoisoeugenoltiglate
H3CO" pseudoisoeugenolisobutyrate
Fig. (9). Phenylpropanoids from Pimpinella species
The other phenylpropanoids tested, anoltiglate, isoeugenolisobutyrate and isoeugenol were completely ineffective. Other uncommon natural derivatives are 2-acylcyclohexane-l,3-diones or p-triketones, typical of hops {Humulus lupulus, Cannabinaceae). The p-acids from hop belong to this class of chemicals and are by-products of hop processing for brewing. The fraction containing these products has been examined in a choice bioassay for its effect on the feeding behavior
432
of T. urticae [273]. The results showed that both the highest concentrations of culupulone, Fig. (10), the chiefs-acid component of the fraction, and the whole p-acid fraction repelled T. urticae and also affected its survival. The greatest difference between the pure compound and the crude fraction treatments was seen in the oviposition of the mites: significantly fewer eggs were found in the whole p-acid fraction. This suggested that culupulone was not the only active component in the pacid fraction. Some secondary metabolites, epitaondiol diacetate, stypetriol triacetate, epitaondiol monoacetate and epitaondiol. Fig. (10), isolated from the brown alga Stypopodium flabelliforme (Dictyotaceae) have been tested on adults of T. urticae [274]. Only epitaondiol showed little acaricidal activity at 500 ppm (14% mortality), while the other compounds resulted inactive at 1000 ppm.
epitaondiol culupulone
ajoene stypetriol Fig. (10). Structure of hop and algal metabolites
Ajoene, Fig. (10), an unsaturated sulfoxide disulfide, is the principal chemical responsible for garlic's anticoagulant properties. It has been also investigated for its acaricidal activity on T. urticae [275]. Complete mortality (100%) by ajoene was observed at 0.075% after 14 h of treatment, a dose comparable with other synthetic acaricides used in the experiment. At lower concentrations (0.05%), it affected female fecundity
433
and only 31.5% of the juvenile stages. These results suggested that ajoene, besides having direct acaricidal effect, could also control resurgence of the pest. Glandular trichomes of wild tomato, Lycopersicon hirsutum f. glabratum (Solanaceae), yielded two methyl ketones: 2tridecanone and 2-undecanone [276]. They are known to cause mortality in several herbivorous insect species; it seems that this kind of chemicals could be considered defensive substances against pollen-feeding animals, as confirmed also by their abundance in wind-pollinated plants [277]. Dutch researchers investigated the effects of these compounds on two strains of T. urticae, collected from tomato and cucumber crops in greenhouses [276]. The two ketones were tested separately, in combination in the ratio found in L hirsutum f glabratum and in several other ratios to detect any synergistic interaction between them. Synergistic effects were not detected. They measured both the direct contact and residual toxicity, as well as the viability of the eggs produced by ketone-treated females. Both compounds showed LC50 values comparable to the formulated acaricide amitraz; 2-tridecanone was slightly more toxic than 2-undecanone, but only against the tomato strain. In the bioassays for the residual effects, no significant mortality occurred, however the mites avoided feeding on the treated surface and the eggs were laid almost exclusively on the untreated area. Furthermore, there was no significant egg viability for most of the treatments. A new plant species. Quassia sp. aff. bidwillii (Simaroubaceae) was discovered in Australia and the MeOH extract of its aerial parts was tested against T, urticae [278]. Because of its effectiveness, subsequent fractionation by RP-chromatography gave the pure active quassinoid derivative chapparinone, which showed a LC5o=47 ppm. Neem extracts, pure constituents (i.e. azadirachtin) and formulated products showed positive results against Tetranichus mites [279-283]. Less polar extracts were considerably more toxic than polar ones or coldpressed neem oil or commercial neem oil, and reduced the fecundity of the mites on treated plants and the survival of nymphs hatched from treated eggs; application of pentane extract or neem oil in sublethal concentrations, caused growth disrupting effects on the nymphal stages and ovicidal effects. Quantification of the insecticidal substance azadirachtin in the extracts revealed that this compound was not the most active principle against the mites [284].
434
Other promising control agents are microbial metabolites. Abamectin was found much less toxic to the useful predatory mite Phytoseiulus persimilis than to the parasite mite T, urticae [285]. A strain of the soil bacterium Streptomyces platensis yielded three new substances having a marked acaricidal activity against T. urticae, AB3217 A, B and C, Fig. (11) [286,287], while another strain of Streptomyces, NKl 1687, produced gualamycin. Fig. (11), that was able to kill 100% of dicofol-sensitive and resistant mites (adults and larvae) at 250 |ig/ml [288].
AB3217A:R=H O
^
^^
11
I
AB3217 B:R= —c—(CH2)4—C-(CH3)2
(f
AB3217C:R= —C—(CH2)2—CH-(CH3)2 ^
H N ^ ^
gualamycin
Fig. (11). Acaricidal agents of bacterial origin
Finally, as altematives to chemical compounds or as part of integrated pest management programs, predatory arthropods or fungal pathogens have been used. Among predators, the most used are phytoseiid mites such as Phytoseiulus persimilis and Neoseiulus fallacis [289-293], while the fungus Neozygites adjarica was tested as pathogen agent [294,295]. The acaricidal properties of some of the previous compounds were
435
attributed to the interference with mitochondrial electron transport due to inhibition of Complex III. Some structurally diverse miticides are able to inhibit Complex I (NADHiubiquinone oxidoreductase), a system distinct from Complex III. Complex I is the first electron transport complex of the mitochondrial respiratory chain. It oxidizes NADH and transfers the electrons via a flavin mononucleotide cofactor and several iron-sulfur clusters to ubiquinone (Q). So, Complex I contributes to the protonmotive force that drives ATP synthesis. Besides many synthetic products, some secondary products from microbial and plant sources exhibit biological activity against agricultural pests because of their action on Complex I. Rotenone and piericidin A, Fig. (12), were known for a long time as high-affinity inhibitors of proton-translocating NADHiQ oxidoreductase [296]. Rotenone is the most widely used inhibitor of Complex I because of its high inhibitory potency and commercial availability. It is the most potent member of the rotenoids, a family of isoflavonoids extracted from Fabaceae plants. All known natural rotenoids have been isolated in the thermodynamically stable cis-B/C ring fusion; synthetic analogues in which B and C-rings planes are almost coplanar were about 100-fold less active than natural rotenone, indicating that the bent form is essential for the activity [297]. This conclusion is supported by the observation that rotenol, in which the whole conformation is not fixed due to opening of the C-ring, is about 200-fold less active than rotenone. Another important feature for the activity is the configuration of the isopropenyl group linked to the E-ring. Cube resin, the roots extract of Lonchocarpus utilis and L. urucu (Fabaceae), is an important acaricide. The four principal active constituents are rotenone, rotenolone, deguelin and tephrosin. Fang and Casida identified further 25 minor rotenoids having variations in the B, D and E-rings, thereby providing a new and unique set of compounds to elucidate structureactivity relationships for the activity on Complex I [298]. The rotenone series and the deguelin series, with modifications in the B, C and D-rings, followed similar overall substituent effects on activity. In particular, the parent compounds rotenone and deguelin were more potent than any of their derivatives. Hydroxylation or methoxylation in the A-D ring system considerably reduced the potency. The trans isomers were 7-100 fold less active than the corresponding cis isomers. Authors affirmed that, considering the potency and the amounts, the four
436
major rotenoids accounted for more than 95% and probably almost all of the biological activity of the cube resin as inhibitor of Complex I. Many kinds of Streptomyces strains produce piericidin homologues. Piericidin A is a very potent inhibitor of Complex I. The natural side chain of piericidin A is not essential for the activity since piericidin B, C and D analogues, in which the region from C-5 to C-13 differs, exhibit activity as high as or only slightly less than piericidin A. On the basis of studies with synthetic analogues, it was concluded that a branched methyl group at C-3 and unsaturation between C-2 and C-3 are important for potent activity [297].
OCHo
OCH OCHo
OCHo
DeguelinR=H Tephrosin R=OH
Rotenone R=H Rotenolone R=OH
HoC
H3CO'
Piericidin A
HO*
Capsaicin
Fig. (12). Complex I inhibitors
Capsaicin, Fig. (12), the pungent principle of Capsicum species
437
(Solanaceae), acts as competitive inhibitor for ubiquinone in Complex I. Methyl capsaicin is more potent than capsaicin, indicating that the phenolic OH is not essential for the activity [297]. Other natural inhibitors of Complex I are annonaceous acetogenins. These compounds belong to a wide group of natural products isolated from several species of the Annonaceae family, which include more than 250 molecules with diverse chemical structures. Among the various classes, it seems that monotetrahydrofuranic derivatives are less potent than other acetogenins [296,299]. CONCLUSIONS The control of parasitic diseases is mainly based on the use of effective drugs, both in agriculture or human and veterinary medicine; for this reason the lack of effective drugs often prevents the control of some parasitic diseases, making them more serious and important. At present, however, the use of commercial drugs involves many problems that strongly limit their use: foremost the drug-resistance problem shown by the most important parasites, the environmental damage and the toxicity of many synthetic drugs. In addition, drug residues in plant and animal food products are important reasons of considerable economic losses for farmers. The European Community's recent law (EC n. 1804/99) regarding biological animal farming, limits the use of synthetic drugs, while the use of homeopathic remedies and phytotherapies is allowed. All these problems are stimulating the search for new and alternative control methods, including the search of effective compounds characterized by smaller environmental impacts in terms of residues and toxicity. Since plant-derived compounds are generally more easily degradable and could show a reduced environmental damage with respect to synthetic drugs, at present the evaluation of the antiparasite activity of plant extracts is being increasingly investigated, as demonstrated by the recent studies that have evaluated and confirmed the effectiveness of many plant compounds on bacteria, fungi, protozoa, helmints and arthropods. Much of present day antiparasite chemotherapy is derived from practices and advances made in the 19-20th Centuries, during which the antiparasite activity of some plants has been scientifically confirmed. Nowadays higher plants are still important sources of new active principles, among which the antimalarial compound artemisinin is one of the most recently introduced.
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Even so, the pharmacological control of some parasitic diseases is still very difficult; among them we can find arthropod-related diseases. Perhaps human and veterinary medicine are the most suitable fields for a real application of natural drugs, in fact the treatment of these pathologies is mostly topical, and particular drug-formulations are not required. Furthermore, generally only a few treatments are necessary to kill all the parasites. In agriculture, in spite of the studies performed to date, these substances are perhaps still far from their effective use: their main usefiil feature, that is their biodegradability, is also their weakness. Often, many products are not able to persist in the environment for a period of time sufficient for pests control. Further studies are necessary to prepare better formulations that allow us to solve this problem. Other important future research topics should concentrate on the evaluation of the toxicity of these compounds, an unknovm feature for many natural compounds. REFERENCES [I]
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4S1
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
453
PODOLACTONES: A GROUP OF BIOLOGICALLY ACTIVE NORDITERPENOIDS ALEJANDRO F. BARRERO, JOSE F. QUILEZ DEL MORAL and M. MARHERRADOR Department of Organic Chemistry, Institute ofBiotechnology, University of Granada, Avda. Fuentenueva, 18071, Granada, Spain ABSTRACT: More than seventy podolactones have been isolated mainly from Podocarpus species. Some of these structures have also been found in filamentous fungi. The different biosynthetic origin of these molecules considering their vegetal or fungic source is discussed in this review. These molecules present a wide range of biological activities: anti-tumor activity, anti-inflammatory activity, fungicide activity, insecticidal activity and plant growth regulatory activity. These biological properties are analyzed in detail, and in the light of their results, structure/activity relationships are discussed. Finally, chemical reactivity, including interconversion reactions, and synthetic approaches to these compounds are summarized.
INTRODUCTION Podolactones are considered to be a group of natural products whose basic skeleton contains a y-lactone between carbons 19-6 and a 6-lactone between carbons 12-14, which are their characteristic functions Fig. (1) [1]. The numbering of the podolactone skeleton has been assigned on the basis of the totarane skeleton from which most of podolactones have been proposed to be derived.
Fig.{l)
The podolactones with a nor- or bisnorditerpenoid structure are mainly found in different species of the Podocarpus plant type [2-3]. Apart from
454
the Podocarpus species, five podolactones have also been isolated from a New Zealand mistletoe, Ileostylus micranthus, which was parasiting P. totara, from which it has been suggested that podolactones were assimilated [4]. A small number of tetranorditerpenoid dilactones isolated from the filamentous fungi Oidiodendron truncatum [5-6], O. griseum [7], Aspergillus wentii [8] and a non-identified species of the Acrostalagmus genus [9] have also been considered as fomiing a part of this group. These natural substances are of great interest due to both their unusual structures and the potent wide-ranging spectrum of biological activities that they possess: antitumor activity [10] in vitro and/or in vivo, antiinflammatory activity [7], fungicidal activity [11], herbivorous manmialian antifeedant activity [12], insecticide activity against house-fly larvae and other insects [13] and, lastly, a potent plant growth regulatory activity, both as inhibitors and as stimulants [14-15]. BIOSYNTHETIC ORIGIN All natural podolactones have been isolated from two types of natural sources, plants related to the genus Podocarpus, from which the majority of podolactones have been described, and the filamentous fungi. This different natural source also possesses a different biosynthetic origin. The first noticeable evidence supporting this difference is that, while podolactones isolated from plants are nor- orfcwnorditerpenoids(i.e. nagilactone C and nagilactone E), the dilactones isolated from fungi have lost four carbons from a diterpene precursor (i.e. oidilactone C).
Nagilactone C
Nagilactone E
Oidiolactone 0
Appearance of podolactones in the extracts of Podocarpus species along with various derivatives with a totarane skeleton (totarol, 12-
455
hydroxytotarol, 19-hydroxytotarol, totaral and 4p-carboxy-19-nortotarol) [16] led the Hayashi's team to postulate the following biosynthetic scheme for these substances (Scheme 1)* The pathway starts with 12hydroxytotarol, which suffers a meta-pyrochatecase-type fission to give a hydroxy-acid, intemiediate which decarbonylates leading to the corresponding a-pyrone, transformation already described in other catechols [17],
Scheme 1. Proposed biogenetic pathway for podoactones isolated from plants.
On the other hand, the C-16 terpenoid dilactones isolated from fiingi have been postulated to have a biosynthetic origin different from that reported for podolactones from plants. Two possible biosynthetic ways were envisaged, one supposing an oxidative loss of four carbons from a diterpene precursor, and secondly, the addition of a C-1 unit to a sesquiterpenoid [18]. The results obtained by administering isotopically labelled acetic and mevalonic acids to an Acrostalamus fungi, together with the isolation from this same fungus of acrostalidic acid, acrostalic acid and isoacrostalidic acid let us to conclude an attribution of a diterpenoid origin for these lactones with the following biogenetic pathway proposal[19] (Scheme 2).
456
.CO2H
-^
CO2H
*
ciS' and frans-communic acid
COgH
CO2H
aerostatic acid
CO2H
'OMe
0
^
"" CO2H III
acrostaiidic acid
^^ 'CO2H isoacrostalidic acid
Scheme 2. Proposed biogenetic pathway for podoactones isolated from fungi.
The route could start from the mixture of cis- and rrans-coimnunic acids (or also from other diterpenes, such as isocupresic acid). The isolation of hydroxyacid I as a natural product from Acrostalagmus reinforces the possiblity of the existence of diene II as a key precursor, not only of isoacrostalidic acid and acrostaiidic acid, but also of dilactone III. The straightforward chemical conversion of I into III in a recent publication by Barrero et al. [20] supports this hypothesis.
457
STRUCTURES OF NATURAL PODOLACTONES Natural podolactones can be classified into three major stractural types depending on the nature of the conjugated lactone system in the B/C ring moiety [21], Fig. (2). Type A: a-pirone [8(14),9(ll)-dienolide], Type B: 7a,8a-epoxy-9(ll)-enolide, Type C: 7,9(1 l)-dienolide.
Type A
TypeB
TypeC
Fig. (2).
Below we can see all the podolactones described up to date, distributed according to the aforementioned classification with an indication of the species from which they were identifed and their bibliographic reference. First, the podolactones isolated from Podocarpus species are presented and, later, the podolactones found in fungi will be listed.
458
Podolactones from plants Type A
nagilactone A 1 P. nagi, P. macrophyllus P. philippinensis, P. polystachyus [M, 22-22]
0
nagilactone B 2
P.nagi[\7\ 0
nagilactone C 3 P. nagi, P. nivalis, P. halii, P. macrophyllus, P. purdeanus, lleostylus micranthus [4,17,25-27] O
inumakilactone E 6 P. macrophyllus, P. polystachyus [24, 26] O
C02Me
hallactone A
7 P. ham [30]
1 -deoxy-2a-hydroxynagiiactone A 8 P./lag/[31]
15-methoxycarbonylnagilactone D
9 P. nag/ [31]
459
10 P. nagi [32]
O 1-deoxy-2p,3pepoxynagilactone A 13 P. nagi [33]
15-hydroxynagitactone D
3p-hydroxynagiiactone A 11 P. nagi [32]
urbalactone 14 P. urbanii [34]
12 P. nag/ [32]
O
R=p-D-glc nagilactoside A 15 P. nagi [35]
O
3-deoxynagilactone C 16 lleostylus micranthus [4]
3-ephnagilactone C 17
P. na^/ [36]
1-deoxynagiiactone A 18 P. nag/[16]
460
2,3-dehydro1-deoxynagilactone A
2,3-dehydronagilactone A
20 P. na^/[16]
19
P. nag/[16]
R=p-D-glc nagilactoside B 21 P. nagi [37]
R*,
epi-sellowin C 22 P. nagi [37]
R=p-D-glc-(1-^6)p-D-glc-nagilactoside E 25 P. nagi [38]
R=p-D-glc-(1 -•Sj-p-D-glcnagilactoside C 23 P. nagi [38]
R=p-D-glc-(1-*-3)p-D-glc-(1-^ 6)-p-D-glcnagilactoside F 26 P. nagi [39]
R=p-D-glc-(1^^6)p-D-glc-nagilactoside D 24 P. nagi [38]
R=:p-D-glc-{1-#-6)p-D-glc-(1-^ 3)-p-D-glcnagilactoside G
27 P. nag/ [39]
461
TypeB
OH
0 inumakilactone A 28 P. macrophyllus, P. philippinensis [40-41,22] O
O
podolactone A 29 P. neriifolius [42-43]
O podolactone B 30 P. neriifolius [42]
SOMe
0
inumakilactone B 31 P. polystachyus, P. macrophyllus P. neriifolius [23,44-45]
^
O
podolactone C 32 P. neriifolius P. milanjianus [45-48] O
podolactone D 33 P. neriifolius [45-47] 0
'%^
O
sellowin B 35 P. sellowii [29,47]
nagilactone E 36 P. nap/ [49]
462
SOaMe
::o I
0Glu(0H)4
inumakiiactone A giucoside 37 P. macrophylus P, philippinensis [40-41,22]
O
nagilactone Q 40 P. sellowii P. milanjianus [50]
2p,3p-epoxypodolide 43 P. nagi [52]
o;
hallactone B 38 P. hallii, P. sellowii P. polystachyus [24,30,47]
podolide
39 P. gracilor [9]
O
16-hydroxypodollde (salignone H) 41 P. saligna [51]
milanjilactone A 44 P. milanjianus [53]
O
2,3-dihydro16-hydroxypodolide 42 P. nagi [52]
salignone I 45 P. saligna [54]
463
K^
O 3-deoxy-2a-hydroxynagiiactone E 46
O
salignone M 47 P. saligna [55]
P. nagi lleostylus micranthus [4,36]
16-hydroxynagiiactone E 48
P. nagi [56]
TypeC
Gluc-0'
O
ponaiactone A
49
P. na/fa// [57]
-O d ' ip-hydroxynagilactone F 52 P. nagi
O ponaiactone A glucoside 50 P. r?a/fa// [57]
nubilactone A 53 P. nubigena [58]
podolactone E 51 lleostylus micranthus P. neriifolius [4,45]
O
3p-hydroxynagilactone F
54 P. naflf/ [33]
464
nagilactone F 55 P. nagi, P. milanjianus, P. sellowii P. macrophilus [49-50,25]
2,3-dehydro-16-hldroxinagilactone F
miianjilactone B 56
57 P. nagi [56]
P. milanjianus [53]
Ha,
nagilactone I 58 P. nagi [66]
2a-hydroxynagilactone F
59 P. nagi lleostylus micranthus [4,36]
465
Podolactones from fungi
-O PR 1388 60 Oidiodendron truncatum Oidiodendron griseum [6,7]
0^ Oidiolactone C (Oidiodendroiide C) 61 Oidiodendron truncatum Oidiodendron griseum [7,11] O
oidiolactone D (oidiodendroiide A) 62 Oidiodendron truncatum Oidiodendron griseum [7,11]
'''OMe
O
LL-21271a 0 LL-Z1271Y 64 63 Acrostalagmus Acrostalagmus Oidiodendron griseum [7,9,59] Oidiodendron griseum [7,9,59]
wentilactone A 65 Aspergillus wentii [8]
0
O wentilactone B 66 Aspergillus wentii [8,20]
oidiodendroiide B 67 Oidiodendron truncatum [11]
Oidiodendron griseum [7]
466
Miscellaneous
OH '''OH
HO'
inumakilactone C 69 P. macrophyllus [44]
inumakilactone D 70 P. macrophyllus [60]
saljgnone A 71 P.sa//flfna[51,61]
OH
"'^^x^OH H / —0
C02Me
0^
V ^ ^ x * \ ^
COaMe
saiignone J 73 P. sa//flfna [61]
saiignone B 72 P. saligna [61]
saiignone K 74 P. saligna [55]
C02Me
saiignone L 75 P. sa//p/7a [55]
nagilactone J 76 P. /lagf/ [62]
O dlhydrodeoxynubilactone A 77 P. saligna [63]
467
BIOLOGICAL ACTIVITY OF PODOLACTONES Anti-tumoral Activity. a) Yoshida Sarcoma. The in vitro bioactivitivity of 29 podolactones, 15 of them natural products, against cultured Yoshida Sarcoma cells [64-65] was investigated by Hayashi's group during the period between 1975 and 1979.
H2O3PO'
468
Table 1 summarizes the obtained results. Podolactones were grouped on the basis of the previously reported structural subgroups in which these natural compounds had been classified. Table 1. Citotoxidty of natural and synthetic podolactones against Yoshida Sarcoma Type A Lactones Nagilactone A (1) Nagilaaone B (2) Nagilactone C (3) Nagilactone D (4) 1 -deoxy-2a-hydroxynagilactone A(8) 3p-hydroxynagilactone A (11) 15-methoxycarbonyl nagilactone D (9) 10 78 79 80 81
TypeB Lactones ICso (x 10-^ M) 3l0 17.2 22.5 3.32 16.4 487.0 21.5 305.0 1460.0 1000.0 138.0 119.0
Nagilactone E (36) NagUactone G (40) 82 23-ciihydro-16hydroxypodolide (42) and 16-hydroxy podolide (41) Inumakilactone A( 28) Inumakilactone B (31) 83 84 85 86 87 88
TypeC ICso (xlO^M) 336 1.48 3.72 14.8 10.4 4.11 18.3 87.0 607.0 4,19 20.6 110.0
Lactones Nagilactone F (55) 89 90 91
ICso (XIQ-^ M)
o
12.2 16.1 18.9
469
Synthetic derivatives 92 and 93, which can not be not included in any of the three A-C subgroups, turned out to be inactive against this cell line.
On the basis of the reported data, the following structure-activity relationships were proposed: i) The dilactones with few or no polar substituents showed strong activity, a factor that could be related to the permeability of these substances through the cell membrane. Thus, the most active compounds (ICso -- 0.015 jig/mL), nagilactone F (55) and 7,8-epoxy-nagilactone F (91), do not have any hydroxyl group. The monohydroxylated dilactones nagilactone D (4), inumakilactone B (29) and nagilactone E (34) are slightly less active, but still maintain a substantial activity (IC5o=0.11-0.14 M-g/mL), while the dihydroxylated lactones nagilactone A (1), nagilactone C (3) and inumakilactone A (26), and the trihydroxylated 10 and 3p-hydroxynagilactone A (11) are, respectively, 10 and 100 times less active than the non-hydroxylated ones. ii) The dienic system conjugated with the y-lactone is one of the most important functional groups for anti-tumor activity. ///) The y-lactone group in positions 4p and 6P is important for the activity, but not essential. iv) The acetylation of hydroxyl groups reduces the activity by 10-50 times, probably due to steric effects. v) The 7,8-epoxy group of the type-B dilactone is crucial, since the hydrogenolysis product of the epoxide ring is completely inactive. vi) Activity decreases when the configuration of the substituent is changed on Ci? from a to p. vii) Oxidation of the hydroxyl groups to ketones or the introduction of a p epoxide in the A ring do not affect activity, but the introduction of an a epoxide in position 2,3 reduces activity up to ten times.
470
b) P-388 Mouse Linfome Podolide was the first dilactone reported to have antitumor activity in vivo against P-388 leukemia in mice and citotoxycity in vitro towards cells derived from P-388 murine leukemia [9]. More detailed reports on the antileukemic activity of nagilactone C (3) and nagilactone E (36) was given by Hayashi in 1975 [64]. Both lactones were effective with a dose of 20 mg/kg/day (T/C 125%). Podolactone C also showed antineoplasic activity against P 388 cells in vivo at 20 mg/kg/day (151% T/C) [48]. Finally, Bloor and Molly reported the cytotoxic activity of five podolactones isolated from a New Zealand mistletoe. The more active lactones were 3-deoxy-2a-hydroxynagilactone E (46), 2ahydroxynagilactone F (59) and podolactone E (51) showing IC50 (jxg/mL) values of 0.06, 0.06 and <0.01 [big/mL, respectively [4]. c) Humane Nasopharynx Carcinoma (9KB) Podolactones have also been reported to be active against the tumoral cell line 9KB. Nagilactone G (40) and nagilactone F (55) exhibit a remarkable in vitro activity against 9KB tumoral cells (ED50 '-lO"^ fxg/ml). Milanjilactone A (44) and milanjilactone B (45) also showed a significative in vitro activity (ED50 = 4 jig/ml y ED50 = 0 . 1 \ig/wl, respectively) [53]. d) Other Cell lines (9KB) Recently, Barrero et al. [20] have tested the activity of natural podolactones LL-Z1271a (63) and 68, and of synthetic derivatives 94-99, the latter being a mixture of isomers, against four tumoral cell lines: P388, A-549 (human lung carcinome), HT-29 (human colon carcinome) and MEL-28 (human melanome) (Table 2). The two natural compounds, as well as 98 showed a potent activity (IC50<1 M^g/mL). Besides, 63 and 68 exhibited a certain selectivity towards the cell lines P-388 and A-549.
471
OMe
Table 2. Cytotoxic activity of different podolactones against four ceD lines IC5o(|ig/ml) Cellline
G
68
94
95
96
97
98
99_
P-388
0.12
0.50
20
2.0
10
>10
0.10
>10
A-549
0.12
0.50
20
2.0
20
>10
0.10
>10
HT-29
0.25
1.0
>20
5.0
>20
>10
0.12
>10
MEL-28
0.25
1.0
>20
2.0
>20
>10
0.12
>10
From these data, certain structure-activity relationships can be inferred. As expected, the presence of the structural moiety 7,9(ll)-dien-12,17olide moiety is necessary to achieve the maximum activity. The closure of the Y-lactone increased the biological activity four times (68 against 95). On the other hand, the presence of methoxy groups at C-14 on dienolides remarkably stressed the activity (it increases from 4 to 5 times). Finally, it is worth-mentioning that 99, (mixture containing natural compound 64) possessing a hydroxy group at C-14, appeared to be inactive.
472
Anti-inflamatory Activity Very recently, in 1999, an European patent was published involving the description of a pharmaceutical composition, which includes terpenoid dilactones isolated from a new strain of Oidiodendrum griseum filamentous fungi, together with some semi-synthetic derivatives from the isolated natural podolactones [7]. This pharaiaceutical composition was reported to be useful for the treatment of IL-1 (interleukin-1) and TNF (tumor necrosis factor)-mediated diseases. IL-1 and TNF are biological substances produced by a number of cells, such as monocytes and macrophages. These substances have been demonstrated to mediate a variety of biological activities thought to be important in immunoregulation and inflammation. A few of the diseases reported to be exarcebated and/or caused by high or unregulated amounts of IL-1 are arthritis, osteoarthritis, tuberculosis, atherosclerosis, rheumatoid arthritis, gout and acute synovitis. Some examples of TNF mediated deseases are rheumatoid arthritis, osteoarthritis, chronic pulmonary inflammatory disease and silicosis. Authors claimed that a therapeutically effective dose for the active compound will range from 0.01 to 100 mg/kg, generally from about 1 to about 5 mg/kg; although the dosage also depends on the illness to be treated. Among all compounds reported in this invention, the following are the ones most useful for the treatment of inflammation.
'OMe
PR 1388 (60)
LL-Z1271a(63)
68
473
Oidiolactone D (62)
Oidiolactone C (61) O
O
OlVIe
OMe OMe 102
Lactones 100-102 were synthesized from natural PR1388 (60). Antimicrobial Activity LL-Z1271a (63) was the first podolactone-type compound reported to exhibit in vitro and in vivo antifungal activity against several experimental fungal infections [9]. Barrero et al. [20] evaluated the minimum inhibitory concentration of this compound together with another seven natural and synthetic podolactone-related structures against selected Gram-positive and Gram-negative bacteria and yeasts (Table 3).
103
474
Table 3. Antiiiiicrobial activity of selected compounds. MIC ( lig^mL). Gram-positive bacteria'
Gram-negative bacteria^
Yeasts'"
A
B
c
D
E
F
G
H
I
63
>25
>25
>25
>100
>100
>100
<3.12
<3.12
<3.12
68
>25
>25
>25
>50
>50
>50
<12.5
<6.25
<3.12
94
25-50
25-50
25-50
>100
>100
>100
25-50
<25
<25
95
<3.12
<3.12
<3.12
50-100
50-100
50-100
<6.25
<3.12
<6.25
96
12-25
50-100
50-100
>100
>100
>100
>100
25-50
>100
98
>25
>25
>25
>100
>100
>100
<6.25
<6.25
<3.12
99
>25
>25
>25
J >100
>100
>100
>25
>25
>100
>100
-
-
Compounds
103
>100
>100
>100
>100
t
^^ >100
' A: Enterococcusfaecalis S 48; B: Bacillus subtilis CECT 397; C: Staphylococcus aureus ATCC 8. ^ D: Salmonella typhymurium LT 2; E: Escherichia coli; F: Proteus s.'' G: Candida albicans CECT 1394; H: Saccharomyces cerevisiae; I: Cryptococum neoformans.
An analysis of the results led to the following conclusions. With respect to tricyclic lactones, maximum activity is shown by compound 95, which showed itself to be selective against Gram-positive bacteria and yeasts. On the other hand, three dienediolides (63, 68 and 98) showed a relevant activity against yeasts, and comparatively (from four to eight times) less effect against Gra/n-positive bacteria. This comparison suggests certain structure-activity relationships. Thus, the presence of the structural moiety 7,9(ll)-dien-12,17-olide moiety is necessary to show the maximum activity against yeasts. For molecules possessing the abovementioned functionalities, the closure of the y-lactone ring results in a remarkable lack of activity against Gram-positive bacteria In 1991, Kubo et al. described that 2a-hydroxynagilactone F (59) showed growth inhibition against Saccharomyces cerevisiae (MIC 800 |Lig/mL) [66]. This same group extended this study to nagilactone C (3) and nagilactone E (36), the most abundant podolactone in P. nagi. These three nagilactones, alone and in combination with a variety of phenylpropanoids, were investigated against C. albicans, S. cerevisiae and P. ovale. The results, listed in Table 4, showed that the activity of the nagilactones was significantly increased by Vi MIC of anethole. For example, the activity of nagilactone E was remarkably increased 128 times when tested against C. albicans [67].
475
Table 4. Antiliiiigal activity of podolactones atone or in combination with V^ MIC of anethoi MIC (fJig/ml) Podolactone alone//plus anethoie Compound
C. albicans
S, cerevisiae
P. ovale
2-hydroxynagilactone F (59)
Not tested
800//6.25
Not tested
Nagilactone E (36)
800//6.25
100//6.25
50//1.56
Nagilactone C (3)
Not tested
1600//400
1600//50
MeO anethoie
Four oidiolactones, podolactone-type metabolites isolated from Oidiodendrum truncata, were tested for antifungal activities against Ustilago violacea, Eurotium repens and Mycotypha microspora. All the substances exerted activity, LL-Z1271a (63) being strongly fungicidal against U. violacea [6]. Recently, Kwai et al. justified their research for antifungal substances after noticing that the incidence of life-threatening fungal infections has steadily increased in inmunocompromised hosts such as immunodefficiency-virus (HIV) infected people and cancer patients [11]. In these cases, Candida albicans is the major oportunistic pathogen, and its resistance to orally taken azoles, the most important antifungals currently known, is attracting much attention. Taking all this into consideration, new drugs capable of stopping the infectious process are required. As a result of their research, Kawai's group isolated five tetranorditerpenoids from Oidiodendrum cf. truncatum, and their activity was tested against 11 strains of pathogenic yeasts and 4 strains of filamentous fungal pathogens (Table 5). The antifungal activity against the pathogenic yeast C. albicans, showed MIC^s of 8 to 32 (xg/mL for LL-Z1271a (63), PR 1388 (60) and oidiolactone C (61), whereas the other two components, oidiolactone D (62) and oidiodendrolide B (68), showed no antifungal up to a concentration of 64 jxg/mL. This trend was the same in other pathogenic yeasts, although the activities differed. In contrast, the tested compounds had almost no activity against the filamentous pathogens. Possibly , the functional moiety responsible for the antifungal activity is the planar six
476
membered-lactone. When the 6-lactone ring is opened or non-planar, the activity decreases. Out of the three tested compounds, LL-Z1271a (63) appeared to be the most active. TaUe 5. Aiitifiingal activity of tetranorditerpenoids isolated fkom O, Truncaium MIC (fig/ml) Test organism
LL-Z1271a
PR 1388
Candida albicans ATCC 90028
8
16
i
32
Catoica/isATCC 90029
8
16
1
32
C. albicans 1463D
8
16
32
C. tropicalis IFM 46816
32
64
64
C parapsibsis IFM 46863
8
16
32
C. dubliniensis CBS 7987
8
8
32
C.kefyrWM 46921
2
2
16
C. guilliermondu IFM 46823
8
32
32
Pichia anomala IFM 47182
2
4
16
Criptococcus neoformans ATCC 90112
4
8
16
Exophiala dermatitidis
16
32
>64
Trichophyton mentagrophytes KCH 1155
32
64
>64
Aspergillus fumigatus IFM 41243
>64
>64
>64
AyZawisIFM41934
64
64
>64
64
64
>64
AmgerH7160B
oidiolactone C
It is also worth mentioning that compounds LL-Z1271a (63) and PR 1388 (60) were also effective against Criptococcum neoformans, the causal agent of cryptococcosis. On the other hand, PR 1388 clearly inhibited the growth of Histoplasma capsulatum, a dimorphic fungus causing histoplasmosis, one of the most severe mycoses. This fact is worth underlining since the fungi responsible for the most severe mycoses, such as Coccidioides immitis (causing cidioidomycosis), Paracoccidioides brasiliensis (causing paracoccidioidomycosis) and Blastomyces dermatitidis (causing blastomycosis), are also dimorphic organisms. So, PR 1388 could be very useful in the search of more efficient drugs against these fungal infections.
477
Insecticidal Activity Species of the Podocarpus genus have been reported to be resistant to many insects, and nor- and bisnorditerpene lactones isolated from these plants, such as nagilactones, have been shown to be responsible for this resistance. Different studies on the effects of naturally occurring lactones on the development of the housefly (Musca domestica) have been reported by Singh et al. [25, 68-69]. These investigations include larval development, pupation and adult emergence and LD50 concentrations (dose required to give 50% total mortality) under defined conditions. Table 6 shows the estimated LD50 concentrations for the housefly, and where sufficient material was available, for the codling moth (Laspeyresia pomonella) and the light-brown apple moth (Epiphyas postvittana). TaMe 6. Toxicity of diffrent lactones to housefly, codling moth and lig^t-brown apple moth LDsoCppm) Compound
Housefly
PodoIactoneA(29)
<250
Nagilactone A ( l )
135.0
Hallactone B (38)
48.2
Nagilactone E (36)
40.8
Podolide (39)
33.9
Sellowin A (34)
13.3
Nagilactone C (3)
IZO
14-Epi-ponolactone A (104)
9.7
Podolactone C (32)
8.2
Hallactone A (7)
3.5
Podolactone E (51)
<1.3
Nagilactone D (4)
0.7
Codling moth
<6.3
Light-brown apple moth
76.4
175.2 7.3
63.8
Podolactone A (29) happened to be the less toxic lactone, and the most active compounds are nagilactone D (4) and podolactone E (51). These podolactones did not produce mortality when applied topically to larvae or adults (5 |Lig per insect), which suggests that these compounds are not
478
toxic by cx)ntact. It appears that they are either toxic by oral ingestion or are antifeedants.
0' 14-epi-ponalactone A (104)
The relationship between lactone structure and toxicity to housefly is also discussed by these authors. All the tested compounds contained an a,p-unsaturated-Y-lactone, which forms ring C, and an electron rich group at C-8; features considered to be essential for plant-cell growth inhibition. The most active compounds have a relatively small, non-polar side chain. The exception is podolactone C (32), but in this case the sulphoxide moiety may contribute to its toxicity. The other structural feature which is reported to strongly influence activity is substitution of ring A. All the more active lactones have a lp,2p-epoxide group, a 3P-hydroxyl and a ylactone joining C-4 and C-6. Apart from structural considerations, the authors claim that the toxicity of the lactones can be considered in terms of their overall polarity. In general, compounds with decreasing polarity show increasing toxicity. Wih respect to the insecticide activity, Podocarpus macrophyllus is widely known in Japan for its resistance to termite attack. Saeki disclosed in 1970 that the termiticidal activity is entirely due to inumakilactone A (28) and another unidentified compound of similar structure, the former being more active [70]. This second compound was later reported to be nagilactone D (4). The methanolic extract of the leaves of P. gracilior (Kenya) caused mortality within 12 days after incorporation into a meridic artificial diet of several lepidopterous pest species. The toxic and growth inhibitory action of nagilactones C (3), D (4) and F (55) and podolide (39) towards these species are shown in Table 7. All tested podolactones are relatively potent growth inhibitors (ED50: 4-30 ppm), while the concentration of the compounds that cause mortality was about two orders of magnitude
479
higher (LD50: 300-2000 ppm), which could be due to a deterrent effect on these compounds [71]. Table 7. Activities of nagilactones C, D and F and podolide towards different lepidopterous agricultuinl pest species. Species
Test compound
LD90 (ppm)
EDw (ppm))
NagilactoneC(3) NagilactoneD(4) NagilactoneF(55) PodoHde(39)
1500
20 4 30
Spodoptera frugiperda
Nagilactone C (3) Nagilactone D (4) Nagilactone F (55) PodoUde(39)
2000 2000 2000
7
Pectinophora gossypieUa
Nagilactone C (3) Nagilactone D (4) Nagilactone F (55) PodoUde(39)
1500
14 4 20 9
Heliothis zea
800
12
200 300
18 6 12
In 1992, Kubo et al. [13] studied the effect of the four most abundant nagilactones in Podocarpus nagi -l-deoxy-2p,3P-epoxynagilactone A (13), l-deoxy-2a-hydroxynagilactone A (8), nagilactone C (3) and nagilactone D (4)- on the feeding and growth of tobacco budwomi larvae, Heliothis virescens. Nagilactone C and D exhibit strong inhibition to the growth of the first instar larvae at 168 and 166 ppm respectively. At these concentrations, none of the larvae fed with nagilactone D reached the third instar, while, when nagilactone C was used, half of the larvae reached the third instar but growth was delayed. Most first instar larvae fed on diets containing l-deoxy-2p,3p-epoxynagilactone A or 1-deoxy2a-hydroxynagilactone A reached the third larval instar but development took longer periods than the control. Finally, when fifth instar larvae were fed on a diet containing nagilactone D at 160 ppm, most of the larvae could not pupate. The three remaining tested compounds were much less active than nagilactone D against H. virescens fifth instar larvae. On the other hand, these investigations also concluded that growth inhibition is caused by feeding deterrence, with nagilactone D being the most potent in inhibiting the mouthpart sensory receptors.
480
Antifeedant Activity Against Mammals The antifeedant activity of these compounds has been described not only for insects, but these dilactones have also been reported as the active principles causing anti-feedant activity for a herbivorous mammal, the guinea pig. Thus, the antifeedant activity of these compounds has been tested employing 1% acetone solutions of natural dilactones. Nagilactone A (1), nagilactone C (3) and l-deoxy-2p,3p-epoxynagilactone A (13) showed activity, whilst their acetate derivatives were inactive. On the other hand, the dilactones were not active with deers, protected species in Japanese forests, which do no eat Podocarpus nagi. The activity of the dilactones seems not simply to have been repellent, but actually poisonous or toxic to the animal, so, the animal supplied only with a sample diet containing ca. 0.5% of a crude extract of the leaves completely rejected the diet for two continuous weeks [12]. Plant Regulatory Activity Since the discovery of the first podolactones, the capacity of these substances to inhibit, not only plant growth, but also the expansion and mytosis of plant cells became apparent [42]. The Galbraith group, which systematically studied this type of activity in different podolactones, published a study of the structure-inhibitory activity of podolactones using a pea-stem growth system [72]. The inhibitory properties in the expansion on hook segments from peas are listed in Table 8. From the analysis of these data, some conclusions were presented. The a,p unsaturated 8-lactone forming ring C seems to be the essential group for the activity. Besides this requirement, a basic centre on Cg is also necessary. All the active compounds possess a double bond or epoxide between C7 and Cs, or a double bond A^^^^\ They also possess a relatively small non-polar lateral chain. The other structural feature which was reported to influence the inhibitory activity is the shape of A ring. The most active lactone, podolactone E (45), has a 1,2-epoxy group and a ylactone joining C-4 and C-6. Finally, the presence of a hydroxy at C-3 does not seem to affect the activity.
481
Table 8. Inhibitoiy activity of naturally occiirriiig lactones on hook segments Crom etiolated dwarf peas % Control growth at concentration given Compound
10'M
10^ M
10^ M
102 (5 X 10^ M)
Inumakilactone C (69) NagilactoneB(2)
81
Nagilactone A (1)
79
Podolactone C (32)
100
70
Podolactone D (33)
98
60
Podolactone B (30)
82
60
Nagilactone C (3)
89
53
Podolactone A (29)
88
37
Inumakilactone A (28)
66
34
Nagilactone D (4)
72
15
Inumakilactone B (31)
47
10
38
0
Podolactone E (45)
82
In 1972, Hayashi's group noticed that podolactones belonging to each of the three above-mentioned structural groups show fine differences in their allelopathic activity [49]. Thus, nagilactones A-D (1-4) (all A type dilactones) inhibited the elongation of the Avena coleoptiles section at 10" ^-10"^ M, however, at 10'^ M, all compounds tumed out to be stimulatory. On the other hand, B type [inumakilactone A (28) and nagilactone E (3^] and C type [ponalactone A (49)] podolactones were exclusively inhibitory in the whole concentration range studied. Experiments with Jerusalem artichoke slices supported the above findings. The studies on the inhibitory effects of podolactones A (29) and E (51) and nagilactone E (36) were extended to a range of bioassays [73]. In some of these assays, the effects of the podolactones were compared to those of lycoricidinol and harringtonolide, plant growth inhibitors (Table 9).
482
TaUe 9.
Concentration of podolactones and related compounds causing significant
(/' = 0.05) inhibition
Concentration for significant effect (i&M)
Bioassay
Germination of radish seed
Harringtonolide Podolactone E (51) Lycoricidinol
10 10 10
Grov/ih of Arabidopsis
Nagilactone C (3) Podolactone A (29)
22 22
Wheat embryo coieoptile
Lycoricidinol Harringtonolide Nagilactone C (3) PodolaaoneE(51)
1 10 100 10
Lettuce hypocotyl (+ GA)
Podolaaone A (29)
27
Lettuce radicle)
Podolactone A (29)
27
Pea tips
Podolactone E (51) Harringtonolide Lycoricidinol
1 10 1
Auxin transport in bean petiole
Podolactone E (51)
10
GA-induced amylase in barley seed
Podolactone A (29)
27
Reduction of mature pikelets in Lolium temulentum
Podolactone A (29) 0.27
In the course of an investigation directed at isolating active chemical components from Podocarpus species, the groups of Hayashi and Kubo [74] noticed that the activity of the crude extract of P. nagi was lower in seed gennination and growth tests could then be explained when the amount of nagilactone E (36) present in the crude extract and strength of the activity of pure nagilactone E was considered. This led to the examination of other components of the crude extract, the known flavonoid epicatechin. Both components were tested employing lettuce and rice seed. When epicatechin was tested alone it caused little change, whereas assays using nagilactone E showed an increase in growth inhibition when the concentration was raised. However, when both compounds were applied together, the co-presence of epicatechin removed the inhibitory effect of nagilactone E, and in some cases even caused growth stimulation. The effect is strongest in the case of rice. So, the inhibitory activity of the root was observed even at 0.1 and 1.0 jxg mL'^ of nagilactone. The presence of epicatechin removed the growth
483
inhibition at 3 \ig mU^ and when 30 \xg mL"^ was applied, a 41% stimulation of root growth was caused. Studying the effect of nagilactones on the growth of lettuce seedlings, Kubo et al. [75] classified the active podolactones isolated from P. nagi into two groups according to their bioactivity. Group I [l-deoxy-2p,3Pepoxynagilactone A (13), l-deoxy-2a-hydroxynagilactone A (8), nagilactone A (1), nagilactone C (3) and 15-methoxycarbonyl nagilactone D (9)] consists of podolactones which stimulate lettuce radicle growth at concentrations between 1 and 10 \ig mL"^ On the other hand, Group II [nagilactone D (4) and nagilactone E (36)] is composed of podolactones which show a significant inhibitory effect on the growth of the lettuce radicle at less than 10 jxg mL"\ It is worth noting that dilactones belonging to Group I were able to stimulate the growth of the radicle at lower concentrations (<10 jxg mL"^) while they were shown to be inhibitory at higher concentrations (>100 \ig mL"^). From the above results, the relationship between the number of hydroxyl groups and the inhibitory effects of podolactones can be inferred, resulting that the less polar compounds possess higher activities. Since all Group I dilactones are type A podolactones, these findings are in accordance with the Hayashi conclusion on the different activity shown by each of the three podolactone structural types. In contrast with Hayashi's reports, findings described in different Japanese patents [76] revealed that all three podolactone structural types are able to stimulate plant growth. Thus, when cucumber seeds are soaked in a 1 ppm type A nagilactone C (3) aqueous solution for 24 hours, and then cultivated for 18 days, they experienced a 54% increase in dry weight of stems and leaves when compared with untreated controls. When type B podolactones were investigated, these authors claimed that the cucumber seeds showed a 30% increase in dry weight of stems and leaves after being soaked in a 1 ppm type B nagilactone E (36) aqueous solution for 24 hours and cultivated for 18 days. Finally, type C dilactones also proved to be stimulant. For instance, tomato seeds were soaked in a 1 ppm aqueous solution of type C nagilactone F (55) for 22 hours, and then cultivated for 5 weeks to show a 31% increase in dry weight of stems and leaves when compared with untreated controls. The aforementioned capability of podolactones to inhibit or stimulate plant growth was again verified in a study carried out by Macias and Barrero's groups [14]. In this study the allelopathic activity of 11 natural
484
and synthetic podolactones on the dicotyledoneae Lactuca sativa, Lepidium sativum^ Lycopersicum esculentum and the monocotyledoneae Allium cepa, Hordeum vulgare and Triticum aestivum was investigated. A selection of the best results is shown in Fig. (3). Fig. (3). Effects of selected dilactones in the gemination, radical and shoot length oil. sativum^ A. cepa and H. vulgare in comparison with the commercial herbicide LOGRAN
^\ l£pi&£^:^im^lt,'
:
10^ M 10' M
i^^"=B
i?^^.^
r^l^ga:feg«j^2^m^jL. , ^;^ Jc
n
10 H I 'iBi:. 10-' M f.^CJ.. 10-^ M |f#>-v;i;;i'
10" M
'or^^hl-
\: lO"" M
I 10-" M 14'
10' M
Ri~
«3
10" M
485
The strong inhibition produced by compounds LL-Z1271a (63), 68 and 98 is noticeable (at a concentration of 10"^ M) in the germination and growth of all the species studied (-90% average). This activity is probably due to the y-lactone ring located between C19 and Q . Likewise, the presence of a metoxyl group at Ci? increases the inhibitory activity at low concentrations. The stimulation effect shown by 68 with dilution over geraiination and growth of monocotyledon species is also noteworthy. These compounds showed the same activity as the commercial herbicide LOGRAN®, and in some cases even greater, which means that they are excellent models for the design of new natural herbicides. In spite of the aforementioned results, the Ohmae group, which has carried out exhaustive studies on the content in nagilactones in forests where P. nagi grows, questions the allelopathic effects of nagilactones [77].
486
CHEMICAL REACTIVITY Before the work reported by Hayashi's group in 1982 [21], the study of podolactones reactivities was limited to the preparation of analogs to facilitate their structure determination. This limited study of reactivity could be explained considering the poor content of podolactones in their natural sources. Thus, to confirm the location of the two hydroxyl groups in inumakilactone A (28), this compound was selectively acetylated, and its diacetate selectively saponificated. The thus obtained 15- and 3monoacetates were oxidized to the corresponding ketones with Jones reagent (Scheme 3) [40].
^ % ^
:o I
mild
*f '''''^^'Oones
^
'*^
HO'
Inumakilactone A (28)
OAc
105
AC2O I NaOAc O
AcO'
Schemes
With respect to the side chain, although some standard reactions failed [78], different correlation reactions assayed to confirm the structure of new isolated members were achieved successfully, some examples include oxidation of the podolactone C sulphoxide group to give the corresponding sulphone (110), as well as the glycol oxidative cleavage of
487
podolactone B 3-acetate (111) with NaI04 to give the cx)rresponding methyl ketone (112).
S02Me mCPBA
Qi
podoiactona C 32
On catalytic hydrogenation with Pt02 inumakilactone A (28) yielded the corresponding dihydro derivative 113 [40]. In contrast, the C ring double bond of inumakilactone B (31) was unaffected when this dilactone was catalytically hydrogenated with Pd-C [44].
ROo OH
inumakilactone A (28)
H2
113
488
inumakilactone B (31)
114
In the earlier studies on the chemical reactivity of podolactones, it was noticed that some standard chemical transforaiations were reported to lead to unexpected results. Some of these outcomes were found in the treatment of nagilactone A (1) and its diacetate (116) with chromic acid and sodium borohydride, respectively [17] (Scheme 4).
Scheme 4
The very same kind of transformation was found upon treatment of nagilactone C-7 acetate with sodium borohydride [57]. Both transformations supposes the first interconversion from type A to type C podolactone groups. In this sense, the particularly limited availability of
489
type C podolactones led Hayashi and Matsumo to focuss their reactivity studies on the formation of these type C podolactones from the more abundant A and B types. In their paper, published in 1982 [20], two main ways were reported to convert type B podolactones into type C. The reductive cleavage of the 7,8-epoxy group experienced by nagilactone C (3) and closely related podolide 39 led, after the corresponding dehydration, to obtain type C podolactones. The second way of conversion of type B dilactones entailed the treatment of nagilactone C or its acetate (119) with alumina to give the acidic derivatives 126 or 127 which upon reduction with sodium borohydride gave the type C 3phydroxynagilactone F (54). Scheme 5 summarizes these results. Type A to type C conversion could be achieved using the previously mentioned transformation of type A 7-acetoxyderivatives with sodium borohydride; unfortunately, the type C dilactones so obtained were epimer at C-14 of the natural dilactones (i.e. 129). A similar type of double bond migration from 8,14 to 7,8 position took place under photolytic conditions with concomitant introduction of a hydroxyl (or a methoxyl) group at C-14. So, the irradiation of nagilactone A diacetate (116) in aqueous THF gave the corresponding 14-hydroxy analog 130, which, after reduction, gave a mixture of type C dilactones, epimers at C14, including 131, the acetoxy derivative of the natural product Iphydroxynagilactone F (52). Transformation of type A to type C dilactones was finally achieved (Scheme 6).
490
0
Podolide (39) TsCI
AcO^
AcO
Scheme 5
491
Scheme 6
Having described the correlation among the different podolactone types, some other transformations were reported, involving mainly the A ring. For instance, the P-epoxide on ring A has been proved to undergo acidic cleavage. However, although some epoxide openings have been reported on inumakilactone A (28) and sellowin A (34) and B (35) [26, 78], these data are not shown here since the structures of these three natural dilactones were corrected after the publication of these transformations [43,47]. Lately, nagilactone D acetate (132) was reported to give, after treatment with HCl, the mixture of chlorohydrins corresponding to the attack of chloride at both extremes of the epoxide (Scheme 7). The same la-chloro-2p-hydroxy chlorohydrin was formed when nagilactone C (3) was heated with rhodium chloride [21].
492
HCI AcO'
132 Scheme 7
Another unusual transformation detected in podolactones was the simultaneous deoxigenation and reduction of nagilactone C (3) to give sellowin C (5) when treated with chromous chloride or Zn-Cu [78]. This result could not be obtained by Hayashi et al. using the complex Cr(C104)2/ethylenediamine in DMF, the 1,2-unsaturated analog 133 being obtained. Compound 133 experienced reduction to natural podolactone 19, transfomiation that entails a double bond migration [21] (Scheme 8). Selective hydrogenation at either the 1,2- or 2,3-position has not been achieved [79], almost surely due to undesired side-reactions, as the above-mentioned saturation of the C ring double bond or the reductive cleavage of the 7,8-epoxide. 0
0
CrClg Zn-Cu
Nagilactone C (3)
Sellowin C
ethylenediamine Cr(CI04)2| DMF 0
Pd-C ^ Hz
Schemes
493
Podolide (39) could be obtained from nagilactone E (36) by treating the alcohol with tosyl chloride in refluxing pyridine. The same elimination was accomplished on heating nagilactone E with phosphorus oxychloride in pyridine. Although the ring A double bond has been reported to show no reactivity, the reaction of podolide and 16hydroxypodolide with mCPBA in the presence of a radical inhibitor takes place stereoselectively, yielding the corresponding a-epoxides [79, 21].
no
I
TsCI/PyrA orPGCIa/PyrA
mCPBA
^ 4,4'-thiobis(6-rt)utyl-mcresol)
Nagilactone E (36)
Scheme 9
Although the epoxidation of the A^ double bond of type C podolactones has been reported as being unsuccessful after several conditions, when C-14 position is not substituted, the epoxidation of this double bond of dilactone 68 is achieved to synthesize oidiolactone C (61) [80].
dimethyloxirane
O Oidiolactone C (61)
494
Ichikawa et al. patented in 1999 a pharmaceutical composition which included natural dilactones from Oidiodedron griseum together with some synthetic derivatives prepared from the more abundant natural compounds [7]. The previously unreported chemical transformations included in this invention are shown in Scheme 10.
495
Synthesis of Podolactones Few routes for synthesizing podolactones are known. Only the syntheses of LL-Z1271a (63), nagilactone F (55) and 3p-hydroxynagilactone F have been described, which are the molecules structurally more simple than their other congeneric types but which, however, are biologically much more active. These syntheses are summarized below. Synthesis of LL-Z1271a a) Starting from Marrubiine In 1973, the group led by Prof. Adinolfi synthetized the antibiotic LLZ1271a (63) [81] starting from ketolactone 139, obtained, in turn, by degradation of the diterpene marrabiin (9% overall yield). This synthesis basically consists of the formation of the 8-lactone C ring by nucleophilic addition to carbonyl group and subsequent lactonization (Scheme 11).. Bromination of 139 in glacial acetic acid, followed by treatment with 1,5-diazabicycle [4.3.0]-5-nonene gives unsaturated ketone 140 in a yield of 85%. The latter's reaction with lithium ethoxyacetylide in THF gives ethynyl-carbynol 141 (90%), which, on being treated with concentrated sulphuric acid in ethanol, gives conjugated ester 142 (65%). The oxidation of 142 with Se02 (2 moles) in glacial acetic acid for between 20 and 40 hours produces a 1:3 mixture of a- and p-acetyllactols (143), respectively (70%). Treatment of 143 with hydrogen chloride in absolute methanol gives compounds 63 and 98 in a proportion of 1:2.5, respectively. COaEt a,b
^ 85%
139
496
OAc
f
'"OMe
OMe
70%
(a) AcOH, HBr; (b) DBN; (c) U O COEt, THF; (d) H2SO4, EtOH; (e) SeOz, AcOH, reflujo; (f) MeOH, HCl. Scheme 11.
b) StartingfromWieland-Miescher Ketone The Welch group tackles stereoselective synthesis of LL-Z1271a using Wieland-Miescher diketone 144 [82] (3% overall yield) (Scheme 4). This synthesis includes, as key steps, the stereoselective introduction of the methyl on carbon 4 in an equatorial position, the foraiation of the ylactone via bromolactonization and the construction of the 6-lactone C ring through a Meyer-Schuster rearrangement. P-Ketoester 145 was prepared starting from Wieland-Miescher ketone in 3 stages with an overall yield of 50%. The reaction of 145 with NaH in HMPA at room temperature for 2 hours, followed by addition of chloromethyl methyl ether gives 146 in 91% yield. Treatment of 146 with lithium in anhydrous liquid ammonia/1,2dimethoxyethane, followed by addition of methyl iodide gives ester 147 in 72% yield. This sequence of reactions allows the highly stereoselective achievement of the metoxycarbonile group's axial stereochemistry in position 4. The synthesis of intermediates 148 and 149 is carried out using conventional methods. Enone 149, after treatment with bromine and then with anhydrous potassium carbonate gives unsaturated y-l^ctone 150 (73%). After hydrogenation of the double bond A^ and reaction with NaH in the presence of ethyl formiate, 151 is obtained in 91% yield. Then, the double bond A^ is regenerated and the aldehyde is protected in the form of acetal (54% yield).
497
Finally, the synthesis is completed by using the Meyer-Schuster rearrangement of the Aren-van Dorp synthesis on enone acetal 152. Treatment of 152 with lithium etoxyacetilide in THF gives an unstable tertiary allylic-propargylic alcohol that, when dissolved in methanol with a catalytic quantity of sulphuric acid produces 63 and its epimer 98 in a proportion of 7:3, respectively, in 42% yield.
7:3 (a) NaH, HMPA; (b) CHaOCHzQ; (c) U, NH3, DME; (d) CH3I; (e) UPrS, HMPA; (f) MeOH, H3O"; (g) MeOH, APTS; (h) Jones; (i) AcOH, Brz; 0) CaCOa, DMA; (k) DCM, Brz; (1) K2CO3, DMF; (m) H2, (Ph3P)3RhCl, benzene; (n) NaH, HCOOEt; (0) Et3N, PhSeCl, THF; (p) AMCPB, THF; (q) ethylene glycol, H2SO4 cat, THF, CaS04; (r) LiC-COEt, THF; (s) H2SO4 5%, MeOH. Scheme 12
498
c) Startingfromcis- and trans-Communic Acids Recently, Barrero et al. have synthesized 63 from cis- and transcommunic acids (153) [14], natural substances present in the cones of Juniperus communis L. Two of the four rings, and three asymmetric carbons have the same configuration as the final molecules. These syntheses were achieved in 10 steps with overall yields of 1% and involves as key steps (Scheme 13): -degradation of the conmaunic acids' side chain, -formation of the 6-lactone by mercuriation-demercuriation, and -allylic functionalization of C-17. Degradation of the side chain of the communic methyl esters is achieved by using metachloroperbenzoic acid and then treating with HIO4. Oxidation of the resulting aldehyde with Jones reagent and esterification with diazomethane gives diester 154 (66%). Treatment of 154 with mercury acetate in reflux toluene quantitatively gives the mercurial derivatives 155. Reaction of this mixture with sodium borohydride in the presence of oxygen and subsequent dehydrogenation with DDQ and p-toluensulfonic acid leads to dienolide 157 (45%). Hydrolysis of the methyl ester and subsequent y-lactonization with lead tetraacetate/light leads to dilactone 6 in 45% yield. Exposure of 68 to Se02 in refluxing dioxane furnishes lactol 99 (100%), which, after treatment with MeOH-catalytic sulphuric acid yields compounds 63 and 98, respectively. C02Me
^COaMe
HgOAc
499
(a) i-m-CPBA, ii-HI04 (73%); (b) Jones reagent (90%); (c) CH2N2 (100%); (d) Hg(0Ac)2, toluene, A (100%); (e) NaBH4, O2; (f) DDQ, dioxane, A (45% yield for steps e and f); (g) H2SO4, H2O (100%); (h) Pb(0Ac)4, hv (45%); (i) Se02, dioxane, A (100%); (j) MeOH, H2S04,(85%). Scheme 13.
Synthesis of Nagilactone F a) Starting from Podocarpic Acid The first synthesis of nagilactone F was carried out by Hayashi et al. [83] starting from (+)-0-methylpodocarpic acid 158 in 23 steps (2% overall yield). The main characteristics of this synthesis include the transformation of the aromatic ring into the 8-lactone ring, the a arrangement (equatorial) of the isopropyl group by photochemical cyclization, as well as the construction of the y-lactone using radical conditions (Scheme 14).
500
(a) Li, NH3, r-BuOH; (b) U3O*; (c) CH2N2; (d) H2, Pd-C; (e) LDA; (f) PhSeQ; (g) H2O2; (h) (i-Pr)2CuU; (i) O3, MciS; 0) Jones reagent; (k) BjHe, THF; (I) APIS; (m) r-BuOK, DMSO; (n) hv, EtOH; (o) DDQ, BF3, dioxane; (p) H2SO4, H2O; (q) Pb(0Ac)4, hv. Scheme 14.
Birch reduction, followed by acid treatment and addition of diazomethane leads to the A^^^^^-enone 159 in 41% yield. Then, the double bond is hydrogenated and, by using PhSeCl and hydrogen hydroperoxide, the double bond A^^ is formed. Treatment of the enone with lithium disopropylcuprate-dimethyl sulfide complex gives an intermediate enolate that is trapped again using PhSeCl. Enone 160 is obtained via oxidative elimination (62%). Ozonolysis of 160, followed by oxidation with Jones reagent, esterification with diazomethane, reduction of the ketone with diborane
501
and treatment with p-toluensulfonic acid gives lactones 161 in a yield of 61%. Then, the double bond A^^^^^ is formed via the selenylation/oxidation protocol. Subsequent treatment with potassium ^err-butoxide in dimethyl sulfoxide gives dienic acid 162 (84%), which is converted into lactone 163 by a photochemical process (90%). The equatorial arrangement of the isopropyl group is understandable if we bear in mind the free rotation of the bond Cs-Cu during the lactonization process and its tendency to avoid interaction with the angular methyl on Cio. Dehydrogenation of 163 with dichlorodicyano-p-benzoquinone in the presence of BF3 gives dienolide 164 (38%). Finally, acid hydrolysis of the methyl ester and subsequent treatment with lead tetraacetate in benzene under irradiation gives 55 in 55% yield. b) Starting from cis- and trans-Communic Acids The same strategy used by Barrero et al. for synthesis of the fungic metabolite LL-Z1271a (63) was used to synthesize nagilactone F (55). Thus, starting from the mixture of communic acids, lactol 99 is achieved, an immediate precursor of both 55 and epimer 165, which are obtained by treatment with isopropylmagnesium bromide. Thus, the synthesis of nagilactone F (55) is completed, with an overall yield of 10%. (Scheme 15). o
nagilactone F (55) 9:1 Scheme 15
165
502
c) Total Synthesis Burke et al. [84] synthetised nagilactone F (55) by a polyenic cyclization initiated with acetal and concluded with vinylsilane, giving an overall yield of 6%. The key steps in this synthesis were the coupling of substrates 166 and 167 with control of the absolute and relative stereochemistry, the cationic biscyclization to form the intermediate tricyclic trans-anti-trans 169 and the formation of the D ring by regioselective intramolecular remote functionalization.
MeaSi
(a) n-BuU; (b) (2-thienyI)Cu(CN)y; (c) BF3-Et20; d) TiCU, Ti(i-Pr0)4; (e) Swem oxidation; (f) AcOH, piperidine; (g) RhCb•3H2O, CaCOa, i-PrOH; (h) PhI(0Ac)2,12, ciclohexane, hv; (i) H2, Pt02; Q) RuOsHaO, NaI04, CH2N2; (k) PhNMeaBra, THF; 0) U2CO3, UBr; (m) DIBALH; (n) TPAP, NMO; (o) UHMDS, TMSQ, PhSeQ; (p) Davis oxaziridine. Scheme 17.
503
Transmetalation of the vinylmercurial derivative with n-BuLi, followed by reaction with 2-lithium thienylcyanocuprate and subsequent addition of BFs-OEta and 167, leads to lactone 168 in 86% yield. Cationic biscyclization of vinylsilane 168 with TiCU and Ti(0i-Pr)4 (at a proportion of 5:1), followed by the cleavage of the remaining acetal (oxidization of the hydroxypropyl ether side chain and subsequent Pelimination of piperidinium acetate) leads to secondary alcohol 169. Isomerization of the double bond, catalyzed by rhodium, followed by remote functionalization of the axial methyl on C4 according to the photochemical conditions established by Suarez et al. [(PhI(0Ac)2, I2] gives tetrahydrofuran 170 in 35% yield. Catalytic hydrogenation of the double bond A^, followed by oxidization using RUO4, esterification with diazomethane and introduction of the double bond A^ leads to compound 171 (67% yield). Treatment of 171 with DIBAL-H, followed by oxidization with tetrapropylammonium perrutenate (TPAP) and NMO and subsequent generation of the double bond A^^^^^ give rise to 55 in 75% yield. Synthesis of(±)'3j3'hidroxinagilactoneF(58) De Groot et al. synthesized (±)-3p-hydroxynagilactone F (54) [85] from compound 172 [86] with an overall yield of 0.2% (13 stages). The key steps in this synthesis were the formation of the 8-lactone by nuclelophilic addition to carbonyl and subsequent treatment with/7-toluensulfonic acid, the introduction of the isopropyl in an a equatorial disposition following a procedure similar to that described by Hayashi et al. and finally the formation of the y-lactone via bromolactonization. Protection of the secondary hydroxyl in 172 and then condensation with 2-methylpropanal leads to enone 173 (65% yield). Addition of dilitioacetate gives rise to compound 174 (78%), which on exposure to ptoluensulfonic acid, and subsequent saponification and light irradiation leads to lactone 175 (57%). Dehydrogenation of 175 with DDQ and 2mesitylensulfonic acid (AMS) gives dienolides 176 and 177 in a proportion of 3:2, respectively (60% yield). These compounds cannot be separated, which means that their thioester derivatives need to be formed, separated and then hydrolyzed again. The overall yield of this process for 176 is 17%. Finally, the formation of the y-l^^tone ring by
504
bromolactonization and subsequent deprotection of the secondary alcohol using boron tribromide gives 3P-hydroxynagilactone F (54).
^
C02CH2SPh 178 52%
^
COaCHaSPh 179
24%
505
MeO^
(a) BaO, Ba(0H)2, DMSO, Me2S04; (b) LDA, (CH3)2CHCHO; (c) UCH2CO2U; (d) TsOH; (e) n-PrSLi; (f) hv, EtOH; (g) DDQ, MSA; (h) r-BuOK, PhSCH2a; (i) chromatographic separation; (j) CF3COOH; (k) piridine, HBr, Br2, AcOH; (1) K2CO3; (m) BBrj. Scheme 18.
Synthesis of Oidiolactone C Starting from Communic Acids The main drawback of the first method described by Barrero et al. [14] for the synthesis of podolactones, lies in the presence of a mixture of three isomers called "communic acids" (153), which requires a chromatographic separation over silicagel impregnated with 20% silver nitrate to eliminate mirceO'Communic acid to allow use of the 2component mixture of cis- and trans-isomors as starting material. Furthermore, some steps in this former synthetic strategy, such as the closure of the 8-lactone or the formation of the dienolide system need improvement. Studying the content in communic acids of the arcestides of different Juniperus and Cupressus sempervirens (Mediterranean cypress), we found the presence of only rrans-communic acid (180) in the arcestides of cypress. Furthermore, this acid could easily be isolated from the acid fraction on the hexane extract by crystallization of its sodium salt. Starting with trans-communic acid, two diffrent strategies were developed, the first one using diene 186 as the key intermediate, which is summarized in Scheme 19.
506
CHO
^COaMe
VfJ^^^^'^'OCOCFa (70%) COaMe 184 ^COaMe
CO2H
186
103
(a) O3, MezS (60%); (b) Jones, (c) CH2N2; (d) SeOj, r-BuOOH (67%); (e) TFAA, DMAP (86%); (f) Pd(PPh3)4 (70%); (g) NaCH3(CH2)2S (85%), (h) Pd(PPh3)4 (72%); (i) LDA, PhSeCl, H2O2 (80%); (j) DMDO (45%). Scheme 19
There are three major achievements in this synthesis [20]. The first consists of the selective degradation of 180 to 181 in 60% yield realized via ozonolysis at low temperature. The second one was the allylic elimination of trifluoroacetate 184 after exposure to Pd(PPh3)4, which resulted in the formation of diene 185 in an acceptable yield (72% based
507
on 70% conversion). The third, and to our judgement, most interesting achievement was the Pd(II)-mediated double lactonization of diacid 186 to form key intemiediate 103 in 70% yield (based on 80% conversion). Fig. (4). This reaction constitutes the first example described of bislactonization of conjugated dienes.
Figure (4)
The second synthetic approach to oidiolactone C (61) is summarized in Scheme 20. This route also conmiences with the ozonolysis of transcommunic acid 180. Now, when this compound was exposed to ozone in excess, keto aldehyde 187 was obtained in 76% yield. The key step in this approach was the y-lactone closure via chemoselective reduction of the lactone moiety on compound 189 through a SN2"mechanism. Compound 189 could be prepared by saponification of the corresponding methyl ester with sodium propanethiolate. Once the primary alcohol is oxidized, the completion of the synthesis of key lactone 103 only requires the allylic oxidation of the C-17 methyl with concomitant closure of the 8lactone. This conversion was achieved with Se02 in refluxing acetic acid to give 103 in 51% yield.
508
103
(a) O3, MezS (76%); (b) Jones, CH2N2; (c) PhSeCl, H2O2 (77% for three steps); (d) MeMgBr (84%);(e) NaS(CH2)2CH3 (83%); (f) LAH (88%); (g) PDC, DMF, CH2N2 (70%); (h) Se02, N B U O O H (51%). Scheme 20
Synthesis of 3p-hydroxy-13,14,15,16-tetranorlabda-7,9(ll)-dien(19,6p),(12,17)-diolide As indicated in the section describing the stractures of natural dilactones, wentilactone B was wrongly assigned when the stracture of 3p-hydroxy13,14,15,16-tetranoriabda«7,9(ll)-dien-(19,6p),(12,17)-diolide was isolated. Our synthesis of this compound allows the reassignment of the structure of wentilactone B. Thus, the hydroxyl group of this natural podolactone should be relocated at C-2 with an a- configuration [87]. Two double cyclization steps were employed in this synthesis. The first involves the construction of the bicyclic skeleton via a Mn(III)-mediated
509
radical cyclization, and the second involves the transformation of this bicyclic intermediate into the tetracyclic podolactone skeleton through a Pd (Il)-mediated bislactonization of the corresponding conjugate diene. COOMe
192
^COOMe
194
193
195R=CH20H 196 R= OHO
.CH2OH
198-
199
197 E.E 198E,Z CH2OH
.COOMe
COOMe
AcO^
205
207 R= COOH 208 R= COOEt
510
-^208 +
(a) (i). PBra, r-BuOMe, CPC, 25 min; (ii) 2. KCN, DMSO, 2 h, 80%; (b) (i) KOH 25%, MeOH, 90^C, 9 h; (ii) CH2N2, ether, QPC, 5 min, 68% of 195 and 196 (ratio 4:1) in two steps; (c) Se02, ^BuOOH, CH2a2, 5 ^C, 6 h, 65%; (d) LAH, THF, 0°C-*rt, 12 h, 86% of 197 and 198 (ratio 4:1); e) (i) NCS, DMS, CH2CI2, O^C, 2.5 h, 85%; (f) CHjCOCHCHsCOOEt, NaH, n-BuLi, THF, 2.5 h, 79%; (g) Mn(OAc)3-2H20, Cu(OAc)2-H20, HOAc, 68%. (h) (i) PDC, DMF, 24 h; (ii) CH2N2, ether, 75% in two steps; (i) (i) NaBH4, MeOH, 0°C, 15 min, 91%; (ii) ClOAc, DMAP, 20 h, 95%; (j) (i). O3, CH2CI2, -78°C, 20 min; (ii) PPhj, -78^C-^rt, 2 h., 91%; (k) (i) PhSeCl, EtOAc, 60 h, (ii) H2O2, Py, CH2CI2, 0°C^rt, 15 min, 77%; (I) Tebbe's reagent, THF, 0°C, 17 min, 69%; (m) NaSCH2CH2CH3, DMF, 50°C, 26 h, 60%; (n) Pd(0Ac)2, p-benzoquinone, HOAc, acetone, 18 h, 78% of 209 based on the amount of 207 in the mixture of 207 and 208; (0) (i) LDA, -78°C, THF, 20 min; (ii) TMSCl; (iii) PhSeCl; (iv) H2O2, Py, CH2CI2, 1 min, 40°C, 36%. Scheme 21
The acyclic precursor 200 was obtained from commercially available geraniol 192. Standard transformations on 192 led to a mixture of diols 197 and 198 in a 4:1 ratio favorable to the desired E,E isomer. Regioselective chlorination of 197 yielded 199. Alkylation of the dianion of ethyl 2-methylacetoacetate with 199 afforded 79% of 200. Oxidative free-radical cyclization of 10 using a 2:1 molar ratio of Mn(0Ac)3 and Cu(0Ac)2 provided 68% of the corresponding bicyclic intermediate. Following a similar process to that described for the synthesis of oidiolactone C, 203 was subjected to ozonolysis and the to the presence of PhSeCl/H202 to give unsaturated ketone 205 which was homologated to diene 206 using Tebbe reagent. Finally The Pd(II)-mediated biscyclization of diacid 207 gave dilactone 209 which was transformed into 210 again using the selenylation/oxidization protocol.
511
ABBREVATIONS IC50 T/C
= =
ED50
=
IL-1 TNF MIC LD50
= = = =
A 50% growth inhibition concentration in vivo. Ratio of life-span of the treted mice to that of the control mice. dose required to be effective against 50% of treated tumoral cells interleukin-1. tumor necrosis factor. mininum inhibition concentration dose required to give 50% total mortality, larvae to adults
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512
[11] Tetranorditerpene lactones, potent antifungal antibiotics for human pathogenic yeast, from a unique species of Oidiodendron, Hosoe, T.; Nozawa, K.; Lumley, T. C; Currah, R. S.; Fukushima, K.;Takizawa, K.; Miyaji, M.; Kawai, K. Chem, Pharm, Bull 1999,47,1591-1597. [12] Nagilactones as an antifeedant from Podocarpus nagi for herbivorous manunals. Hayashi, Y.; Kim, Y.; Hayashi, Y.; Chairul. BioscL Biotech, Biochem. 1992, 56, 1302-1303. [13] Nagilactones from Podocarpus nagi and their effects on the feeding and growth of tobacco budworm. Zhang, M.; Ying, B. P.; Kubo, I. / . Nat Prod. 1992, 55, 1057-1062. [14] (a) Enantiospecific syntheses of the potent bioactives nagilactone F and the mould metabolite LL-Z1271a. An evaluation of their allelopathic potenciaL Barrero, A. P.; Sanchez, J. P.; Elmerabet, J.; Jimenez-Gonzalez, D.; Macias, P. A.; Simonet, A. M. Tetrahedron 1999, 55, 7289-7304. (b) Natural and synthetic podolactones with potential use as natural herbicides. Macias, P. A.; Simonet, A. M.; Pacheco, P. C; Barrero, A. P.; Cabrera, E.; Jimenez-Gonzalez, D. J. Agric, Food Chem. 2000,48,3003-3007. [15] a) Plant growth accelerators containing nagilactones. Imanaka, Y. JP 63243004 A2, 1988. b) Plant growth stimulants containing norditerpene compounds. Imanaka, Y. JP 63243005 A2, 1988. c) Plant growth stimulants containing norditerpene compounds. Imanaka, Y. JP 63243006 A2,1988. [16] Norditerpene dilactones from Podocarpus nagi. Ying, B-P; Kubo, I. Phytochemistry 1993,34,1107-1110. [17] Structures of nagilactone A, B, C and D, novel nor and dinorditerpenoids. Hayashi, Y.; Takahashi, S.; Ona, H.; Sakan, T. Tetrahedron Lett. 1968,17, 20712076. [18] Biosynthesis of a Cie-terpenoid lactone, a plant growth regulator. Kakisawa, H.; Sato, M.; Ruo, T.; Hayashi, T.J. Chem. Soc. Chem. Commun. 1973,166-167. [19] Structures of three new Ci6-terpenoids from an Acrostalamus fungus. Sato, M.; Kakisawa, S.J. Chem. Soc. Perkin 1.1976,2407-2413. [20] The First Synthesis of the Antifungal Oidiolactone C from rran^-Communic Acid: Cytotoxic and Antimicrobial Activity in Podolactone-related Compounds. Barrero, A.F.; Arseniyadis, S.; Quflez del Moral, J. P.; Herrador, M. M.; Valdivia, M.; Jimenez, D.J. Org. Chem. 2002,67, 2501-2508. [21] Reactions and interconversion of norditerpenoid dilactones, biologically active principles isolated from Podocarpus nagi. Hayashi, Y.; Matsumoto, M. J. Org. Chem. 1982,47,3421-3428. [22] Cristal and molecular structure of nagilactone A diacetate. Hirotsu, K.; Higuchi, T.; Shimada, A.; Hayashi, Y. Bull. Chem. Soc. Jpn. 1975,48,1157-1162. [23] Constituents of Podocarpus philippinensis. Chen, Y.P. Chung Kuo Nung Yeh Hua Hsueh Hui Chih 1975, 66,587-588. [24] A study of the constituents of Podocarpus polystachyus R. Br. Hsu, H.Y.; Chen, Y.P.; Huang, T.Y.; Sato, M.; Ruo, T.I.; Kakisawa, H. Tai-wan Yao Hsueh Tsa Chih 1975,27,59-63. [25] Insect-control chemicals from plants. II. Effects of five natural norditerpene dilactones on the development of the housefly. Singh, P.; Fenemore, P.G.; Russell, G.B. Aust. J. Biol. Sci. 1973,26, 911-915
513
[26]
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Structure of the further norditerpenoids of Podocarpus macrophyllus, Inumakilactone A glucoside, a plant growth inhibitor and inumakilactone E. Hayashi, T.; Kakisawa, H.; Ito, S.; Chen, Y.P.; Hsu, H-Y. Tetrahedron Lett. 1972,33,3385-3388. Nagilactone C from Podocarpus purdieanus Wenkert, E.; Chang, CJ. Phytochemistry 191 A, 13,1991. Configurations of nagilactones C and D. Ito, S.; Kodama, M.; Sunagawa, M.; Honma, H.; Hayashi, Y.; Takahashi, S.; Ona, H.; Sakan, T.; Takahashi, T. Tetrahedron Lett, 1969, 2951-2954. Hidrophilic chemical constituents of Podocarpus sellowL Sanchez, L.: Wolfango, E.; Brown, K.S.; Nishida, T.; Durham, L.J.; Duffiels, A.M. An, Acad, Brasil, Cienc, 1970,42, 77-85. Structures of hallactones A and B, insect toxins from Podocarpus hallii. Russell, G. B.; Fenemore, P. G.; Singh, P. J, Chem. Soc. Chem, Commun, 1973,166-167. New congeners of cytotoxic nor-diterpenoid dilactones in Podocarpus nagi: two Ci9 lactones from seed extract. Hayashi, Y.; Yuki, Y.; Matsumoto, T.; Sakan, T. Tetrahedron Lett, 1977,41,3637-3640. New congeners of cytotoxic nor-diterpenoid dilactones in Podocarpus nagi: three highly polar components with a-pyrone ring. Hayashi, Y.; Yuki, Y.; Matsumoto, T.; Sakan, T. Tetrahedron Lett. 1977,34,2953-2956. New congeners of cytotoxic nor-diterpenoid dilactones in Podocarpus nagi: C19 lactones of an a-pyrone type and a 7:8,9:ll-dienolide type. Hayashi, Y.; Matsumoto, T.; Sakan, T. Heterocycles 1978,10,123-131. Biflavonoids, norditerpenes, and a nortriterpene from Podocarpus urbanii, Dasgupta, B.; Burke, B.A.; Stuart, K.L. Phytochemistry 1981,20,153-156. Chemical constituents of Podocarpaceae. I. Cytotoxic constituents from Podocarpus nagi, Xu, Y.; Fang, S. HuaxueXuebao 1989,47,1080-1086. Two nor-diterpene dilactones from Podocarpus nagi. Kubo, L; Ying, B.P. Phytochemistry 1991,30,1967-1969. Two new diterpene dilactones from Podocarpus nagi. Xu, Y.; Fang, S. Zhiwu Xuebao 1993,35,133-137. Three diterpene dilactone glycosides from Podocarpus nagi. Xuan, L-J; Xu, MY; Fang, S-D. Phytochemistry 1995,39,1143-1145 Two new diterpene dilactone glycosides with a trisaccharide moiety from Podocarpus nagi. Xu, Y.M.; Xuan, L.J. Stud. Plant Set 1999, 6, 399-402. Structure of inumakilactone A, a bisnorditerpenoid. Ito, S.; Kodama, M.; Sunagawa, M.; Takahashi, T.; Imamura, H.; Honda, 0. Tetrahedron Lett. 1968, i 7, 2065-2070. Crystal and molecular structure of the bisnorditerpenoid inumakilactone. Godfrey, J.E.; Waters, J.M. AusL J. Chem. 1975,28, 745-753. The structures of podolactones A and B, inhibitors of expansion and division of plant cells. Galbraith, M.N.; Horn, D.H.S.; Sasse, J.M.; Adamson, D. J. Chem, Soc, D, 1970,170-171. Podolactone A p-bromobenzoate. Stereochemistry and absolute configuration. Poppleton, B. J. Cryst Struct Commun, 1975,4,101-6.
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Structures of inumakilactones B and C, lactones of diterpenoid origin. Ito, S.; Sunagawa, M.; Kodama, M.; Honma, H.; Takahashi, T. J. Chem, Soc, Chem. Comm. 1971,91-93. Plant growth inhibitory lactones from Podocarpus neriifolius. Structure of podolactone £". Galbraith, M.N.; Horn, D.H.S.; Sasse, J.M. Experientia 1972, 28, 253-254. Podolactones C and D, terpene sulphoxides from Podocarpus neriifolius. Galbraith, M.N.; Horn, D.H.S.; Sasse, J.M.J. Chem. Soc. D. Chem. Comm. 1971, 1362-1363. Structures of norditerpene lactones from Podocarpus species. Arora, S.K.; Bates, R.B.; Chou, P.C.C.; Sanchez L., W.E.; Brown, Jr., K S.; Galbraith, M.N. J. Org. Chem. 1976,41,2458-2461. Revised structure of podolactone C, the antileukemic component of Podocarpus milanjianus Rendle. Cassady, J.M; Lightner, T.K.; McCloud, T.G.; Hembree, J.A.; Bym, S.R.; Chang, C.J. Org. Chem. 1984,49, 942-945. Structures of nagilactone E and F, and biological activity of nagilactones as plant growth regulator. Hayashi, Y.; Yokoi, J.; Watanabe, Y.; Sakan, T.; Masuda, Y.; Yamamoto, R. Chem. Lett. 1972, 9,759-762. The cytotxic norditerpene lactones of Podocarpus milanjianus and Podocarous sellowii. Hembree, J.A.; Chang, C; Mclaughlin, J.L.; Cassady, J.M.; Watts, D.J.; Wenkert, E.; Fonseca, S.F.; De Paiva Campello, J. Phytochemistry 1979, 18, 1691-1694. Salignona A and Salignona H: two diterpene dilactones. Watson, W.H.; 2^bel, v.; Silva, M.; Bittner, M. Acta Cristallogr. Sect. B 1982,38 (2), 588-592. New congeners of cytotoxic nor-diterpenoid dilactones in Podocarpus nagi: three new components of 7,8-epoxy-enolide type. Hayashi, Y.; Matsumoto, T.; Yuki, Y.; Sakan, T. Tetrahedron Lett. 1977, 48, 4215-4218. Milanjilactones A and B, two novel cytotoxic norditerpene dilactones from Podocarpus milanjianus Rendle. Hembree, J.A.; Chang, C; McLaughlin, J.L.; Cassady, J.M. Experientia 1980,36,28-29. Lignan and norditerpene dilactone constituents of Podocarpus saligna. Matlin, S. A.; Bittner, M.; Silva, M. Phytochemistry 1984,23, 2867-2870. Norditerpene dilactones from Podocarpus saligna. Matlin, S. A.; Prazeres, M. A.; Bittner, M.; Silva, M. Phytochemistry 1984,23, 2863-2866. Congeners of norditerpene dilactones from Podocarpus nagi. Ying, B.P.; Kubo, I.; Chairul; Matsumoto, T.; Hayashi, Y. Phytochemistry 1990,29,3953-3955. Structure of the norditerpene ponalactona A and its glucoside, plant growth inhibitors. Ito, S.; Kodama, M.; Sunagawa, M.; Koreeda, M.; Nakanishi, K. J. Chem. Soc. Chem. Comm. 1971,855-856. Diterpenoids of Podocarpus nubigena. Silva, M.; Bittner, M.; Sammes, P.G. Phytochemistry 1973,12,833-836. Structure and chemistry of antibiotic LL-Z1271a, an antifungal carbon-17 terpene. EUestad, G.A.; Evans, R.H.; Kunstmann, M.P.; Lancaster, J.E.; Morton, G.O. J. Am. Chem. Soc. 1970,92,5483-5489. Structure of inumakilactone D. Kodama, M.; Kabuto, C; Sunagawa, M.; Ito, S. Tetrahedron Lett. 1977,33, 2909-2910.
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[61] Isolation and structure detennination of norditerpene dilactones from Podocarpus saligna. Matlin, S.A.; Prazeres, M.A.; Mersh, J.D.; Sanders, J.K.M.; Bittner, M.; Silva, M.J. Chem, Soc, Perkin Trans, 11982,2589-2593. [62] A bisnorditerpene dilactone from Podocarpus nagi. Kubo, I.; Ying, B.P. Phytochemistry 1991,30,3476-3477. [63] Constituents of Podocarpus saligna. Silva, M.; Hoeneisen, M.; Sammes, E.G. Phytochemistry 1972,11, 433-4. [64] Antitumor activity of podolactones. Hayashi, Y.; Sakan, T.; Sakurai, Y.; Tashiro, T. Gann 1975, 66,587-588. [65] Antitumor activity of norditerpenpoid dilactones in Podocarpus plants: structureactivity relationship on in vitro citotoxicity against Yoshida Sarcoma. Hayashi, Y.; Matsumoto, T; Tashiro, T. Gann 1979, 70,365-369. [66] An antifungal norditerpene dilactone from Podocarpus nagi. Kubo, I.; Himejima, M.; Ying, B.P. Phytochemistry 1991,30,1467-1469. [67] Combination effects of antifungal nagilactones against Candida albicans and two other fungi with phenylpropanoids. Kubo, I.; Muroi, H.; Himejima, M. J. Nat. Prod. I99i,56,220-226. [68] Insect-control chemicals from plants. Nagilactone C, a toxic substance from the leaves of Podocarpus nivalis and Podocarpus hallii. Russell, G.B; Fenemore, P.O.; Singh, P. Aust. J. Biol. Sci. 1972,25,1025-1029. [69] The insecticidal activity of some norditerpene dilactones. Singh, P.; Russell, G.B.; Hayashi, Y.; Gallagher, R. T.; Fredericksen, S. Entomol. Exp. Appl. 1979, 25,121-127. [70] Termiticidal substances from the wood of Podocarpus macrophyllus. Saeki, I.; Sumimoto, M.; Kondo, T. Holzforschung 1970,24, 83-86. [71] Multichemical resistanse of the conifer Podocarpus gracilior (Podocarpaceae) to insect attack. Kubo, I.; Matsumoto, T.; Klocke, J.A. J. Chem. Ecol, 1984, 10, 547-559. [72] Structure-activity relations of podolactones and related compounds. Galbraith, M.N.; Horn, D.H.S.; Ito, S.; Kodama, M.; Sasse, J. M. Agr. Biol. Chem. 1972,36, 2393-2396. [73] Some physiological efeects of podolactone-type inhibitors. Sasse, J.M.; Wardrop, J.J.; Rowan, K.S.; Aspinall, D.; Coombe. B.G.; Paleg, L.G.; Buta, J.G. Phisiol. Plant, 19S2,55,51-59. [74] Epicatechin can cause the seedling growth inhibitor, nagilactone E, to induce growth stimulation. Kubo, I.; Matsumoto, T.; Hanke, F.J.; Taniguchi, M.; Hayashi, Y. Experientia, 1985,41,1462-1463. [75] Effects of nagilactones on the growth of lettuce seedlings. Kubo, I.; Sutisna, M.; Tan, K. S. Phytochemistry 1991,30,455-456. [76] a) Plant growth regulators containing norditerpenes. Imanaka, Y. JP 02022204 A2, 1990. b) Plant growth regulators containing norditerpenes. Imanaka, Y. JP 02022205 A2, 1990. c) Plant growth regulators containing norditerpenes. Imanaka, Y. JP 02022206 A2,1990. [77] The plant growth inhibitor nagilactone does not work directly in a stabilized Podocarpus nagi forest. Ohmae, Y.; Shibata, K.; Yamakura, T. /. Chem. Ecol., 1999,25,969-984.
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Some chemical transformations of norditerpene lactones from Podocarpus sellowii. Brown, K.S.; Sanchez, W.E. Tetrahedron Lett, 1974,8,675-678. Reactions of citotoxic nor-diterpenoid dilactones in Podocarpus nagi: modificatios of ring A functional groups. Hayashi, Y.; Matsumoto, T.; Hyono, T.; Sakan, T. Chem, Lett., 1977,1461-1464. Preparation of bioactive podolactones via a new Pd-catalysed bislactonization reaction. Synthesis of oidiolactone C. Barrero, A. F.; Quflez del Moral, J. F.; Cuerva, J. M.; Cabrera, E.; Jimenez-Gonzalez, D. Tetrahedron Lett. 2000, 41, 5203-5206. Synthesis of the terpenoid antibiotic LL-Z1271a. Adinolfi, M.; Mangoni, L.; Barone, G.; Laonigro, G. Gazz. Chim, Ital. 1973,103,1271-1279. A stereoselective total synthesis of the antifungal mold metabolite 7a-methoxy3a,10b-dimethyl-l,2,3,3aa,5aa,7,10bp,10ca-octahydro-4H,9Hfuro[2',3',4':4,5]naptho [2,l-c]pyran-4,10-dione. Welch, S.C; Hagan, C.P.; White, D.H.; Heming, W.P.; Trotter, J.W. J. Am. Chem. Soc. 1977, 99:2, 549556. Total synthesis of nagilactone F, a biologically active norditerpenoid dilactone isolated from Podocarpus nagi. Hayashi, Y.; Matsumoto, T.; Nishizawa, M.; Togami, M.; Hyono, T.; Nishikawa, N.; Uemura, M. /. Org. Chem. 1982, 47, 3428-3433. Enantioselective synthesis of nagilactone F via vinylsilane-terminated cationic cyclization. Burke, S.D.; Kort, M.E.; Strickland, S.MS.; Organ, H.M.; Silks, L.A., III. Tetrahedron Lett. 1994,35,1503-1506. Total synthesis of (±)-3p-hidroxinagilactone F. Reuvers, J.T.A.; de Groot, A.J. J. Org. Chem. 19S6,51,4594-4599. Stereoselective synthesis of (±)-3,4,4a,5,6,7,8,8a-octahydronaphtalen-l(2H)-ones via homogeneous hydrogenation of (±)-5,6,7,8-tetrahydronaphthalenones. Reuvers, J.T.A.; de Groot, A.J.J. Org. Chem. 1984,49,1110-1113. A convenient synthesis of ring A-functionalized podolactones. Revision of the structure of wentilactone B. Barrero, A.F.; Quflez del Moral, J. F.; Herrador, M. M.; Valdivia, M. Org. Lett 2002,4,1379-1382.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol, 28 © 2003 Elsevier Science B.V. All rights reserved.
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ANTITUMORAL ACTIVITY OF LIPIDS A STUDIES IN ANIMAL MODELS AND CANCER PATIENTS DANIELE REISSER, NOLWENN GAUTHIER, ALENA PANCE, JEAN-FRANCOIS JEANNIN Cancer Immunotherapy Research Laboratory, Ecole Pratique des Hautes EtudesJNSERM U517, Faculty ofMedicine, 7 Bd Jeanne d 'Arc, 21079 Dijon, France, ABSTRACT: LPS consist of an 0-specific chain, a core and a lipid A. It brgan to be used as a tumor therapy since the XEX century, but it became clear in the 1980s that the biological activity of LPS is due to their lipid A component. LPS and lipid A interact with membrane receptors on target cells which initiate signal transduction. The end result of the intracellular signaling is mainly the activation of N F K B and AP-1, which then induce the expression of diverse genes such as cytokines, adhesion molecules, etc. Thus the antitumoral effect of LPS and lipid A is indirect and relies on the induction of an immune response, both innate and acquired. These components aflfect tumor development mainly inhibiting tumor blood flow and inducing necrosis as well as apoptosis of the tumor cells. Cancer treatments with LPS and lipid A have been tested in animal models as well as at the clinical level, either alone or as adjuvants in therapeutic vaccines. In animals, these treatments have achieved certain efficacy, which depends on the type of molecule and the protocol used. In general, an increased survival has been obtained, accompanied in some cases of tumor regression and cure. In clinical trials, the induction of an immune response has been evidenced, but the results are so far too preliminary to permit a definite conclusion. Nevertherless, these components represent a hope for cancer patients and now it is necessary to extend the studies to clinical phase III trials.
INTRODUCTION Cancer has become the second cause of death in industrialized countries. Numerous advances in molecular biology and immunology have contributed to a better knowledge of carcinogenesis, tumor growth, and tumor-host interactions. They have opened new therapeutic strategies, such as gene therapy, even though a number of obstacles to their successful application remain. Therefore the therapeutic advances in the
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short term will probably consist in more conventional concepts. The immunological unresponsiveness of patients to their growing tumors is an important aspect, therefore strategies to overcome this unresponsiveness are an important approach in cancer therapy. An antitumoral immune response can be induced in vivo with endotoxins. Important progress in lipid A chemistry has been achieved, which together with preclinical studies done in the past decade, allowed clinical assays that will be reviewed here. The chemistry of endotoxins and lipid A will be briefly introduced from an historical point of view. The biological properties of lipid A relevant to their antitumoral activities will be reported with particular attention to specific receptors and their signal transduction. Lipid A antitumoral activities as well as their mechanisms of action in animal models and cancer patients will be reviewed. fflSTORICAL BACKGROUND Richard Pfeiffer [1] introduced the term endotoxin, in contrast to toxins or exotoxins, to name toxic bacterial components not excreted from the living bacteria but released during bacteriolysis. The first indications about their composition were given by Boivin in the years 1935-1945 [2,3]. He purified substances composed of polysaccharides and lipids with only small amounts of proteins named lipopolysaccharides (LPS) by Shear and Turner [4]. Now we know that LPS are constituents of the outer membrane of Gram-negative bacteria. The chemical and biological properties of LPS were extensively studied by Westphal and Liideritz [5]. They proposed in the sixties, that all LPS consist of 3 regions: O-specific side chain, core and lipid A as a common architecture. The O-specific chain, usually composed of a polymer of repeated oligosaccharide units, is a structure specific for any given strain of bacteria, which is responsible for the antigenic properties of LPS. The core is an oligosaccharide structurally less variable, which contains at least one Kdo (2-keto-3-deoxyoctulosonic acid). A majority of core regions are characterized by the additional presence of heptoses (L-glycero-Dmannose-heptose). Some of the cores have common segments, and constitute the link between the O-specific chain and the lipid A. Lipid A
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is the lipidic component and is the structurally most stable part of LPS. Lipid A consists of a diglucosamine backbone acylated by ester-linked and amide-linked long chain fatty acids. The nature of the backbone glycosyl residues is the same for a large number of LPS. The precise structure of the lipids AfromEscherichia coli (E. coli) and Salmonella typhimurium (S, typhimurium) were obtained at the beginning of the years 1980 by Takayama, Kusumoto, Galanos and Rietschel [6-8]. The first synthetic lipids A were chemically synthesized by Kusumoto and collaborators in 1985 [9]. Since then it became feasible to prepare non-toxic derivatives of lipid A. A link between bacteria and tumor ther^ was found early, at the beginning of the XVni century [10]. By the end of the XIX century, Coley [11] developed a treatment for cancer with a mixture of bacterial toxins. In 1943 Shear and Tumer [4] found that the antitumor effect of Coley's toxin was due to endotoxins, and after several decades it was shown that the biological activity of LPS was due to the lipid A [5]. We investigated the structures of lipids A with regard to their antitumor activities [12], finding that the optimum in vivo activity is obtained with diglucosamines acylated by 3 long chain fatty acids. BIOLOGICAL PROPERTIES OF LIPID A Lipid A in vivo LPS and lipids A alike, when injected in vivo interact with diverse serum proteins which can be divided in 3 groups, depending on their capacity to modify their activities. The first group of proteins which decrease the activities of LPS consist of high-density lipoprotein (HDL), low-density lipoprotein (LDL), the bactericidal permeability-increasing (BPI) protein [13], and the cationic protein CAP37 [14]. The proteins which do not modify their activities are albumin, lactoferrin [13], transferrin, hemoglobin [15], and lysozyme. Finally some proteins such as LPS binding protein (LBP) augment their activities [16,17]. A few hours after its injection, circulating lipid A is found mainly in liver, lung as well as in the spleen, adrenals and kidneys [18]. In these
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organs and in the blood, lipid A will bind target cells via binding proteins and receptors. Lipid A signaling pathway Different receptors for LPS have been identified, of which the scavenger receptor and the CDl 1/CD18 receptor mediate uptake and detoxification of LPS. In contrast CD14, a glycosylphosphatidylinositol (GPI)-linked protein, initiates LPS signal transduction. Membrane-bound or soluble CD 14 can bind LPS in the absence of LBP, however LBP increases this binding. Lipids A also bind LBP, scavenger receptor [19], CD11/CD18 [20] and CD14 [21]. Of these, CD14 is likely to be the main pathway for initiating downstream signaling events, although in some cases soluble LPS-binding proteins permit lipid A signaling independently [22]. Membrane bound CD 14 receptors are found on the surface of monocyte/macrophages. The soluble CD 14 is capable of binding to CD14-negative cells such as endothelial [23] or smooth muscle cells [24] and of inducing several signaling pathways. CD14 lacks transmembrane and intracellular domains, probably acting through the binding of distinct transmembrane proteins, such as the Toll Like Receptors (TLR), which transduce the signal. A general agreement is that TLR4 mediates the lipid A signaling pathway initiated by the binding of lipid A to CD 14, since TLR4-deficient mice are unresponsive to LPS in vivo [25]. In contrast, the role of TLR2 in lipid A (and LPS) transduction is debated [26]. TLR4 requires the presence of the MD-2 surface molecule to transduce the lipid A signal and probably of one additional molecule. MD-2 is coexpressed with TLR4 on the cell surface. The TLRs have an intracellular domain (TIR) which binds to a homologous domain in the adaptor protein, MyD88. This protein contains a death domain which interacts with the death domain of the mammalian Interleukine-1 Receptor- (IL-IR)-Associated kinase (IRAK). IRAK binds another adaptor, TRAF6 which seems to bridge 2 signaling pathways: a) through the adaptor TAB2 activates the nuclear factor (NF)kB-inducing kinase (NIK) and IkB kinase (KK); b) through the recently identified Evolutionarily-Conserved Signaling Intermediate in the Toll pathway (ECSIT) protein, activates another cascade of kinases:
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MEKK-1, ERK, p38, and JNK/SAPK. The results are the phosphorylation of IkB and its dissociation from NFKB, thus permitting the translocation of NFKB to the nucleus, as well as the activation of the AP-1 transcription factors Jun and Fos. Furthermore, the LPS signal transduction involves the activation of G proteins, of phospholipases C and D, the formation of diacyl-glycerol (DG) and inositol triphosphate (IPS). DG mediates the stimulation of protein kinase C (PKC) and IPS induces an increase of cytosolic Ca^. The LPS signaling pathway also involves tyrosine kinases, constitutive nitric oxide (NO) synthase (cNOS), cGMP-dependent protein kinase, Ca^ channels, calmodulin and calmodulin kinase [27,28], as well as the MAP kinases [29] ERKl, ERK2 and pS8 [2S]. The intracellular events in response to LPS are due to lipid A because they are inhibited by polymyxin B which is known to bind lipid A [27] and they are reproduced by lipids A [S0,S1]. The capacity of lipids A to activate a signaling pathway depends on their structure and is species-dependent. Thus, the lipid A analog lipid IVa and the Rhodobacter sphaeroides lipid A (RSLA) are LPS antagonists in human macrophages [S2], while acting as LPS agonists in hamster macrophages [SS]. On the contrary, in mouse macrophages, lipid IVa is a LPS agonist and RSLA a LPS antagonist [21]. CD14 is not involved in the species dependence [21] but TLR4 is responsible for the species-specific recognition of the lipid A structure [SS]. Lipids A first induce the expression of early inflammatory genes such as tumor necrosis factor- (TNF)-a, EL-lp, type 2 TNF receptor, IP-10, DS, D8 and D2 genes [S4]. Then further genes are activated such as other cytokines and receptors, adhesion molecules, acute-phase proteins, tissue factors, as well as the inducible NOS (NOS H). These cascades of events initiated by lipid A provoke in their target cells complex responses in vivo, whose relevance in the host response to tumor growth is reviewed below. Lipid A tolerance Lipid A can induce a state of hyporesponsiveness to its own effects (or to LPS) in animals or humans. This effect is called LPS tolerance and by
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analogy lipid A tolerance [35-37]. LPS tolerance can be divided in early and late tolerance which is mediated by anti-LPS antibodies. However, there is no late tolerance to lipids A because lipids A are not immunogenic. In vivo, after a sublethal dose of lipid A, a transient period of hyporesponsiveness follows during which a lethal dose of lipid A or LPS is well tolerated in mice, rats and humans [37-40]. The decrease of lipid A toxicity e.g. death rate and weight loss, is probably due to stabilization of blood pressure, coagulation and temperature, as well as a decrease of cytokine concentrations in serum, such as TNF-a, IL-lp, IL-6, and colony stimulating factors (CSF) [35,38,41-44]. Pulmonary and hepatic levels of IL-ip, IL-6, TNF-a, G-CSF, IL-Ra, IL-10, interferon-(IFN).y, macrophage-inducing protein (MlP)-la, MIP-ip, monocyte chemoattractant protein (MCP)-l, and NOS11 mRNAs in mice submitted to septic shock are lower when they have been pretreated with lipid A [45]. Induction of lipid A tolerance is concomitant with corticosterone production and does not occur in adrenalectomized mice [46,47]. Glucocorticoids down-regulate many genes including TNF-a, IL-lp, and IL-6 [48,49], which is mediated by the inhibition of NFKB nuclear translocation and activity. If some molecules are decreased in tolerant animals, others are increased such as the TNF receptor 2 and IL-10, which are able to antagonize the effects of TNF-a or to inhibit the expression of numerous cytokine genes. Lipid A tolerance is due to CD 14 positive cells, mainly monocytes/macrophages. We have shown that when rats are injected with different lipids A, their peritoneal macrophages produce high amounts of TNF-a, IL-ip, IL-6 and NO in vitro after the first i.p. injection, but are no longer active after 2-5 i.p. injections. Furthermore they do not respond in vitro to lipid A or LPS stimulation for at least 2 weeks after the last injection, while normal macrophages do (unpublished results). The production of CSF and IFN-y also decreases during lipid A tolerance [42]. In vitro, mouse as well as human monocytes/macrophages can be tolerized to lipid A [50-52]. The in vitro induced tolerance lasts one or two days during which the CD 14 receptor is not down-regulated [52-54]. Uncoupling of the CD 14 receptor to downstream signaling pathways, or a specific blockade of the transduction, is unlikely to occur as explained
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below. We have shown that the tolerisation of Raw 264.7 murine macrophages to LPS [52] and Hpid A (unpubHshed results), is induced at the transcriptional level. An increased level of IxBa has been reported as a mechanism for the down-regulation of IL-lp in tolerized monocytes [55]. Diminished levels of TNF-a and NO have been ascribed to a predominance of the p50 homodimeric form of NFKB, which represses transcription in the nuclei of macrophages tolerized to LPS [50,52]. This coincides with the fact that in tolerant cells the level of the pi05, which is the proform of p50, is increased, and then processed [56]. In addition, the secretory leukocyte protease inhibitor (SLPI) is a LPS inhibitor induced by LPS in macrophages [55] which suppresses the LPS-induced activation of NFKB. Thus SLPI inhibits the production of TNF-a and NO, constituting another mechanism of LPS tolerance. STUDIES IN ANIMAL MODELS Animal models permit the investigation of the antitumoral effect of lipids A and LPS [57]. We will first consider the acute effects observed after the first injection of lipids A or LPS. In each case, their relevance to an antitumoral activity will be discussed. Acute effects of LPS and lipids A Effect on general physiology
The hallmark of a bacterial infection is fever due to LPS pyrogenicity. It may have some relevance in cancer as hyperthermia is tested as a cancer treatment on its own, as reviewed by Christophi et al. [58]. This effect is mediated by cytokines (mainly BL-lp) produced in response to LPS. In himians, LPS can induce coagulation [59] which might involve TNF-a [60]. The importance of coagulation for timior irrigation and therefore its effect on tumor growth is difficult to evaluate. However, Parr et al. [61] showed that it is involved in but not sufficient for tumor regression induced by LPS in mice, since lymphocytes were also necessary.
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LPS are known to activate complement via the alternative pathway. Regression of subcutaneous MH134 tumor in C3H/He mice was shown to implicate the complement as part of the antitumoral effect of LPS [62] but this effect has not been since documented. LPS can provoke a multi-organ failure, due to the secretion of acutephase reactants such as platelet-activating factor (PAF), TNF-a, and LBP. Multi-organ failure is also due to the secretion of prostaglandin E2 (PGE2) and inflammatory cytokines such as TNF-a, EL-ip, IL-6, whose production may increase the secretion of the acute-phase reactant, LBP [63], thus constituting an amplifying loop. The presence of a tumor may modify this host response as will be discussed below. LPS are toxic for rabbit endothelial cells in vivo [64] and Choi et al. [65] showed that LPS in vitro toxicity for human endothelial cells was Fas-Associated Death Domain (FADD)-dependent. LPS are also involved in the activation of endothelial cells, increasing their expression of adhesion molecules. Lipids A increase the expression of Intercellular Adhesion Molecule (ICAM)-1 by human endothelial cells [66] which, by enhancing the adhesion of leukocytes to the endothelium, plays a role in the inflammatory process. In Long Evans rats, the increase in ICAM-1 expression is mediated by IL-lp and TNF-a after LPS instillation in the airways [67]. Is adhesion an important phenomenon during the metastatic process? Taichman et al. [68] showed that LPS facilitate the adhesion of several human tumor cell lines on endothelial cells in vitro. In this regard, LPS could be prometastatic by increasing the arrest of tumor cells in capillaries. On the contrary, Jibu et al. [69] showed that LPS promote the detachment of tumor cells from lung endothelium, thus having an antimetastatic effect. LPS also increase the production of NO in bovine endothelial cells, via a protein tyrosine kinase pathway [70]. The antimetastatic effect of the NO secreted by endothelial cells has been shown in vivo by Rocha et al. [71] in a model of murine lymphoma. Finally, activated endothelial cells release cytokines, whose role will be discussed later.
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Effect on innate immunity
As components of bacterial cell walls, LPS and lipids A are first recognized by the cells of the innate immune system and are able to activate them. The increased expression of adhesion molecules by the endothelium may activate polymorphonuclear neutrophils (PMN) in rabbits [72]. During endotoxic shock, activated PMNs release their granule content and secrete both proinflammatory and cytotoxic molecules. Pickaver et al. [73] were the first to show PMN cytotoxicity against tumor cells. We showed that PMNs are toxic for PROb colon tumor cells [74] in BDIX rats. In vivo, PMNs have been implicated in the Schwartzman reaction [75], and may be involved in LPS-induced tumor necrosis. PMNs, when activated by LPS, synthesize and release NO. The role of NO in tumor growth will be discussed later. The decrease in tumor growth after intradermal injections of LPS is attributed to the induction of TNF-a secretion by PMNs both in intradermal tumors (Meth A sarcoma in BALB/c mice, MH-134 hepatoma in C3H/He mice, Lewis Lung carcinomas in C57BL/6 mice) and pulmonary Meth A metastases [76,77]. Historically, since the 1970s, the first cells considered as good candidates in the fight against cancer were macrophages. Macrophages were shown to destroy tumor cells non-specifically in vitro in the presence of LPS [78]. Mannel et al. [79] considered them as the effector cells in the regression induced by LPS of a fibrosarcoma in C3H mice. In v/vo, the role of macrophages is controversial and difficult to prove: they are considered either as friends [80] or foes as their tolerization is concomitant with the antitumoral effect of lipid A [81]. However, we showed that no correlation exists between the capacity of LPS and lipids A to activate macrophages from BDIX rats in vitro and their in vivo antitumoral activity to PROb tumors except for ILl-p production [12,82,83]. In any case, the importance of macrophages has been emphasized because they are the main producer of TNF-a in response to LPS. Natural killer (NK) cells are mononuclear cells able to lyse tumor cells without prior sensitization to specific antigens [84]. When NK cells
526
are activated in vitro, they differentiate into Lymphokine-activated killer (LAK) cells with a broader panel of target cells. NK cells from mice which had received an injection of LPS from E. coli or lipid A from Porphyromona gingivalis (P. gingivalis) have an increased in vitro activity [85]. In vivo, GLA-27 reduced metastasis in (C57BL/6X DBA/2)F1 mice via NK cell activation when injected one day before B16-F10 melanoma tumor cells [86]. Furthermore, pretreatment with ONO-4007 (Ono Pharmaceutical Co, Osaka, Japan) increased NK activity in the liver of C57BL/6 mice and decreased the nimiber of EL4 hepatic metastases via the secretion of the NK-activating cytokine IL-12, byKuppfercells[87]. Effect on cytokine production
One of the most important effects of lipids A on tumor growth occurs through the modulation of cytokine production. The main cytokine whose production is induced by lipids A, is TNFa. Historically, this was the first mechanism described to explain the efficacy of LPS on tumor regression in experimental models. Gratia and Linz [88] in guinea pig liposarcoma, and Schwartzman and Michailovski [87] in mouse sarcoma, observed that filtrates of bacteria cultures induced hemorrhage and necrosis within the tumors. However, Andervont [89] tested bacterial filtrates on several murine tumors, and found that not all of them underwent haemorrhagic necrosis, while Seligman et al. [91] concluded that in BALB/c mice bearing Meth A sarcoma the in vivo effect of LPS is not direct, but can be attributed to a circulating factor [92]. Similarly, necrosis of Meth A tumors in BALB/c mice treated by lipids A may depend on TNF-a [93]. However the phenomenon is not clearcut as injections of antibodies directed against TNF-a did not prevent tumor regression [94]. Consistent with these results, we have shown that lipid A induces regression of TNF-ainsensitive cells [81]. For a complete regression, an acquired immune response is necessary since tumor regression does not occur in T celldeficient mice [95] or nude rats [96]. Moreover, to achieve tumor regression, several injections of the lipids A: SDZ MRL 953 (Sandoz, Vienna, Austria), DT-5461 (Daiichi Pharmaceutical Co, Tokyo, Japan),
527
or OM-174 (OM Pharma, Geneva, Switzerland) are needed, and these protocols induce tolerisation of rat macrophages which then no longer produce TNF-a (unpublished results). As is implied by its name, the first TNF-a-dependent mechanism described was the induction of tumor necrosis in vivo through its role in tumor vasculature. However the mechanisms of the in vitro toxicity of TNF-a to tumor cells imply apoptosis rather than necrosis [97]. Tumor necrosis in SCED (severe combined immuno-deficiency) mice treated with LPS does not lead to the rejection of tumors [98]. Furthermore, necrosis and tumor regression must be dissociated since anti-IFN-y antibodies inhibit LPS-induced regression of Meth A sarcoma in mice, but not the necrotic hemorrhage attributed to TNF-a. It is now accepted that the antitumoral effect of TNF-a is indirect and dependent on acquired immune response. Matsumoto et al. [99] reported that, while TNF-a itself has no effect on hepatoma KDH-8 tumor cells in vitro, the antitumoral effect of the lipid A ONO-4007 against KDH-8 tumors in vivo is inhibited by anti-TNF-a antibodies in WKAH rat, showing an indirect effect of TNF-a. IFN-y is a cytokine produced by lymphocytes, whose production can be induced by LPS. This effect may be indirect, as macrophages activated by lipid A produce IL-12 and/or IL-18 that are IFN-y inducing cytokines [100-101]. Furthermore, human endothelial cells treated in vitro with LPS, induce the secretion of IFN-y by T lymphocytes [102]. Its requirement for tumor regression was evaluated in vivo by Dighe et al. [98] who showed that DFN-y-unresponsive Meth A fibrosarcoma is not rejected by BALB/c mice treated with LPS. However, IFN-y might act in combination with TNF-a, as shown in C57BL/6 mice bearing B16 melanoma [103]. Particularly in combination with IL-ip and TNF-a, IFN-y induces the expression of NOS 11 and the production of NO [104]. Other IFNs may also be involved as the lipid A DT-5461 acts through DFN-a/p as well as IFN-y [105]. Another cytokine whose secretion can be induced by LPS or lipid A is EL-ip. It is principally secreted by macrophages, but a variety of other cell types release it, such as dendritic cells, endothelial cells, NK cells and lymphocytes. It may be directly cytotoxic to tumor cells on its own
528
[106] or in association with IFN-a, as is the case for B16 melanoma in C57BL/6 mice in vivo [103]. Other cytokines are involved in the effect of lipid A on tumor growth. OM-174 decreases TGF-P mRNA and protein in rat PROb tumors [107]. ONO-4007 increases TNF-a, IL-ip, IL-12, and 11-6 while decreasing TGF-P levels [108]. The secretion of XL-12 by dendritic cells is also induced by microbial products Uke LPS [109]. These cytokines have further effects on other cell types, which could be considered as indirect effects of LPS. In this respect, 11-6 and IL-12 increase LAK induction [110], Long term effects of LPS and lipids A Effect on bloodflow and angiogenesis
Some long term effects of LPS in vivo are vasodilation and hypotension. These phenomena are of great relevance in cancer since they modulate tumor blood flow and consequently the availability of oxygen and nutrients needed for tumor growth. They also modulate access of circulating toxic cytokines and immune cells to the tumor. According to MacPherson and North [111], LPS act at two levels: general by decreasing cardiac output and intratumoral by affecting the endothelium. Injection of DT-5461 decreases blood flow in Meth A fibrosarcoma subcutaneous nodules in BALB/c mouse [112] and in VX2 carcinoma liver nodules in the rabbit [113]. This decreased blood flow involves TNF-a, IFN-a/p and IFN-Y [112], and cannot be reproduced by intravenous (i.v.) injections of TNF-a [113]. Another effect of LPS may be the inhibition of tumor angiogenesis. This was evidenced in melanoma B16- bearing C57BL/6 mice treated i.v. with the lipid A GL-60, after an IFN-y injection [114]. It was confirmed in the same model using DT-5461 alone injected i.v. [94]. This effect of LPS is likely to be due to the production of TNF-a. This cytokine may be toxic for the endothelium [64], inhibiting at the same time the motility and proliferation of endothelial cells [59]. Sato et al. [115] showed that TNF-a inhibits tumor-induced angiogenesis in a rabbit comeal pocket assay. On the contrary, Vukanovic and Isaacs [116] concluded that TNF-
529
a secreted by intratumoral macrophages in response to LPS stimulates angiogenesis in primary prostatic cancer in the rat. Furthermore, NO, which is also induced by LPS, has been suspected of increasing neovascularisation [117]. But LPS also induce the in vivo production of IL-18 [118] which inhibits angiogenesis in mouse T241 fibrosarcoma [119]. Effect on NO production
NO was recognized as a mediator of macrophage cytotoxicity in vitro [120] but its role in tumor growth is not unequivocal. NO was shown to be involved in tumor-induced immunosuppression [121,122]. Tumor cells engineered to produce NO show a reduced proliferation in vitro but the cells producing the highest levels of NO present the highest growth rate in nude mice [117]. On the other hand, an inverse correlation was found between NO production and melanoma K1735 lung metastasis formation in C3H/HeN mice [123] or ESbL hepatic metastasis formation in the DBA/2 mouse [71]. The antitumoral effect of IL-12 in the MCA 207 subcutaneous fibrosarcoma in C57BL/6 mice [124] and of IL-10 in the mammary carcinoma 66-1 in BALB/c mice [125] involves NO production. The effect of the NO produced by human tumor cells on tumor growth in nude mice may depend on the p53 status of these cells [126]. All these controversial results could be due to the utilisation of different models. Using the same model, we have also found opposing effects of NO on PROb tumor growth in BDIX rats, but they depend on the NO-producing cells. Without treatment, the NO produced by macrophages upon activation by tumor cells inhibits T cell proliferation, thereby promoting tumor growth [122]. Treatment with lipid A induces the tolerance of macrophages, causing them to produce no further NO. On the contrary the NO produced by the tumor cells is autotoxic and its production upon activation by the lipid A treatment is concomitant with tumor regression [81,104]. Effect on acquired immunity
LPS can also have a role in the acquired immunity against cancer.
530
Recent studies emphasize the role of dendritic cells in the onset of an efficient primary immune response [127]. On one hand, tissue or blood dendritic cells phagocytose and process soluble or particulate antigens such as apoptotic bodies. After maturation they efficiently present cognate antigens to naive lymphocytes. In this respect, by inducing the maturation of dendritic cells in BALB/c and CBA/J mice [128], LPS injected i.v. can induce an immune response against tumors. For an efficient immune response, T cells must not only encounter their cognate antigen, but also receive simultaneous costimulation. According to Matzinger [129], costimulatory factors are released by antigen-presenting cells (APC) in the presence of a danger signal frequently delivered by bacterial products. Therefore LPS or lipid A may allow specific lymphocytes to differentiate and kill tumor cells they would have otherwise ignored. LPS can be directly mitogenic for T cells [130], but the antitumoral activity of lymphocytes depends on antigen recognition by their TCR in the context of the major histocompatibility complex (MHC) class I or II. Though LPS enhance it, T lymphocyte activity requires APC [131]. The effect of LPS on T lymphocytes has been shown to depend on monocytes independent of MHC, but to be due to the secretion of costimulatory signals and IL-12 in humans [132]. In vivo, LPS induces principally the proliferation of CD8+ T lymphocytes, but also that of CD4+ T and B lymphocytes through the activation of APC and secretion of IFN a/p in C57BL/6 mice [133]. The importance of lymphocytes in cancer treatments based on LPS had been evoked by Parr et al. [61]. Their role was later emphasized in A/J mice bearing SA-1 sarcoma or BALB/c mice bearing Meth A fibrosarcoma, treated with LPS [95], and in BDDC rats bearing PROb colon carcinoma, treated with bacterial extracts [96]. Furthermore, SCID mice treated with intraperitoneal (i.p.) injections of LPS are unable to reject Meth A fibrosarcoma [96]. However, lipid A treatment was efficient in nude mice bearing human pancreatic tumors [134]. A well-known effect of LPS in mice is mitogenicity to B lymphocytes. Lipid A from P. gingivalis is shovm to be mitogenic in vitro even to splenic B cellsfi-omLPS-unresponsive C3H/HeJ mice [31]. The lipid A ONO-4007 is mitogenic to spleen cells from BALB/c mice [135]. The role of antibodies in tumor regression has solely been documented in the
531
Studies of Sinkovics et al. [136] implicating antibodies in leukemia regression in LPS-treated mice. The relevance of the antibody response in the antitumoral effect of LPS remains controversial, as will be discussed later concerning data obtained in clinical studies. Tumor effect on the host response to LPS and lipid A The importance of tumor-induced immunosuppression as a limitation of the efficacy of immunotherapy has been demonstrated [137]. In tumorbearing animals, macrophages produce suppressive molecules: macrophages from sarcoma-bearing BALB/c mice secrete TGF-P, IL-10 and PGE2 [121], and macrophages from BDIX rats bearing colon carcinoma produce NO which inhibits lymphocyte proliferation [122]. The presence of a cancer may increase the susceptibility of its host to LPS as was shown in AB6F1 mice bearing SA-1 sarcoma [138]. This effect was attributed to the activation of macrophages in a model of fibrosarcoma in BALB/c mice [139]. Compared to macrophages from normal mice, these macrophages produce less GM-CSF [140], and more TNF-a [141] in the presence of LPS in vitro. Furthermore, macrophages from C57BL/6 mice bearing Lewis lung carcinoma produce less NO, and more PGE2 [142] in the same conditions. In rats, adherent splenic cells from WKAH rats bearing KDH8 hepatoma produce more TNF-a than cells from normal rats when treated by ONO-4007 [135]. Finally, Dalton lymphoma cells impair the PKC metabolism of C3H/He mouse macrophages treated with LPS [143]. In vivo, TNF-a production in spleen and liver as higher in C3H/He mice bearing MM46 or MH134-tumor, after an i.v. injection of ONO4007 [144]. In Sprague Dawley rats, the presence of Ward colon carcinoma worsens LPS damage of the kidney (measured as blood urea), liver (measured as blood alanine aminotransferase) and lung (measured as blood partial oxygen pressure), decreases blood albumin (a witness of leakage), increases plasma levels of nitrite-nitrate and, consequently, LPS lethality [145]. On the contrary, an LPS treatment with low effect on normal Dark agouti rats normalizes metabolic parameters in the blood of mammary carcinoma-bearing animals [146]. The effects of lipid A on tumor are summed up in Fig. (1).
532
LYMPHOCYTE!
Fig. (1). Effect of lipid A on tumor Lipid A acts ( ^ > ) on distinct cell typesfrominnate immunity (monocytes/macrophages, NK cells, PMNs, dendritic cells) and adaptive immunity (lymphocytes). • ) other cells or directly the tumor, These cells produce cytokines or NO ( C 3 ) which target ( resulting in toxicity ( • « ^ ) . Lipid A can also inhibit angiogenesis, bloodflowin the tumor and the secretion of immunosuppressive TGF-p by the tumor cells.
533
Treatments in animal models LPS and lipids A treatments
LPS treatments In animals, the first in vivo experiments were performed with bacterial extracts in a guinea pig sarcoma model by Gratia and Linz [88], and with LPS in mouse primary subcutaneous tumors by Shear and Turner [4]. The antitumoral effect of LPS on the growth of subcutaneous or intramuscular tumors has been extensively investigated [61,147-153]. On ascitic tumors, treatment with LPS was shown to be efficient in some cases [153-155] while failing in others [61,156]. We tested the effect of LPS in a model of peritoneal carcinomatosis (solid tumor) induced by PROb colon cancer cells in syngeneic BDIX rats. We showed that i.p. injections of LPS from E. coli can cure 20 % of the rats [157]. Comparing the effect of LPS from different strains in this model, we found that the efficacy depends on the bacterial strain and on the structure of the lipid A used. Whatever the lipid A used, we have shown a correlation with in vitro macrophage secretion of IL-ip but not with NO, TNF-a or IL-6 [83]. Lipids A treatments Here, we will emphasize on treatments with lipid A, considering only curative treatments beginning after tumor cell injection. After a review of the literature, we will detail the effects and mechanisms of DT-5461, ONO-4007 and OM-174, the three lipids A which have been mostly documented. Parr et al. [61] showed that lipid A has the same antitumoral effect as whole endotoxin preparations on murine L5178Y lymphoma. The effects of LPS and synthetic lipid A treatments were compared by Shimizu et al. [158-161] on Meth A fibrosarcoma in BALB/c mouse. The antitumoral activity of different lipids A has also been investigated. Ribi et al. [162] used an extract from S, typhimurium containing lipid A, which when injected directly into hepatocarcinoma line 10 tumors in guinea pigs shows an antitumoral effect. This activity is attributed to a monophosphoryl diglucosamine derivative of lipid A [163]. Synthetic lipid A analogs also proved to be active in this system [164], as well as
534
when injected i.p. in Meth A fibrosarcoma-bearing BALB/c mice presensitized with Propionibacterium acnes. The i.v. injection of the monoglucosamine GLA-27 slows the growth rate of RL-1 lymphoma and Meth-A sarcoma in BALB/c mice [165]. Shimizu et al. [160] compared several lipid A analogs with regards to their antitumoral activity using Meth A tumors in BALB/c mice. Antitumoral activity is not correlated with mitogenicity of C3H/He mice splenocytes and NO production in Swiss mice macrophages, but is correlated with macrophage TNF-a production. A synthetic lipid A was shovm to inhibit the growth of tumors induced in nude mice by the injection of ML\ paca-2 or Panc-1 human pancreatic tumor cells, likely through TNF-a secretion by macrophages [134]. Association of lipids A with other immunomodulators Treatments with lipids A were tested in association with diverse immunomodulators. Intravenous injections of GLA-60 in association with IFN-Y were found to reduce B16 melanoma lung metastases in C57BL/6 mice [114]. DT-5461 injected i.v. in association with indomethacin increases the survival of BALB/c mice bearing peritoneal, liver and lung C26 colon carcinoma [166] through the inhibition of angiogenesis. MDP (muramyl dipeptide), a Mycobacteria derivative, potentiates the antitumoral effect on Meth A fibrosarcoma in BALB/c mice, of several lipids A (A-171, A-172, 56, A-606, A-607, A-608) injected i.v., but the associations were less efficient than LPS alone [160,167]. MDP also increases the efficacy of DT 5461 in the same model [158]. The increase was correlated with an in vitro mitogenic effect of DT 5461 on spleen cells, as well as the production of NO and TNF-a by macrophages. In 1996, the same team tested several lipid A analogues, finding that the association with MDP showed no better efficacy than LPS on Meth A sarcoma in BALB/c mice. In these mice, cyclophosphamide injected 7 days prior an ONO-4007 treatment in order to inhibit the immunosuppressive response, enhanced the efficacy of the lipid A [108]. Treatment with the lipid A DT 5461 The synthetic diglucosamine compound DT-5461 has been reported to reduce the weight of various tumors including murine Meth A
535
fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis Lung carcinoma, and C38 colon carcinoma, but not that of C26 colon carcinoma [168] or L5178Y lymphoma [169]. The tumor size was reduced by a necrotic process. DT-5461 i.v. injections induced TNF-a secretion in subcutaneous Meth A tumors in BALB/c mice [112] as well as in B16-BL6 tumors in C57BL/6 mice, decreased angiogenesis, and reduced the number of spontaneous metastases [94]. The effect of DT5461 was accompanied by a reduced blood flow in the tumor which could be reversed by antisera directed against TNF-a, IFN-oc/|3, and IFNY [105,112]. No effect on the life span of animals bearing ascitic tumors was observed. The antitumoral effect of i.v. injections of DT-5461 in a rabbit hepatic carcinoma [113] was associated with blood flow reduction in the tumor area. In vitro, it was shown that in the murine macrophage cell line J774, DT-5461 enhanced cytokine production using LPS receptors [30], and that in the murine macrophage cell line RAW 264, signal transduction involved phosphorylation of MAP kinases [170]. Treatment with the lipid A ONO-4007 The monoglucosamine compound ONO-4007, injected i.v. slowed the growth of subcutaneous MM46 murine mammary carcinoma in C3H/He mice, increasing TNF-a secretion by intratumoral macrophages [80]. It induced TNF-a production by spleen cells from BALB/c mice bearing intradermic murine Meth-A fibrosarcoma, as well as their proliferation in vitro [135]. Intravenous injections increased the survival of WKAH rats bearing KDH-8 hepatocarcinoma subcutaneous tumors, but had no effect on rats bearing KMT-17 fibrosarcoma, or SST-2 mammary adenocarcinoma [171]. In WKAH rats, ONO-4007 acted by inducing the production of TNF-a [99], as was confirmed in C3H/He mice bearing MM46 mammary carcinoma or MH134 hepatoma [144]. The efficacy of this lipid A may be limited to TNF-a-sensitive tumors in WKAH rats [172]. While 3 i.v. injections every 7 days inhibited TNF-a production in liver and blood, no tolerization was found in tumors [99]. A similar effect was seen in hamsters bearing pancreatic carcinoma [173]. The prolongation of survival of WKAH rats bearing c-WRT-7 myelomonocytic leukemia by i.v. injections of ONO-4007 can be explained by a differentiating effect [174] that could not be reproduced
536
by a cytokine (BL-la, IL-6 or TNF-a) treatment. Recently, Mizushima et al. [175] showed that subcutaneous 13762NF mammary tumors, but not peritoneal or lung tumors, were cured by i.v. injections of ONO-4007 in F-344 rats. The efficacy of ONO-4007 was enhanced in mice bearing Meth A fibrosarcoma when cyclophosphamide was injected 7 days prior treatment in order to inhibit an immunosuppressive response [106]. Subcutaneous injections of ONO-4007 increased vascular permeability in the skin of normal mice by increasing the production of TNF-a and ILip [176]. In the same conditions, i.v. injections induced NO production in the lungs [177] but these effects were not studied in the context of tumor growth. Treatment with the lipid A OM-174 We investigated the antitumoral activity of OM-174 in a model of peritoneal carcinomatosis induced by PROb colon cancer cells in syngeneic BDIX rats. These cells are chemoresistant [178], NK-resistant [179] and TNF-a-resistant [81]. Without treatment, all rats die of their tumors. Treatment of peritoneal carcinomatosis (solid tumors) always started after the formation of macroscopic nodules up to 3 mm. The cumulative volume of these numerous nodules corresponds to the volume of a large tumor. An equivalent stage of tumors in himians cannot be resected and have always a fatal evolution. OM-174, which is a triacylated diglucosamine, has a partial structure ofE, coli lipid A [180]. Repeated i.v. injections of OM-174, every 2 days, cured 90 - 100 % of the rats. Such a success has never been obtained with a treatment of this kind. Tumor disappeared via the apoptotic pathway without an inflammatory reaction. OM-174 is not toxic to tumor cells in vitro^ therefore it does not induce tumor cell apoptosis directly. The establishment of a specific immune response was evidenced with a Winn-type assay, e.g. the protection of naive rats against a tumor by the injection of spleen cell from rats cured of the same tumor. Treatment efficacy depended on the number and frequency of injections which indeed induced the tolerance of macrophages to OM-174 decreasing their TNF-a production [81]. After a peak following the 2 first injections, TNF-a in tumors returned to basal levels. Moreover, PROb cells are resistant to TNF-a in vitro, in consequence in our model, TNF-a is probably not involved in tumor regression. On the contrary, the efficacy
537
of both DT-5461 in CDFl mice [169], and ONO-4007 in BALB/c mice [99], depended on TNF-a. In our model, during lipid A-induced txmior regression, NOS II mRNA and protein levels were induced in tumor cells with the concomitant production of NO [104]. Neither OM-174 nor TNFa induced NO production by tumor cells in vitro, whereas NOSII expression is induced by IFN-y and IL-ip in these cells [122]. Therefore, the in vivo NOS II induction may be indirectly due to the presence of IFN-y and IL-ip in the tumors of treated animals [96]. Accordingly, we determined that the treatment with OM-174 causes IFN-y and IL-ip accumulation in tumors, at the mRNA and proteins levels (unpublished results). The NO thus produced is autotoxic for tumor cells provoking their apoptosis. Moreover, treatment with OM-174 inhibited the synthesis of TGF-pi by PROb tumor cells [107], therefore abrogating its immunosupressive role [181]. Furthermore the inhibition of TGF-pl enhanced the synthesis of NOSII [107], thus increasing the autotoxic effect of NO on tumor cells. Therapeutic vaccination
Lipids A are also used in therapeutic cancer vaccination to cure tumors. In this case, lipids A are used as adjuvants, e.g. administered simultaneously with tumor extracts or tumor antigens, to increase the immunogenicity of the vaccine or to inhibit the tumor-induced tolerance. Therapeutic vaccines were tested in BALB/c mice bearing TA3-Ha mammary carcinoma. The treatment consisted of 4 subcutaneous injections, at 3-6 days intervals, of Detox [a commercial preparation of cell wall skeletons from Mycobacterium phlei and non-toxic monophosphoryl lipid A from Salmonella minnesota (S. minnesota) in squalane oil and Tween 80 from Ribi Immunochemical research, Montana, USA] mixed with Thomsen-Friedenreich (TF) antigen coupled with KLH (Keyhole Limpet Hemocyanin) performed 5 days after the tumor cell injection. This vaccination achieved the survival of 25 % of the mice. Pretreatment of mice with cyclophosphamide in order to inhibit any suppressive response, increased survival to 50 % when the treatment began 5 days after tumor cell injection, and to 90 % when the treatment began 2 days after tumor cell injection. Both antibody as well as delayed-
538
type hypersensivity (DTH) responses were obtained. Moreover, lymph node cells were protective in a Winn-type assay [182], In C3H/HeN mice bearing MH134 hepatoma, monophosphoryl lipids A from P. gingivalis or S, minnesota Re 595 increased the survival of mice when administered in combination with timior cell lysates and Freund*s incomplete adjuvant [31]. Conciusion In conclusion, various lipid A have been tested as treatments for tumorbearing animals, using different routes (intratumoral, intraperitoneal, intravenous, intradermic). While the intradermic route permits the use of greater doses without toxicity [76,77], most of the studies were performed using several i.v. injections of lipid A. Optimum doses range from 1 to 5 mg/kg for the 3 lipids A DT 5461, ONO 4007 and OM-174 in rats and mice. In our model of colon carcinoma in rats, we showed that the i.v. treatment is more efficient than an intraperitoneal one [81]. In general, most studies showed that the treatments increase survival, or slow the growth of established tumors in mice [80,93,94,99,105,112,135,144,160,168], rats [12,81,96,107,171174,181], and rabbits [113]. To our knowledge, only two laboratories reported the total cure of established tumors. Mizushima et al. [175] showed that ONO-4007 cures subcutaneous tumors but not intraperitoneal or lung ones. Onier et al. [81] reported that OM-174 cures 90-100 % of rats bearing peritoneal carcinomatosis consisting of a large number of nodules between 1 and 3 mm, while all untreated rats die of their cancer. Lipids A are generally considered to act through TNF-a secretion [112]. For instance ONO-4007 was shown to be efficient only on TNF-asensitive tumors [171,172], therefore, only well vascularized timiors can be affected. However, in our model, we showed that TNF-a is probably not involved since this cytokine peakes after the first two injections and then retums to basal levels before tumor regression. The efficacy of OM174 relies on an indirect induction of an autotoxic production of NO by the tumor cells [104]. Perhaps a more important aspect is the immunogenicity of tumor cells. After apoptosis or necrosis of tumor
539
cells, apoptotic bodies or debris can be phagocytosed by macrophages and dendritic cells. These cells will then present the immunogenic peptides to helper T lymphocytes which will induce a specific immune response. CLINICAL STUDIES The aim of phase I trials is to determine the toxicity of potential drugs. The following phase II trials are designed to study the pharmacological properties and the potential effectiveness of a drug. The aim of a phase III trial is to study the efficacy and safety of a particular protocol. Treatments with LPS Several phase I trials have been performed with LPS from Salmonella abortus equi administered i.v. in patients who suffered from disseminated cancer. White blood cell number decreased after each injection and retumed to basal level by 24 hours. There were no changes in coagulation parameters, and no disseminated intravascular coagulation was observed. After the first injection of LPS, increases in TNF-a concentration and IL-6 activity in serum were detected. However LPS tolerance which is accompanied by a decrease in TNF-a and IL-6 production depended on the intervals between repeated injections, but it was not determined whether it was a benefit or a draw-back. Injections of IFN-y prevented this decrease in TNF-a and IL-6, and ibuprofen attenuated LPS toxicity [183,186]. In a phase II trial, patients with colorectal cancer or non-small cell lung cancer, LPS showed a low grade toxicity and induced a reduction of TNF-a concentration in serum. One complete remission, stable for 36 months, was achieved [187]. In another phase I trial, i.d. injections of LPS from Pantoea agglomerans were given to patients who suffered from disseminated cancer and received cyclophosphamide, and ibuprofen, to attenuate fever. Increases in the serum concentrations of TNF-a, IL-6 and G-CSF were observed, without tolerance [188].
540
The low response of cancer patients to LPS treatments may be due to low maximal tolerated dose (MTD), 4 ng/kg. In order to avoid this problem, several trials were performed with lipids A. Treatments with lipids A A phase I trial was conducted with monophosphoryl lipid A (MPLA) prepared from S, typhimurium or S, minnesota injected i.v. in patients with disseminated cancer. Fever, chills andfetiguewae Ihe most OMnmoti side eflFects and the dose of 250 \x^jr^ i.e. (250x1.7): 65=6 jugl^ was estimated acceptable [189]. Another trial used i.v. injections of the synthetic lipid A SDZ MRL 953 in patients with disseminated cancer who received ibuprofen. The most common toxicity was fever, and the MTD was not reached. The lipid A had no significant effect on the serum concentrations of TNF-a, EL-ip, IL-8, G-CSF and IL-6. White cell number increased within 12 hours after the first injection, mainly due to PMNs, and then retumed to normal after 48 hours [37]. In a recent phase I trial the synthetic lipid A analog ONO-4007 was given by i.v. injections to patients with cancer unresponsive to the standard therapy. The limited systemic toxicity disappeared within 24 hours. The MTD was defined as 125 mg/patient [e.g. (125:65=2 mg/kg]. The lipid A increased serum concentrations of TNF-a and IL-6, without affecting the concentrations of GM-CSF, IFN-y and neopterin. There was a significant drop in lymphocyte counts after injections, but no effect on clotting parameters [190]. The results of phase I trials of LPS and lipids A in cancer patients show that the tested lipids A are approximately 30,000 to 500,000 fold less toxic than the LPS (table 1). The MTD in humans ranging from 6 |Lig/kg to 2 mg/kg, is lower than or close to the optimal doses of lipids A observed in rodents. Humans are more sensitive to lipid A than rodents, therefore it is possible that similarly to the toxic dose, the effective dose for humans is lower than for rodents. To this day the very few existing phase n trials cannot answer this question. These trials are summed up in Table 1.
541
Table 1. List of tfie clinical tiiab performed with LPS or lipids A injected to cancer patients.
Phase Vosika et al. Cancer Immunol Immunother 1984 Engelhardt et al JBiolRespModif 1990 1 Engelhardt et al. Cancer Res 1991 1 Mackensen et al. Blood 1991 1 Mackensen et al. Eur Cytokine Netw 1992 Ottoetal. Eur J Cancer 1996 1 Goto et al. Cancer Immunol Immunother 1996 1 Kiani et al. Blood 1997 1 DeBonoetal, Clin Cancer Res
1
2000
Product
Tumor
Route
MTD
Tested 1 Parameters CC
I
Lipid A Diverse (Salmonella)
i.v.
250 jig/m^ 6^g/kg
I
LPS (Salmonella)
Diverse
i.v.
4ng/kg
CC, CK
Diverse LPS (Salmonella)
i.v.
8ng/mg
CC, CK
LPS (Salmonella)
Diverse
i.v.
CK
LPS Diverse (Salmonella)
i.v.
CC,CK
CC, CK
I
II
LPS (Salmonella)
Diverse
i.v.
I
LPS (Pantoea)
Diverse
s.c.
I
Diverse Lipid A SDZMRL 953 1 Diverse Lipid A ONO-4007
I.v.
>1800ng/kg
CK
> 39.6 ng/kg
CC, CK
125 mg/patient 2mg/kg
1
CK
'
CC = cell count, CK = cytokines, i.v. = intravenously, s.c. = subcutaneously, MTD = maximum tolerated dose
Therapeutic vaccines Several trials of therapeutic vaccination used the adjuvant property of lipid A to enhance the vaccination efficacy against human tumors, which are often considered as weakly immunogenic, or even tolerogenic. The first trials of therapeutic vaccination against cancer using lipid A as adjuvant were performed on melanoma patients with 0.25 ml Detox. The composition of commercial vaccines used as therapeutic vaccines in humans is given in Table 2. Some trials used a pretreatment vsdth 300
542
mg/m of cyclophosphamide to inhibit an eventual suppressive response to the vaccine. Table 2 . Composition of commercial vaccines used as therapeutic vaccines in cancer patients.
DETOX (Ribi Immunochem Research, Inc., Hamilton, Montana, USA) 0.25 ml: 250 mg cell wall skeleton from Mycobacterium phlei, 25 mg monophosphoiyl lipid A from Salmonella minnesota R595 prepared as an oil-in-water emulsion with 2% squalane and 0.4% Tween 80 in 2X noraaal saline. Melacine (Ribi Immunochem Research, Inc., Hamilton, Montana, USA) Homogenates of melanoma tumor cell lines mixed with Detox. THERATOPE sTn-KLH (Biomira Inc., Edmonton,AB, Canada) 100 mg sTn-KLH emulsified in 0.25 ml Detox. (sTn = sialyl-Tn = DAcNeu2-6aGalNAc-0-Ser/Thr). OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA, USA) 1.2 ml: 100 mg/ml recombinant PSA + liposomes of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol + 200 mg /ml monophosphoiyl lipid A from Salmonella minnesota R595.
Melacine
The first phase I trial, performed by Mitchell et al. [191] and several further phase I and II trials by the same group, studying different parameters [192,193] are summed up by Mitchell et al. [194]. Homogenates of 2 melanoma cell lines, mixed with Detox were injected s.c. to melanoma patients. Toxicity was minimal and local. There was no correlation between the antibody response against melanoma determinants and the clinical response. The remissions were correlated with the presence of cytotoxic T lymphocyte (CTL) precursors. Most of the CD8+ and CD4+ isolated clones lysed MHC-matched melanoma target cells. Histological studies of regressing lesions showed the presence of CD3+ lymphocytes, mostly CD4H-, perivascularly and at the periphery of the tumor as well as the presence of macrophages throughout the lesions. Cyclophosphamide did not improve the number of responding patients, however lESf-a given to non-responding patients led to a clinical response. The vaccine, referred to as Melacine (Ribi Immunochemical Research, Inc.), contains homogenates of melanoma cells mixed with 0.25 ml Detox. In a multicenter phase II trial, with patients with
543
disseminated melanoma, the toxicity was moderate, mostly local. PreCTL number increased, and the expansion of CD8+ T cells was correlated with increased survival Clinical responses (remissions and disease stabilizations) were obtained [195]. The efficacy of Detox was contrasted with that of other adjuvants. Helling et al. [196], treated melanoma patients with cyclophosphamide, comparing Detox, BCG and the saponin QS-21 in a vaccine containing the ganglioside GM2, conjugated with KLH. Detox toxicity was only local. The antibody response was not increased by Detox, in contrast to the other adjuvants. Schultz et al. [197] used vaccines made of materials shed from melanoma cell lines, mixed with Detox or alum. Local side effects occurred in the Detox group. In this trial, an antibody response was present and more frequent in the Detox group than in the alum group while there was no difference in DTH. However, the disease-free survival was lower in the Detox groups than the alum group. Eton et al. [198] mixed irradiated melanoma cells with Detox. Toxicity was only local. Peripheral blood mononuclear cell cytotoxicity against autologous melanoma cells was correlated with survival, but no NK cell cytotoxicity occured. Two major responses were obtained, not correlated with DTH response. Jheratope
Detox was also used in immunotherapy directed against other types of cancers. Vaccines generally used sialylated (s)Tn, which are mucin epitopes expressed on epithelial tumors, conjugated with KLH. This vaccine was commercialized as Theratope (Biomira Inc., Edmonton, Canada). In a phase I study O'Boyle et al. [199] injected the vaccine to colorectal cancer patients. Toxicity was only local, and an antibody response was observed. Several trials were performed on breast cancer patients treated with cyclophosphamide. Little local toxicity was found, and an antibody response was evidenced. Partial clinical responses and disease stabilizations were obtained. [200-202]. Adluri et al. [203] compared vaccines using Detox or QS-21 in an adjuvant therapy for colorectal cancer patients. Toxicity was mostly local. An antibody response was
544
induced against synthetic epitopes but not against natural antigens. No DTH response was detected. A phase II trial was performed on metastatic breast cancer patients with or without cyclophosphamide [204]. The results showed a local toxicity. Cyclophosphamide was not efficient. An antibody response was evidenced. No complete remission was obtained. On the other hand, a randomized trial with breast, ovarian and colorectal cancer patients, showed an increase in the antibody response by cyclophosphamide. This antibody response was correlated with increased survival, when antibody levels to mucins were low before immunotherapy. The beneficial role of this response might be due to a blockage of immunosuppressive mucins [205-207]. In another study involving patients with metastatic breast, colorectal and ovarian cancer, increased anti-sTn titers were correlated with better survival. Even if before treatment, elevated titers of antibodies against the mucin MUCl, were correlated with a poor response to immunotherapy, CTL precursors to the MUCl were detected in carcinoma patients [208]. A vaccine using 10 |Lig/ml MUCl-KLH mixed with Detox was injected s.c. in breast cancer patients treated with cyclophosphamide. A weak antibody response, and an ex vivo CTL response against HLA-matched adenocarcinoma cell lines were seen. No correlation with the clinical outcome was available [209]. Theratope was given to patients with breast or ovarian cancer who received peripheral blood stem cell rescue after chemotherapy. Toxicity was mostly local. In vitro, NK activity which was low before immunization returned to normal values, toxicity against cells bearing sTn antigen appeared, and lymphocytes responded to sTn, by proliferation and IFN-y production. Antibodies against sTn were detected in 16 patients, while the anti-MUC-1 antibody titer decreased [210]. The remissions were longer in treated patients and there was a tendency to a decreased risk of relapse [211]. Another vaccine, formulated by mixing irradiated cells from colon carcinoma cell lines with Detox was injected to patients with colorectal metastatic adenocarcinoma, with or without DL-la [212]. The vaccine induced local toxicity, and fatigue which was increased in the group treated with IL-la. DTH occurred in both groups. No clinical response was available.
545
Since point mutations of the ras proto-oncogene are often found in cancer, a vaccine was made with mutated ras peptides mixed with Detox. In a phase I study, CD4+ proliferation and CD8+ cytotoxicity specific to the mutated peptide, were observed. The side effects were minimal and one patient showed a stabilisation of the disease [213]. OncoVax
Trials of therapeutic vaccination against prostate cancer used OncoVax-P (Jenner Biotherapies, Inc, San Ramon, California). OncoVax-P consists of 200 ng monophosphoryl lipid A (similar to that used in Detox) added to 1 ml liposomes and 100 ^g PSA (prostate-specific antigen). Patients received injections by different routes (intramuscular, intravenous or subcutaneous) according to the trial, with or without GM-CSF, IL-2 or BCG and cyclophosphamide pretreatment. No serious side effects were seen. DTH and antibody responses were achieved. Vaccination increased the PSA-reactive T cell frequency as determined by IFN-y secretion, but no toxicity against PSA-expressing target cells was detected. The most effective strategy could not be determined, and no conclusion about the clinical efficacy of the treatment was possible [214,215]. Conclusion
The phase I and phase II trials of therapeutic vaccines (Table 3.) show a weak toxicity of lipids A, furthermore they show a stimulation of the acquired antitumoral immune response. CTL responses correlate better than antibody responses to clinical outcomes, in agreement with current concepts of antitumor immunity. Phase HI trials are now necessary to determine the effective protocol.
546
Table 3.
List of clmical trials performed with lipids A as adjuvant of therapeutic vaccines administered to cancer patients.
Phase 1 Adjuvant 1 Mitchell et al. Cancer Res, 1988 Mitchell etal., AfmNYAcadSci,\99^
1
Schultzetal. Vaccine, 1995 | Elhot et al. Semin Surg Oncol, 1993 1 Eton et al. Clin Cancer Res, 1998 Longenecker et al. Ann NY Acad Sci, 1993 Adluri et al. Cancer Immunol Immunother, 1995 1 Miles et al. Brit J Cancer, 1996 Reddish et al. Cancer Immunol Immunother, 1996 MacLean et al. J Immunother, 1996 MacLean et al. J Immunother, 1996 1 MacLean et al. J Immunother, 1997 Sandmaier et al. J Immunother,\999 Holmberg et al. Bone Marrow transplant, 2000 1 Reddish et al. Int J Cancer, 1998 Kleif et al. J Immunother, 1999 Woodlock et al. J Immunother, 1999 1 Harris et al. 1 Semin Oncol, \999
Antigen
Tumor
I
Detox
Cell material 1
Melanoma
1
Detox
Cell material
Melanoma
II I
Detox
Cell material 1
Detox 1 Cell material
Melanoma Melanoma
II
Detox
Cell material]
Melanoma
I
Detox
Cell material
Melanoma
CTL,DHT
Breast
AR
AR,CTL AR,DTH
Detox
sTn-KLH j
I
Detox
sTn-KLH
Colorectal
AR,DTH
II
Detox
sTn-KLH
Breast
AR
Detox
sTn-KLH
AR
Detox
sTn-KLH
Theratope
sTn-KLH
Breast, ovarian, colorectal Breast, ovarian, colorectal Breast
Theratope J sTn-KLH
Breast, ovarian
AR
sTn-KLH
Breast, ovarian
I
Theratope
AR AR
1 sTn-KLH
Theratope
MUCl-KLH 1
Detox I
Detox
I
Detox
m\
QncoVax-P
1 Breast, ovarian Breast
Ras
Colorectal, pancreas, lung Cell material 1 Colorectal PSA
1
Prostate
1
AR = antibody response, CTL = cytotoxic T lymphocytes, DTH = delayed type hypersensitivity, PR = proliferative response
Tested i parameters AR,DTH, CTL AR,CTL
AR,CTL, PR CTL AR, CTL CTL, PR DTH AR,DTH
547
CONCLUSION LPS immunotherapy was the first immunotherapy for cancers assayed in patients in spite of its toxicity. The standardisation of animal models of cancer, the discovery of the LPS composition and of lipid A activity, the discovery of lipid A structure leading to its chemical synthesis, and the synthesis of lipid A derivatives far less toxic than the natural lipids A, restarted research in this field. At the same time, advances in immunology allowed a better understanding of the mechanisms of action of LPS and lipids A in whole organisms. Most of the articles report a significant enhancement of the life span of treated animals. However recent results show that it is now possible to definitely cure animals bearing large tumors while the untreated counterparts die of their cancer. The most effective structure so far consists of diglucosamine acylated by 3 long chain fatty acids, and the substitution of the diglucosamine backbone is now under investigation. The best treatments consist of repeated i.v. injections, where frequency is an important parameter, and the optimal dose is not necessarily the maximal one. With the same lipid A, the best treatment schedule changes from one animal model to the other. Comparative studies have not been performed to elucidate if species and tumor origin and/or the immunogenicity of tumor cells and their immunosuppressive effect are important parameters at the origin of these differences. Three lipids A have been more intensively studied in animal models, all of them having indirect effects, mediated in vivo by the immune system. For two of them, DT-5461 and ONO-4007, TNF-a is an important mediator acting at the vascular level that provokes tumor necrosis. For the third one, OM-174, the treatment induces the accumulation of IFN-y and DL-lp in tumors, which activate NOS II transcription in tumor cells that produce autotoxic NO, which then provokes the apoptosis of tumor cells. At the same time this treatment inhibits the production of TGF-pl by tumor cells which reduces the TGFpi induced immunosuppression and enhances NO production. Acquired immune response, probably completes the tumor regression started by the apoptosis process and, most probably induces specific memory. Important questions have to be answered to facilitate the definition of protocols for humans. For example, is the lipid A tolerance of
548
macrophages an important parameter for treatment effectiveness ? The answer will influence the choice of doses and frequency of injections, in order to determine whether to increase progressively the dose of lipid A injected to each patient or not. Several lipids A have been tested in cancer patients: MPLA, SDZ MRL 953, and ONO-4007 were injected i.v. in phase I trials. The maximal tolerated dose found is lower than or close to the optimal dose defined in animals. Humans are more sensitive to lipid A than rodents so it is possible that similarly to the toxic dose, the effective dose is lower in humans than in animals. Because it requires small amounts of lipid A generally injected s.c, the adjuvant effect of lipid A has been largely investigated in cancer patients, but only with MPLA. Phase I and phase II trials show weak toxicity of different vaccines with MPLA and the development of an immune response. Phase III are now necessary to find an effective protocol. Therefore it is now to soon to know or to predict whether the lipids A will become an antitimioral medicine. A great deal of data are now available, which justify and impose the necessity of phase III trials. New efforts have to be made quickly because of the thousands of patients who will die in the near future. ABBREVIATIONS APC = antigen-presenting cells; BPI = bactericidal permeabilityincreasing protein; CSF = colony stimulating factor; CTL: cytotoxic T lymphocytes; DG = diacyl-glycerol; DTH = delayed-type hypersensivity; ECSIT = Evolutionarily-Conserved Signaling Intermediate in Toll pathway; FADD = Fas associated death domain; G-CSF = granulocyte colony stimulating factor; GPI = glycosylphosphatidylinositol; HDL = high density lipoprotein; ICAM = intercellular adhesion molecule; i.d.: intradermal; IKK == IkB-inducing kinase; IL-lp = interleukine-lp; IFN-y = interferon-y; IP3 = inositol triphosphate; IL-IR = IL-1 receptor; IRAK = IL-lR-associated kinase; i.p. = intraperitoneal; i.v. = intravenous; KLH = Keyhole Limpet Hemocyanin; LAK = Lymphokine-activated killer; LBP = LPS binding protein; LDL = low density lipoprotein; LPS ==
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lipopolysaccharide; MCP = monocyte chemoattractant protein; MDP = muramyl dipeptide; MIP = macrophage inflammatory protein; MHC = major histocompatibility complex; MPLA = monophosphorylated lipid A; MTD = maximum tolerated dose; NIK = K-inducing kinase; NFKB = nuclear factor KB; NK = natural killer; NO == nitric oxide; NOS == nitric oxide synthase; PAF = platelet-activating factor; PGE2 = prostaglandin E2; PKC = protein kinase C; PMN = polymorphonuclear neutrophils; PSA = prostate-specific antigen; RSLA = Rhodobacter sphaeroides lipid A; s.c. = subcutaneous; SCID == severe combined immuno deficiency; SLPI = secretory leukocyte protease inhibitor; TF = ThomsenFriedenreich; TLR = Toll Like Receptor; TNF-a = Tumor necrosis factor-a.
ACKNOWLEDGEMENTS The authors thank Conseil Regional de Bourgogne, association pour la Recherche contre le Cancer (ARC), Ligues contre le Cancer de Bourgogne et de Haute-Mame and Fondation pour la Recherche Medicale for theirfinancialsupport.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistryy Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.
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Prevention of Cancer Chemotherapy Drug-Induced Adverse Reaction, Antitumor and Antimetastatic Activities by Natural Products YOSHIYUKI KIMURA Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan. ABSTRACT: Although it has recently been thought that a number of medicinal plants and foodstuffs have antitumor and antimetastatic activities, the basis for this hearsay is unclear. Therefore, to clarify whether natural products have antitumor and antimtastatic actions, I have been using biochemical and pharmacological approaches to study the natural products isolated from various medicinal plants and foodstuffs. In the review, we will introduce the biological and pharmacological actions of various components isolated from some medicinal plants and foodstuffs on tumor growth and metastasis in tumor-bearing mice. Chitosan and fish oils prevented the adverse reactions such as gastrointestinal toxicity and myelotoxicity caused by cancer chemotherapy drugs without interfering the antitumor activity of chemotherapy drugs. Stilbenes derivatives isolated from Cassia or Polygonum species inhibited the tumor growth and metastasis to the lung in highly metastaic tumor-bearing mice. Furthermore, I found that stilbenes inhibited the angiogenesis in in vivo and in vitro models.
INTRODUCTION Cancer is the largest single cause of death in both men and women, claiming over 6 million lives each year v^orldwide. Cancer chemotherapy drugs such as 5-fluorouracil (5-FU) derivatives, cisplatin (CDDP), mitomycin, doxorubicin, taxisol, etc. have been used extensively for the treatment of certain types of cancer. However, with these treatments, severe gastrointestinal toxicity with diarrhea and mucosis, and hematologic toxicity with leukopenia and immunosuppression, appear to be dose-limiting factors. Furthermore, the removal of malignant tumor by surgical operation, radiation therapy and/or adjuvant therapy with cancer chemotherapy drugs may be curative. However, the removal of certain cancers, for example, breast carcinoma, colon carcinoma and osteogenic sarcoma, may be followed by the rapid growth of distant metastases to lung, liver etc. Therefore, efforts are underway to develop new modulators that inhibit the adverse reactions without loss of antitumor activity and new drugs having antitumor and antimetastatic activities without adverse
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reactions.
In this review, I describe the following articles.
1) Prevention by Natural Products of Adverse Reactions Induced by Cancer Chemotherapy Drug without Loss of Antitumor Activity. a) Prevention by Chitosan of Myelotoxicity, Gastrointestinal Toxicity and Immunocompentent Organic Toxicity Induced by S-Fluorouracil (5-FU) [1] or Doxorubicin [2] without Loss of Antitumor Activity in Tumor-Bearing Mice, Chitin and chitosan are polymers with molecular weight of about 1000 kDa, and contain more than 5000 acetylglucosamine and glucosamine units, respectively. Chitin is widely distributed in natural products such as the protective cuticles of crustaceans and insects, as well as being found in the cell walls of some fungi and microorganisms, and is usually prepared from the shells of crabs and shrimps. Chitin is converted to chitosan by deacetylation with 45% NaOH at lOO'^C for 2 h. Though chitosan is reported to augment the natural killer activity, the antitumor activity of chitosan is not clear yet. First, I examined the antitumor effects of chitosan, but it had no effect. Gastrointestinal toxicity and myelotoxicity are caused by the 5-FU after the phosphorylation in the digestive tract and bone marrow tissue. To clarify whether chitosan enhances the antitumor activity of 5-FU or doxorubicin and prevents the adverse reactions induced by 5-FU or doxorubicin, I examined the antitumor activity and adverse reactions, such as myelotoxicity, immunocompetent organ toxicity, and gastrointestinal toxicity of combined treatment with chitosan and 5-FU or doxorubicin in sarcoma 180-bearing mice. 5-FU (12.5 mg/kg twice daily) plus chitosan (150, 375 and 750 mg/kg twice daily) inhibited the tumor growth as well as 5-FU alone. Chitosan (150 and 750 mg/kg twice daily) blocked the reduction of blood leukocyte number caused by 5-FU administration, and it prevented the injury of the small intestinal mucosa membrane and delayed the onset of diarrhea induced by 5-FU "Fig. (1), Fig. (2) and Fig. (3)". Furthermore, chitosan (750 mg/kg twice daily) prevented the reduction of spleen weight induced by 5-FU in sarcoma 180-bearing mice "Fig. (4)", and the reduction of lymphocyte, CDS^ and NKl.l.^ T cell numbers induced by 5-FU "Fig. (5)". Intraperitoneal doxorubicin (5 mg/kg on days 1 and 8 after inoculation of tumor cells) significantly inhibited tumor volume and tumor weight, compared with sarcoma 180-bearing mice. Similarly, doxorubicin plus chitosan (200 and 800 mg/kg twice daily) also inhibited the tumor growth, compared with tumor-bearing mice "Fig. (6)". On the other hand, a remarkable reduction in body weight of mice after 8 days was observed in the mice receiving intraperitoneal doxorubicin compared with tumor-bearing mice. Oral administration of chitosan (400 and 800 mg/kg
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21
-a
0 I5-FU(12.5mg/kgx2) + + + + Chitosan(mg/kg x2) 150 375 750 5-FU(12.5 mg/kg x2) + Chitosan(ing/kg x2) Fig. 3. Inhibitory effect of chitosan on gastrointestinal toxicity P»g -*• Inhibitory effect of chitosan on immunocompetent organ (reduction ofsucrase activity in small intestinal mucosa) toxicity (reduction of spleen weight) induced by 5-FU in induced by 5-FU in sarcoma 180-bearing mice. sarcoma 180-bearing mice. 0.0"-
-V—1—J—^^^1.
Results are expressed as means ± S.E. of 9 mice.
I H
KtsnlXs are expressed as means ± S.E. of 9 mice.
I
I sarcoma 180-bearing mice 5-FU
^ ^
Chitosan
H
I sarcoma 180-bearing mice ^'^
[ X J Chitosan
P<0.05
•a
I
I
I 5-FU(12 5mg/kgx2) Chitosan(ing/kg x2)
mm
E
8
5-FU(12.5 mg/kg x2) Chitosan(mg/kg x2) 750
Fig. 1. The combined antiitumor activity of 5-FU and chitosan in sarcoma 180-bearing mice. 5-FU or 5-FU plus chitosan was administered orally twice "^"y for 8 days. On day 9, blood and tissues were obtained. Results are expressed as means ± S.E. of 9 mice.
Fig. 2. Inhibitory effect of chitosan on myelotoxicity of 5-FU in sarcoma 180-bearing mice. Results are expressed as means ± S.E. of 9 mice.
562
• CDS"^ T Cell
H
Nkl.l.'^TCell p<0.05
4.0
3.0
H
+
WD
s
2.0
o
0)
u +
1.0
00
Q U 0.0
5-FU(12.5mg/kgx2) Chitosan(mg/kg x2)
+ 150
Fig. 5. Combined effects of S-FU and chitosan on the numbers ofCD8^ Tcells in spleen ofC57BL/6 mice.
+ 750 andNKLl.^
5-FU or 5-FU plus chitosan was administered orally twice daily for 7 days. On day 8, the mice were killed by cervical dislocation and their spleens were quickly removed. CD8"^ and NKl. 1 ."^ cell populations were analyzed by flow cytometry. Results are expressed as means ± S.E. of 5 mice.
twice daily) prevented the doxorubicin-induced reduction in body weight after 12 days "Fig. (7)". In addition, the intraperitoneal administration of doxorubicin to tumor-bearing mice reduced the weight of small intestine and the sucrase activity of its mucosal membrane. The oral administration of chitosan (400 and 800 mg/kg twice daily) prevented the doxorubicin-induced reduction in small intestine weight and sucrase activity "Fig. (8)". Therefore, it is concluded that the combination of chitosan and 5-FIJ or doxorubicin might be useful for the prevention of adverse reactions such as gastrointestinal toxicity, immunotoxicity and myelotoxicity, caused by 5-FU or doxorubicin without loss of antitumor activity. Orally administered chitosan was retained for a long period in the small intestine, suggestive of possible diffusion of chitosan into the
563
intracellular space of villi in the small intestine. Therefore, it seems likely that the protective mechanism for the 5-FU or doxorubicm-treated gastrointestinal toxicity by the orally administered chitosan may be due to the formation of 5-FU- or doxorubicin -chitosan complex m the sntiall intestinal mucosa through the diffusion of chitosan into the small mtestmal villi without interfering the anitumor activity of 5-FU or doxorubicin.
I
it
I
I
I.. • • I
I
10
12
14 Day
I
j DXR(5mg/kg,ip) DXR(5mg/kg,ip) TDXF Sarcoma 180 Inoculation Fig. 6. The combined effects doxorubicin and chitosan on tumor volume in sarcoma 180-bearing mice.
it
I
I
I
i
I
10
12
14
Day
, DXR(5mg/kg,ip)DXR(5mg/kg,ip) yoxR SarcomalSO inoculation Fig. 7. The combined effects ofdoxorubicin and chitosan on body weight in sarcoma 180-bearing mice.
Results are expressed as means ± S.E. of 10-20 mice •P<0.05. Significantly different from sarcoma 180-bearing mice.
I. I I DXR (5 mg/kg/wcek, ip)
(mgfltg X 2/
DXR (5 mg/kg/wcek. ip)
(mg/kg x 2/diy, po)
Fig. 8. The preventive effects of chitosan on doxorubicin-induced gastrointestinal toxicity in sarcoma I l-bearing mice. a) small intestine weight; b) sucrase activity in small intestinal mucosa Results are expressed as means ± S.E. of 10-20 mice.
b) Prevention by Carp Extract of Myelotoxicity and Gastrointestinal Toxicity Induced by 5-FU without Loss of Antitumor Activity in
564
Tumor-Bearing Mice [3]. Carp (Cyprinus carpi) has been used in Korea, China and Japan as a health food source. In ancient Chinese medicine, carp was eaten as diuretic, and as a remedy for eye fatigue. In Japan, carp meat and blood have traditionally been eaten as a tonic. Although it has recently been thought that carp extract has antitumor activity, the basis for this hearsay is unclear. Therefore, to clarify whether carp extract has antitumor effects, the antitumor effect of carp extract, and the combined effect of 5-FU plus carp extract on antitumor activity and adverse reactions were investigated in sarcoma 180-bearing mice. As shown in "Fig. (9)", carp extract had no effect on survival time in ascites-type sarcoma 180-bearing mice, indicating that carp extract did not possess direct antitumor activity. Next, I attempted to investigate the combined effect of 5-FU and carp extract on antitumor activity in solid-type sarcoma 180-bearing mice. Carp extract was found to prevent the occurrence of myelotoxicity as determined by the reduction of leukocyte number, and of gastrointestinal toxicity, as indicated by the reduction of the weight of the small intestine, induced by 5-FU without loss of the anitumor activity of 5-FU "Fig. (10), Fig. (11) I m
I sarcoma 180-bearing mice S-FU
f y j | S-FU + carp extract
P<0.05
Sarcoma ISO-bearing mice Carp extract (50 mg/mouse x 2/day) Carp extract (100 mg/mouse x 2/day)
0.0 5.FU(12.5mg/kgx2)
• 12
16
20
Day
Fig. 9. Effect of carp extract on survival time in asccites-type sarcoma 180-bearing mice. Carp extract was administered orally twice daily until death, starting 12 h after implantation of tumor cells.
p^o.o.
Mlii
Carp extract (m^mouse x 2)
Fig. 10. Antitumor effect of 5-FU and 5-FU plus carp extract. 5-FU or 5-FU plus carp extract were administered orally twice daily for 8 days. On day 9, blood and tissues were obtained. Results are expressed as means ± S.E. of 10 mice.
and Fig. (12)". These findings indicate that carp extract could be beneficial as health food source for the prevention of adverse reactions such as myelotoxicity and gastrointestinal toxicity induced by the cancer chemotherapy drug 5-FU.
565
I I
I sarcoma ISO-bearing mice I 5-FU
r y j
I sarcoma 180-bearing mice
JIH
5-FU
r i / J 5-FU + carp extract
5-FU + carp extract
I
5-FU(12.5mg/kgx2)
.
+
Carp extract (mg'mousex2)
+ 50
+
5-FU(12.5mg/kgx2)
100
Carp extract (mg/mousex2)
Fig. 11. Inhibitory effect of carp extract on myelotoxicity (reduction of leukocyte number) induced by 5-FU. Results are expressed as means ± S.E. of 10 mice.
.
+
+
+
50
100
Fig. 12. Inhibitory effect of carp extract on gastrointestinal toxicity (reduction of the weight of the small intestine) induced by 5-FU. Results are expressed as means ± S.E. of 10 mice.
c) Fish Oils Enhance S-FU-Induced Antitumor Activity without a Loss of Adverse Reaction of5'FU[4J. Fish oils that contain high amounts of the n-3 polyunsaturated fatty acids eiocosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3) have been suggested to decrease the risk of development of cardiovascular disease. Freshwater fish oil carp oil are not rich in n-3 polyunsaturated fatty acids, but tuna oil is rich in n-3 polyunsaturated fatty acids such as EPA and DHA "Table (1)". Table 1. Tlie fatty acid components of carp oU and tuna oil. Fatty acid composition (%)
Carp oil
Tuna oil
Myristic acid n4:0)
2.0
-
Palmitic acid (16:0) Palmitoleic add (16:1 n-T) Stearic add (18:0)
23.8 7.8
17.9
3.3
3.8
Oleic add (18:1 n-9)
36.6
17.5
Linoleic add (18:2 n-6)
18.2
3.7
-
Arachidonic add (20:4 n-6)
-
1.9
Eicosapentaenoic add (EPA)
0.9
7.7
2.3
26.1
5.1
21.4
(20:5 n-3) Docosahexaenoic add (DHA) (22:6 n-3) Others
566
First, I examine the antitumor activity of the oral administration of two fish oils (carp oil and tuna oil) in sarcoma 180-bearing mice. Carp oil and tuna oil (0.2 and 0.4 ml/mouse) had no effects on tumor growth "Table (2)". Table 2. Effects of carp oil and tuna oil on the weights of body and tumor in sarcoma 180-bearing mice. Mean ± SE Final body weight (g) Sarcoma
180-bearing
mice
Final tumor weight (g) (T/C)
35.5 ± 1.06
3.29 ± 0.78
(100%)
36.8 ± 0.89
2.26 ± 0.54 (68.7%)
37.5 ± 0.55
2.40 ± 0.77 (72.9%)
38.7 ± 0.68
2.54 ± 0.80 (77.2%)
36.1 ± 1.21
2.59 ± 0.73_(78.7%)
(Control) Carp oil (0.2 ml/mouse) (0.4 ml/mouse) Tuna oil (0.2 ml/mouse) (0.4 ml/mouse)
Results are expressed as mean ± SE of 10 mice in each group.
h)t Sarcoma 18(M)earing mice (control) 1000 W
1
Sarcoma 18&4)earmginice(coiiirol)
1000
5-FU(12.5mg/kg)
5-FU(12.5mgAg)
5-FU + tuna oiK0.2ml) 5-FU + tuna oiK0.4ml)
800 V
5-FU + carp cMl(0.2inJ/inousc) S-FU + carp oil(0.4 mlAnousc)
' %
i^
\ 600 k
1
/
!
r'
'
h-
400 1
^^ 1•
200 1
*Jm
^m* 2
4
0' 6
8
10
12
14
Day
0
2
4
6
8
10
12
14
Day
Fig. 13. Effects of the combination of5-FUplus carp oil (a) and 5-FUplus tuna oil (b) on tumor growth in sarcomaJSO- bearing mice. 5-FU, 5-FU plus carp oil or 5-FU plus tuna oil was admnistered orally daily for 14 days. Results are expressed as means ± S.E. of 9-10 mice. *P<0.05, Significantly different from sarcoma 180-bearing mice.
Next, I examined the combined effects of 5-FU plus two fish oils (carp oil and tuna oil) on the antitumor activity and adverse reactions compared to the effects of 5-FU alone (12.5 mg/kg). I found that carp oil (0.4 ml/mouse) or tuna oil (0.2 or 0.4 ml/mouse) enhanced the ability of 5-FU (12.5 mg/kg) to prevent tumor growth, without increasing adverse
567
400
5-FU (12.5 mg/kg)
300
0
-
S-FU + carp oil (0.4 ml/moouse) 5-FU + tuna oil (0.2 ml/mouse)
I
200
20
40
100
60
120
Time (min) Fig. 14. 5-FU levels in the plasma of mice after oral co-administration of 5-FU plus carp oil or 5-FUplus tuna oil Results are expressed as means ± S.E. of 5 mice in each group. •P<0.05, Significantly different from the administration of 5-FU (12.5 mg/kg) alone.
reactions such as myelotoxicity (the reduction of number of leukocytes, platelet and red cells) (data not shown) and immunocompentent organ toxicity (the reduction of spleen and thymus weights) "Fig. (13) and Table (3)". Table 3. Effects of the combinatioii of 5-FU |dus carp oil or tuna oil on the weights of spleen and thymus sarcoma 180-bearing mice. Animal No.
Mean:tSE Spleen (mg)
Thymus (mg)
Sarcoma 180-bearing mice (Control)
10
190.0 ± 30.5
47.95 ± 5.42
5-FU(12.5 mg/kg)
10
149.0 ± 16.8
50.56 ± 3.43
5-FU -H caip oil (0.2 ml/mouse)
10
159.0 ± 23.1
36.69 ± 4.19
10
158.0 ± 10.3
49,53 ± 3.74
10
132.0 ± 4.90
42.23 ± 6.00
9
152.2 ± 9.25
50.33 ± 3.61
+ carp oil (0.4 ml/mouse) 5-FU + tuna oil (0.2 ml/mouse) + tuna oil(0.4 ml/mouse)
Results are expressed as mean ± SE of 9 -10 mice in each group.
As shown in "Fig. (14)", the 5-FU levels in the blood of mice were about 133.8 ± 27.5 and 285.0 ± 19.3 ng/ml, respectively at 5 and 15 min
568
after the oral administration of 5-FU (12.5 mg/kg) and then decreased rapidly. On the other hand, the blood 5-FU levels after the co-administration of 5-FU plus carp oil (0.4 ml/mouse) were 99.7 ± 42.4, 48.9 ± 15.6, 39.9 ± 17.2 and 18.6 ± 9.75 ng/ml, respectively, at 5, 15, 30 and 60 min. The blood 5-FU levels after the co-administration of 5-FU plus tuna oil (0.2 ml/mouse) were 376.4 ± 172.1, 182.6 ± 113.0, 33.9 ± 2.10 and 22.8 ± 5.73 ng/ml, respectively, at 5, 15, 30 and 60 min. The area under the curve (AUC) (0-120 min) of blood 5-FU levels was reduced by the oral co-administration of 5-FU with carp oil or tuna oil. Apparent Tmax was shortened by the oral co-administration of 5-FU with carp oil or tuna oil. However, AUC (0-4 h) of [6-^H]5-FU incorporation into RNA fraction of tumor after the co-administration of 5-FU plus carp oil or tuna oil was similar to that of 5-FU alone "Fig. (15)". These results suggest that the co-administration of 5-FU plus carp oil or tuna oil enhanced the 5-FU-induced antitumor activity without adverse reactions.
li
^H.5-FU(12.5 mg/kg, 18.5MBq/kg) - • —
^H-S-FU + carp oil (0.4 ml/mouse) ^H-S-FU + tuna oil (0.2 ml/mouse)
OL 0.0
0.5
1.5
2.0
2.5
3.0
3.5
4.0
Time (h)
Fig. 15. [6'^H] 5'FU incorporation into RNAfractionsof tumor after oral co-administration of [6'^H]5'FUplus carp oil or [6-3 H]5-FU plus tuna oil Results are expressed as means ± S.E. of 5 mice in each group. *P<0.05, Significantly differentfromthe administration of 5-FU (12.5 mg/kg, 18.5 MBq/kg) alone.
569
2) Isolation of Antitumor Substance from Basidiomycete Fungus Agaricus blazei and its Mechanism [5]. The Basidiomycete fungus Agaricus balzei Murill has traditionally been used as a health food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis. It has been reported that A. balzei is used by 300,000 to 500,000 persons for the prevention of cancer and/or as an adjuvant with cancer chemotherapy drugs after the removal of a malignant tumor. The hot water extract of A. blazei has potent antitumor activity in sarcoma 180-bearing mice, and the antitumor substance was postulated to be the p-(l-6)-glucan fraction. However, the antitumor effects of lipid fractions have not been well studied. I examined the antitumor activities of various substances isolated from the lipid fraction of A. blazei via oral administration to identify the active substances. Tumor growth was retarded by the oral administration of the lipid fraction extracted from A. blazei with a chloroform/methanol (1:1, v/v) mixture at a dose of 800 mg/kg during the 20 days treatment in sarcoma 180-bearing mice. On the other hand, tumor growth was not affected by the oral administration of the acetone-insoluble fraction. The acetone-soluble fraction, which had higher antitumor activity, was divided into two fractions through treatment with n-hexane. The n-hexane-soluble and -insoluble fractions (800 mg/kg) also inhibited the tumor growth "Table (4)". Table 4. Effects of various lifnd fractions of chloroform : methanol extract (l:l^v/v) (a), acetone- soluble and -insoluble fractions (b), and n-hexane-soluble and -insoluble fractions (c) on tumor volume at 20 d and tumor weight (a) \^rious fractions
lipid
Control Chloroform methanol extract
at 21 din sarcoma 180-bearing mice^ Tumor volume mm^ 4826.9±1150.6 1087.3± 567.6*
Inhibition
% -
Tumor weight mg
Inhibition
% -
77.5
4470.0±870.6 844.2±425.1*
81.1
Inhibition
Tumor weight
Inhibition
mg 827.7±381.2
% -
(b) "Various lipid fractions
Tumor volume mm^
Control
928.81250.9
% -
Acetone-soluble
124.1+83.6*
86.6
108.6+83.4*
86.8
649.1+140.9
30.1
490.5±382.3
40.7
fraction -insoluble fraction
570
(c) Various lipid
Tumor volume
Inhibition
Tumor weight
Inhibition
fractions
mm^
mg
Control
766.9±302.9
% -
812.0±277.2
% -
Hexane-soluble
152.0±74.6*
80.2
163.9±150.7*
79.8
fraction -insoluble
75.7±24.6*
90.1
54.6±21.4*
93.3
fraction ^Various lipid fractions (800 mg/kg) were orally administered for 20 day in sarcoma 180-bearing mice. Inhibition ratio (%) was measured as tumor volume or tumor weight of various lipid fraction-treated mice/ tumor volume or tumor weights of control mice. Each value represents the means ± S.E. of 10 mice. *P<0.05, Significantly different from sarcoma 180-bearing mice.
The substance with the antitumor activity in the n-hexane-insoluble fraction was isolated through silica gel column chromtography, eluted with an acetonitrile/methanol (3:2) mixture and identified as ergosterol "Fig. (16)".
Ergosterol F i g . 16. Antitum
or substance
of Agaricus
blazei
The oral administration of ergosterol to sarcoma 180-bearing mice significantly reduced tumor growth at doses of 400 and 800 mg/kg "Fig. (17)" without adverse reactions, such as the decreases in body, epididynal adipose tissue thymus, and spleen weights and leukocyte numbers induced by cancer chemotherapy drugs (data not shown). To clarify the antitumor activity of ergosterol, I examined the effects of ergosterol on tumor-induced angiogenesis using two in vivo models. Intraperitoenal administration of ergosterol at doses of 5, 10 and 20 mgy^g for 5 consecutive days inhibited the neovascularization induced by Lewis lung carcinoma cell (LLC)-packed chambers "Fig. (18)". Moreover, I examined that the inhibitory effects of ergosterol on Matrigel-induced neovascularization. Female C57BL/6 mice were subcutaneously inoculated with Matrigel containing acidic fibroblast growth factor (aFGF)
571
and heparin with or without ergosterol Ergosterol inhibited the Matrigel-induced neovascularization "Fig. (19) and Table (5)". 4000
h
Sarcoma 180-bearingmice (Control) + Ergosterol (lOOmg/kg) + Ergosterol (200mg/kg) + Ergosterol (400 mg/kg) H- Ergosterol (800 mg/kg)
3000
4
2000 h
s 1000
Days after inoculation Fig. 17. Effects of oral administration of ergosterol isolated fromAgaricus blazei on tumor growth in sarcoma 180'bearing mice. Solid-type sarcoma 180 was prepared by subcutaneous transplantation into the right abdomen of mice on day 0. The indicated amounts of ergosterol were administered orally for 20 consecutive days, starting 12 h after the implantation of tumor cells. Results are expressed as means ± S.E. of 10 mice in each group. *P<0.05, Significantly different from sarcoma 180-bearing mice (control).
572
LLC-induced Angiogenesis ^ ,^^ „^ _ ,,^^ (Control) + Ergosterol (5 mg/kg) + Ergosterol (10 mg/kg) Fig. 18. Effects of ergosterol on neovacidarization in C57BL/6 mice bearing LLC cell-packed chamber. Chambers packed with LLC cells were subcutaneously implanted into a dorsal air-sac of C57BL/6 mice on day 0. Ergosterol was intraperitoenallyadministered from days 1 to 5. + Ergosterol (20 mg/kg)
Table 5. Effects of ergosterol on the weights and hemoglobin contents in the gels 5d after implantation of Matrigel
supfdemented a FGF and heparin^ Treatment
Matrigel weight mg
Hemoglobin content mg/Matrigel
Matrigel alone Matrigel + a FGF (1 ng/ml) + heparin (64 units/ml) (Control) Matrigel, a FGF, heparin
103.16 ± 10.15* 371.60 ± 39.75
2.6 ±0.68* 21.0 ± 4.00
185.58 ± 44.40*
6.4 ± 1.86*
108.84 ± 9.69*
3.8 ±0.58*
+ ergosterol (400 ng/ml) Matrigel, a FGF, heparin + ergosterol (800 pig/ml) ^C57BL/6 mice were each injected subcutaneously with 0.5 ml of Matrigel supplemented 1 ng /mL of a FGF and 64 U/ml of heparin in the presence or absence of ergosterol (400 \k% or 800 (ig/ml). The mice were killed on d 5 with an overdose of pentobarbital, and the gels were removed, weighed, and then the hemoglobin contents in the gels were determined. Each value represents the means ± S.E. of 5 mice. *P<0.05, Significantly different from Matrigel/aFGF/heparin mixture-treated mice.
From these results, it seems likely that the antitumor activity of ergosterol might be due to direct inhibition of angiogenesis induced by solid tumors.
573
Matrigel atone
Matrigel a FGF{1 ng/ml) Heparin(64 unrts/ml)
1 cm Matrigel/a FGF/Heparin + Ergosterol (400^g/ml) (800^g/ml)
Fig. 19. Photograph of Matrigel 5 daysfater subcutaneous injection with 0,5 ml of Matrigel alone (a), Matrigel supplemented with aFGF and heparin in the presence or absence of ergosterol.
3) Antitumor and Antimetastatic Activities by Natural Products in Ibmor-Bearing Mice. a) Isolation of Active Substances from Cassia garretiana Heartwood on Primary Solid Tumor Growth and Lung Metastasis in Lewis Lung Carcinoma (LLC)-Bearing Mice and These Mechanisms [6, 7].
574
The heartwood of Cassia garrettiana Craib (Leguminosae) is a Thai drug called "Sa mae sarn". It has been used as a mild cathartic in folk medicine. Although it has recently been thought that extracts of C. garrettiana heartwood have antitumor activity, the basis for this hearsay is unclear. In addition, the pharmacological effects of this drug have not been fully investigated and the active substances have not been isolated. Therefore, to clarify whether the heartwood of C. garrettiana has antitumor effects, here I first examined the effects of a methanol extract of C garrettiana heartwood on tumor growth and lung metastasis in LLC-bearing mice. A methanol extract (500 mg/kg twice daily) of the heartwood of C. garrettiana inhibited the tumor growth and metastasis to the lung in LLC-bearing mice "Table (6)". Table 6.
Effects of MeOH extract of Cassia garrettiana heartwood on tumor volume and lung metastasis
in LLC-bearing mice No. of
Tumor volume (mm^
Lung metastasis
Animals
Mean ± S.E.
(no. of colonies) Mean ± S.E.
LLC-bearing mice (control)
8
1410.3 ± 764.5
4.75 ± 1.92 (6/8)
+ MeOH ext.
8
482.3 ±195.1*
2.00 ± 1.25 (3/8)
(500 mg/kg X 2) Values are expressed as means ± S.E. of 8 mice of each group. * P<0.05, Significantly different from LLC-bearing mice (control).
PR OH OH Piceatannol: R=H Piceatannol acetate: R=COCH3 OH Cassigarcl A Fig.20. Antitumor and antimetastaic substances of Cassia garrettiana heartwood
Compounds 1 and 2, possessing antitumor and antimetastatic activities, were isolated from the methanol extract. Compounds 1 and 2 were identified as cassigarol A and piceatannol (3, 3', 4, 5'-tetrahydroxy stilbene), respectively, based on the ^H-NMR spectral data and products
575
formed by oxidation with potassium permanganate "Fig. (20)". I examined the effects of the cassigarol A, piceatannol and piceatannol acetate on tumor growth in LLC-bearing and on lung metastasis in primary tumor-removed mice. 2500 r
LLC-bearing mice(control) + Cassigarol A(50mg/kg x 2/day) + Cassigarol A(100mg/kg x 2/day)
2000
1500
Q
1000
500
0
i
0
i
i
2
i
i
i
4
i
i
6
i
i
8
i
i
10
i
i
12
14 Day
Fig. 21. Effects of cassigarol A on tumor growth in LLC-bearing mice. Solid-type LLC was prepared by subcutaneous transplantation into the right backs of mice on day 0. Cassigarol A (50 or 100 mg/kg) was administered orallt twice daily for 14 days. Results are expressed as means ± S.E. of 4-7 mice in each group. *P<0.05, Significantly different from LLC-bearing mice (control).
As shown in "Fig. (21)", cassigarol A at the dose of 50 mg/kg x 2/day, significantly inhibited the tumor growth on day 13 whilst at the dose of 100 mg/kg X 2/day, it significantly inhibited the tumor growth on days 11, 13 and 15, as compared to the growth in LLC-bearing mice. On the other hand, piceatannol did not affect the tumor growth during the experimental period (through day 14), but piceatannol acetate (100 mg/kg twice daily)
576
significantly inhibited the tumor growth on day 12 "Fig. (22)' L L C - b e a r i n g m i c e (control)
LLC-bearing mice (control) piceatannol a c e t a t e ( 5 0 m g / k g x 2 / d a y )
— piceatannol(50mg/kg x 2/day) piceatannol(100mg/kg x 2/day) 0
4 6 8 10 1 14 Day 2 Fig. 22. Effects of piceatannol (a) and piceatannol
piceatannol a c e t a t e ( 1 0 0 m g / k g x 2 / d a y ) 2
2
acetate
4
6
(b) on tumor growth
8
10
12
in LLC-bearing
14
Day
mice.
Results are expressed as m e a n s ± S.E. o f 4 - 7 m i c e in e a c h group. * P < 0 . 0 5 , Significantly dififerent fi'om L L C - b e a r i n g m i c e (control).
On day 15, tumor weight reduced by piceatannol acetate (100 mg/kg twice daily), but piceatannol did not affect "Fig. (23)". g H
LLC-bearing mice
j g a l piceatannol-treated mice Y^
piceatannol acetate-treated mice
100 . »
p<0.05
1 I 1
i LLC-removed mice + Cassigarol A(50mg/kg x 2/day)
0 Piceatarmol (mg/kg x 2/day)
'
^/^ 5U
^r^ luu
Piceatannol acetate (mg/kg X 2/day) "
Fig. 23. Effects of piceatarmol and piceatannol in LLC-bearing mice.
+ Cassigarol A(100mg/kg x 2/day)
(A
6
8
10 12
14
16
Day
-
Survival time 50
100
acetate on tumor
weight
Results are expressed as means ± S.E. of 4-7 mice in each group. *P<0.05, Significantly diffaent from LLC-bearing mice (control).
Fig. 24. Effects of cassigarol A on survival time and survival rate in carcinectomized mice On day 15, the solid tumor tissues were removed imder anesthetic pentobarbital and w e i r e d . Thereafter, cassiagarol A w a s again administered orally twice daily for 17 days, starting 24h after resection of tumor tissues on day 0. T h e survival time and numl>er of surviving tumor-removed mice were determined.
577
Cassigarol A, piceatannol and piceatannol acetate, at the dose of 50 and 100 mg/kg X 2/day, prolonged the survival time and increased the survival rate as compared to those in tumor-removed mice "Fig. (24) and Fig.
p<0.05 -•••BlfBa- • Q - - - ! l -
30 h
73 40 • carcinectomized mice • piceatannol (SOmg/kg x2/day)i • piceattannol(100mg/kgx2/day) 20
piceatannol acetate(50mg/kg x 2/day) piceatannol acetate(100mg/kg x 2/day) 0»-
Carcinectomized mice 0
2
4
6
8 10 12 14 16 18 20 22
Casigarol A
50 mg/kg x2
100 mg/kg x2
Survival time (day) Fig. 25. Effects of piceatannol and piceatannol acetate on survival time and survival rate in carcinectomized
Fig. 26. Effeas of cassigarol A on the number of colonies oflung metastasis in carcinectomized mice.
Chi day 15, the solid tumor tissues were removed under anesthetic pentobarbital and weighed. Thereafter, piecatannol, piceataimol acetate "Fig. (25)" or casssigarol A 'Tig. (26)" was again administered orally twice daily for 17 days, starting 24 hours after resection of tumw tissues on day 0. The survival time and number of surviving tumor-removed mice were determined. Resuhs are expressed as means ± S.E. of 4-7 mice in each group.
Cassigarol A, piceatannol and piceatannol acetate inhibited the increases of metastasis to the lung "Fig. (26) and Fig. (27)". Furthermore, to clarify the antitumor and antimetastatic activities by cassigarol A, piceatannol or piceatannol acetate, 1 examined the inhibitory effects of the above active substances on the formation of capillary-like networks (angiogenesis) of human umbilical vein endothelial cells (HUVECs). Cassigarol A inhibited the angiogenesis of HUVECs at concentrations of 10 to 100 JAM "Fig. (28)". Piceatannol also inhibited the angiogenesis of HUVECs at concentrations of 10 to 100 jiM, but its acetate did not affect "Fig. (29)". Therefore, it is suggested that the antitumor and/or antimetastatic activities of cassigarol A, piceatannol might be due to the inhibition of tube formation (angiogenesis) of vascular endothelial cells.
578
I
I Normal mice H
Carcinectomized mice
Carcinectomized mice treated with piceatamiol Carcinectomized mice treated with piceatamiol acetate 15 r
£
p<0,05
10 p<0.05
o \p<0.05
*5 cd
s
OL
Normal mice
piceatamiol (mg/kg X 2/day) piceatamiol acetate (mg/kg X 2/day)
Carcinectomized mice 50
100 50
100
Fig. 27. Effects ofpiceatannol andpiceatannol acetate on the number of metastatic colonies in the lungs of carcinetomized mice. On day 15, the solid tumor tissues were removed, and then cassigarol A was again administered orally twice daily for 17 days, starting 24 h after resection of tumor tissues on day 0. On day 18, the surviving tumor-removed mice were killed and the metastasis to the lung were observed. Results are expressed as means A} S.E. of 4-7 mice in each group
579
n
CMitrol
H i Piceatannol
[2
Piceatannol acetate
p<0.05
11
10
50
100
i!
20
«
10
Piceatannol (>xM) Piceatannol acetate (M M)
Cassigarol A ( M M ) Fig. 28. Effects ofcassigarol A on Matrigel-induced capillarylike tube formation by HUVEC
pig. 29. Effects of piceatannol andpiceatannol acetate on Matrigel-induced capillary-like tube formation by HUVEC.
HUVECs were seeded on the Matrigel in 270 (d of DMEM supplemented with 20% FBS and incubated with the indicated amounts of cassigarol A, piceattanol or piceatannol acetate for 24 h in a humidified 5% CO2 atmosphere. Results are expressed as means ± S.E. of 4 experiments.
b) Stilbene Derivatives loslated from the Roots of Polygonum Species Inhibit Primary Solid-Tumor Growth and Lung Metastasis in LLC-Bearing Mice and These Mechanism [8, 9]. Stilbenes are naturally occurring phytoalexins found in medicinal plants of Polygonum species znd Rheum species (Polygonaceae) [10,11]. Among OH HO Resveratrol
CH2OH
23,5,4 '-Te trah ydroxys tilb e ne 2-0-D-ghicoside
OH
Fig. 30. Stilbenes isolated from the roots of Polygonum species
580
the stilbene derivatives, resveratrol (3,4',5-trihydroxystilbene) and resveratro-3-O-D-glucoside (piceid) are also found in grapes and red wine. Previously, I found that piceid and 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside reduced the elevation of lipid levels [12], and that 2,3,5,4'-tetrahydroxystilbene-2-0-D-glucoside strongly prevented liver damage induced by high lipid peroxidized diets [13]. Furthermore, I reported that resveratrol strongly inhibited the formation of 5-lipoxygenase products, 5-hydroxy-6,8,ll,14-eicosatetraenoic acid, leukotrienes B4 and C4, and the cylcooxygenase product thromboxane B2 from arachidonic acid [14-16]. I found that the inhibitory effect of resveratrol on arachidonic acid-induced platelet aggregation [14]. Resveratrol and its derivatives have been further shown to strongly inhibit the degranulation of human polymorphonuclear leukocytes [16]. Although resveratrol is reported to contain a cancer chemopreventive agent, the inhibitory action by resveratrol on distant metastases to other organs from primary solid-tumors is as yet unproven. In this review, I describe the effects of three stilbenes "Fig. (30)" isolated from medicinal plants and grapes on tumor growth and lung metastasis in mice bearing highly metastatic LLC tumors. 2,3,5,4'-Tetrahydroxystilbene-2-0-D-glucoside and piceid inhibited tumor growth time-dependently after oral administration of 150 or 300 mg/kg twice daily, respectively "Fig. (31)". These stilbenes also inhibited lung metastasis in LLC-bearing mice "Table (7)". Table 7. Effects of 2^,5,4'-tetrahydroxystilbene-2-O-D-glucoside and piceid on cancer metastasis* lo iung, sfdeen and thymus in LLC-bearing Mice Metastasis to Lung(%)
spleen (mg)
Thymus (mg)
0/4 (0)
71.72 ± 3.10
56.04 ± 9.21*
3/5 (60)
139.8 ± 42.18
27,33 ± 7.70
5/5(100)
145.9 ± 53.19
18.64 ± 9.10
1/5 (20)
126.4 ± 35.38
46.31 ± 8.34
(100 mg/kg X 2/day)
5/5(100)
129.9 ± 45.55
30.79 ± 5.26
(300 mg/kg X 2/day)
1/5 (20)
78.74± 11.58
42.78 ± 7.81
Normal LLC-bearing mice (Control) + 2,3^,4'Tetrahydroxystilbene2-O-D-glucoside (50 mg/kg X 2/day) (150 mg/kg X 2/day) + Piceid
Values are expressed as means ± S.E. from 4 - 5 animals. *P<0.05, significantly different from LLC-bearing mice (control mice).
581
a)
b)
LLC-bearing mice (control)
3500
3500
+ 2,3,5,4'-tetrahydroxystilbene2-O-D-glucoside (50 mg/kg X 2/day) LLC-bearing mice (control) + 2,3,5,4'-tetrahydroxystilbene2-0-D-glucoside (150 mg/kg X 2/day) I
E I
Piceid (100 mg/kg x 2/day) Piceid (300 mg/kg x 2/day)
2500 X
2000
2
1500
> o
1000
2000
1500
1000
I
8
12
t i
16
I
I
20
24
28
32
Day
Fig. 31. Effects of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside growth in LLC-bearing mice.
8
12
16
20
24
28
32
Day
(a) and piceid (h) on tumor
Solid-type LLC was prepared by subcutaneous transplantation into the right hind paw on day 0.2,3,5,4'-Tetrahydroxystilbene-2-6>-D-glucoside or piceid was administered orally twice daily for 32 consecutive days, starting 12 h after tumor implantation. Results are expressed as means ± S.E. of 4-5 mice in each group. *P<0.05, Significantly different from LLC-bearing mice (control).
2,3,5,4'-Tetrahydroxystilbene-2-0-D-glucoside or piceid administered orally may be converted to an aglycone form of resveratrol by hydrolysis. Therefore, I examined the effects of resveratrol on tumor growth and lung metastasis in LLC-bearing mice, DNA synthesis of LLC cells, and capillary-like tube formation (angiogenesis) of vascular endothelial cells. Tumor growth and final tumor weight were significantly inhibited by the intraperitoneally administered resveratrol at doses of 2.5 and 10 mg/kg "Fig. (32)". Resveratrol (2.5 and 10 mg/kg) significantly reduced the number of tumor cell colonies that metastasized to the lung compared the LLC-bearing mice "Fig. (33)". Resveratrol inhibited the DNA synthesis in LLC cells with a 50% inhibitory concentration (IC50) of 6.8 yM "Fig. (34)". Treatment with 100 ^M resveratrol for 24 h increased apoptosis to 20.6 ± 1.35 from 12.1 ± 0.36% in LLC cells. In addition, resveratrol decreased the S phase population at concentrations of 50 and 100 \kM. The proportion of LLC cells in the G2/M phase of the cell cycle was increased by the treatment of 50 ^M resveratrol "Table (8)". HUVEC formed capillary-like networks on Matrigel 24 h after seeding. Resveratrol dose-dependently inhibited angiogenesis of HUVEC at 5 to 100 fxM "Fig. (35)". These findings suggest that the mechanism of antitumor and antimetastatic actions of resveratrol might be due to the
582
a) LLC-bearing mice (control) + Resveratrol(0.6 mg/kg) + Resveratrol(2.5 mg/kg) + ResveratroK 10 mg/kg)
5000 Y
3000
1000
0
2
4
6
8
10 12 14 16 18 20 Day
Fig. 32. £;|^c/5 ofresveratrol on tumor volume (a) andfinaltumor weight (b) in LLC-bearing mice. Solid-type LLC was prepared by subcutaneous transplantation into the backs on day 0. Resveratrol was administered intraperitoneally once daily for 21 consecutive days, starting 12 h after implantation of the tumor cells. Results are expressed as means ± S.E. of 7 mice in each group. *P<0.05, Significantly different from LLC-bearing mice (control).
inhibition of DNA synthesis in LLC cells and the inhibition of angiogenesis of vascular endotheUal cells. Table 8. Effects ofresveratrol on apoptosis Go/Gl, S and G2/M phase of cell cycle in LLC cells^ % of total cells Concentration
Apoptosis
Go/Gi
S
Gz/M
(HM) None
12.1±0.36
50.0±L97
35.2±L72
14.8±0.29
(5)
9.43±0.51
44.6±0.75
37.9±L25
17.5±0.49
(10)
9.65±0.61
39.9±1,54
43.8±2.12
16.3±0.70
(50)
12.5±0.97
46.8±L15
22.1+1.03*
31.1±0.26*
(100))
20.6+L35*
52.9±L16
29.2+0.27*
17.8±1.42
Resveratrol
^\^lues are expressed as means ± S.E. of 3 experiments. *P<0.05, Significantly different from medium alone.
583
10 r
o
8
o
;7
8 6
cd
0 ^ Resveratrol 0.6 2.5 10 mg/kg Fig. 33. Effects of resveratrol on the numbers of colonies ofLLC cells metastasizing to the lung on day 22 in LLC-bearing mice. Results are expressed as means ± S.E. of 7 mice in each group. *P<0.05, Significantly different from LLC-bearing mice.
584
& 20
3 ,
10
100
1000 luuu
Resveratrol ( M M ) Fig. 34. i ^ c / s of resveratrol on ^H-thymidine incorporation into DNA ofLLC cells. Results are expressed as means ± S.E. of 4 experiments. *P<0.05, Significantly diflFerent from medium alone.
-
Q
5
JO
5Q
100
Resveratrol ( M M ) Fig. 35. Effects of resveratrol on Matrigel-induced capillarylike networkformation by HUVEC. Results are expressed as means ± S.E. of 4 experiments. *P<0.05, Significantly different from medium alone
585
REFERENCES [1] Kimura, Y. & Okuda, H. Prevention by chitosan of myelotoxicity, gastrointestinal toxicity and immunocompetent organic toxicity induced by 5-fluorouracil without loss of antitumor activity in mice. Jpn. 7. Cancer Res, 1999, 90, 765- 774. [2] Kimura, Y.; Sawai, N.; Okuda, H. Antitumor activity and adverse reactions of combined treatment with chitosan and doxorubicin intumor-bearing mice. / . Pharm. Pharmacol 2001,53, 1373-1378. [3] Kimura, Y & Okuda, H. Prevention by carp extract of myelotoxicity and gastrointestinal toxicity induced by 5-fluorouracil without loss ofantitumor activity in mice. J. Ethnopharmacol 1999, 68, 39-45. [4] Kimura, Y; Takaku, T.; Nakajima, S.; Okuda, H. Effects of carp and tuna oils on 5-fluorouracil-induced antitumor activity and side effectsin sarcoma 180-bearing mice. Lipids 2001,36, 353-359. [5] Takaku, T.; Kimura, Y; Okuda, H. Isolation of an antitumor compound from Agaricus blazei Murill and its mechanism of action./. Nutr, 2001,131,1409-1413. [6] Kimura, Y; Baba, K.; Okuda, H. Inhibitory effects of active substances isolated from Casssia garrettiana heartwood on tumorgrowth and lung metastasis in Lewis lung carcinoma-bearing mice (Part \). Anticancer Res. 2000,20, 2899-2906. [7] Kimura, Y; Baba, K.: Okuda, H. Inhibitory effects of active substances isolated from Casssia garrettiana heartwood on tumor growth and lung metastasis in Lewis lung carcinoma-bearing mice (Part 2), Anticancer Res, 2000,20, 2923-2930. [8] Kimura, Y & Okuda, H. Effects of naturally occurring stilbene glucosides from medicinal plants and wine, on tumor growth and lung metastasis in Lewis lung carcinoma-bearing mice. J. Pharm, Pharmacol, 2000,52, 1287-1295. [9] Kimura, Y & Okuda, H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice. / . Nutr, 2001, 131, 1844-1849. [10] Kubo, M.; Kimura, Y; Shin, H.; Haneda, T.; Tani, T.; Namba, K. Studies on the antifrmgal substance of crude drugs (II). On the roots of Polygonum cuspidatum Sieb.et Zucc. (Polygonaceae). Shouyakugaku Zasshi, 1981, 35, 58-64. [11] Kimura, Y; Kozawa, M.; Baba, K.; Hata, K. New constituents of roots of Polygonum cuspidatum, Planta Med. 1983, 48, 164-169. [12] Arichi, H.: Kimura, Y; Okuda, H.; Baba, K.; Kozawa, M.; Arichi, S. Effects of stilbene componets of the roots of Polygonum cuspidatum Sieb. Et. Zucc. On lipid metabolism. Chem. Pharm, Bull 1982,30, 1766-1770. [13] Kimura, Y; Ohminami, H.; Okuda, H.; Baba, K.; Kozawa, K.; Arichi, S. Effects of stilbene components of roots of Polygonum species on liver injury in peroxizied oil-fed rats. Planta Med 1983,49, 51-54. [14] Kimura, Y; Okuda, H.; Arichi, S. Effects of stilbenes on arachidonate metabolism in leukocytes. Biochim Biophys, Acta 1985,834, 275-278. [15] Kimura, Y; Okuda, H.; Arichi, S. Effects of stilbene derivatives on archodoudlc metabolism in leukocytes. Biochim, Biophys, Acta 1985,837, 209-212. [16] Kimura, Y; Okuda, H.; Kubo, M. Effects of stilbenes isolated from
586
medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes. 7. £mop/iarmaco/. 1995,45, 131-139.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 28 © 2003 Elsevier Science B.V. All rights reserved.
587
BIOLOGICALLY ACTIVE TRITERPENE GLYCOSIDES FROM SEA CUCUMBERS (HOLOTHUROIDEA, ECHINODERMATA) HUGO D. CHLUDIL, ANA P. MURRAY, ALICIA M. SELDES AND MARTA S. MAIER* Departamento de Quimica Orgdnica, Facultadde Ciencias Exactasy Maturates, Universidad de Buenos Aires, Pabell6n2, Ciudad Universitaria, (1428) Buenos Aires, Argentina ABSTRACT: Sea cucumbers are characterized by their content in holothurins, triterpenoid glycosides that are responsible for the toxicity of these echinoderms. Nearly 100 holothurins isolated in the last twenty years are grouped into three main aglycone structural types: 3p-hydrox>iiolost-9(ll)-ene, 3p-hydrox5*iolost-7-ene and nonholostane based aglycones. This communication offers a general view of the structural characteristics of these saponins and the spectral features in their ^H- and ^^C-NMR and FAB-MS spectra. Recent advances in the unambiguous spectroscopic characterization of the triterpenoid skeleton, the substitution patterns and the complete structure of the oligosaccharide chain are discussed.
INTRODUCTION The phylum Echinodermata (Greek echinos^ spiny; derma^ skin) comprises some of the most familiar seashore animals. There are about 7,000 living species widely distributed in all oceans at all depths. The phylum is divided into five classes: Holothuroidea (sea cucumbers or holothurians), Asteroidea (starfishes or sea stars), Ophiuroidea (brittle stars), Crinoidea (sea lilies and feather stars) and Echinoidea (sea urchins). Triterpenoid and steroid oligoglycosides are predominant and characteristic secondary metabolites of sea cucumbers and starfishes and are responsible for their general toxicity [1-6]. Both classes of echinoderms contain also glycosphingolipids, such as monohexosylceramides (cerebrosides) and gangliosides [7]. Brittle stars contain sulfated polyhydroxysteroids [4,8,9] and only two sulfated steroidal monoglycosides have been reported in the brittle star
588
Ophioderma longicaudum [10]. On the contrary, there is no report of steroid or triterpenoid glycosides in the classes Echinoidea and Crinoidea. Several reviews concerning the structures, taxonomic distribution, evolution and biological activities of sea cucumber triterpenoid oligoglycosides have been published [11-14]. The purpose of the present communication is to offer a general view^ of the methods applied in the structural elucidation of these complex molecules, focusing on recent examples of cytotoxic, antifungal and virucidal triterpenoid oligoglycosidesfromour laboratory. TRITERPENOID GLYCOSIDES Triterpenoid saponins are typical metabolites of plant origin, but extensive investigation on marine organisms as sources of new bioactive metabolites has shown that triterpenoid glycosides are widely distributed in sea cucumbers. It has been suggested that these saponins have a defensive role due to their membranotropic action [11]. Penta- and tetraglycosides containing a norlanostane triterpenoid have been encountered rarely also in sponges [15]. Most of the triterpenoid glycosides isolated so far from holothurians present a sugar chain of two to six monosaccharide units linked to the C-3 of the aglycone, which is usually based on a "holostanol" skeleton [3{3,205-dihydroxy-5a-lanostano-18,20-lactone] (1), Fig.(l) [1]. o^
^o
22
24
R—a 30
31
Fig. (1). Structure of hypothetical holostanol
Only quinovose, glucose, 3-0-methylglucose, xylose and 3-0methylxylose are present in the carbohydrate moieties of these glycosides. The &st monosaccharide unit is always xylose, while 3-0-methylglucose and 3-0-methylxylose are always terminal. In comparison to steroidal
589
oligoglycosides from starfishes which always contain a sulfate group attached to C-3 of the aglycone, sixty percent of the triterpenoid glycosides isolated so far from sea cucumbers have sulfate groups linked to the monosaccharide units of the oligosaccharide chain. Although most of them are monosulfated oligoglycosides, several di- and trisulfated glycosides have been isolated, mainlyfromthe order Dendrochirotida. Triterpene glycosides are specific for different taxonomic groups of sea cucumbers and represent good models for studies on biochemical evolution [16]. They have a wide spectrum of biological effects: antifungal, cytotoxic, hemolytic, cytostatic and immunomodulatory activities [12]. These biological activities are a consequence of their membranotropic action against any cellular membrane containing A^sterols. Triterpene glycosides form complexes with these sterols that lead to the development of single ion channels and larger pores, which cause significant changes in the physico-chemical properties of membranes [13]. Sea cucumber cell membranes are resistant to their own toxins due to the presence of A^- and A^'^ ^-sterols, sulfated A^-sterols and p-xylosides of sterols instead of thefreeA^-sterols [17]. CHEMICAL STRUCTURES Nearly 100 different chemical structures of these toxins have been published in the last 20 years. Most of these triterpenoid oligoglycosides contain an aglycone based on a "holostanol" skeleton and two main series can be distinguished: glycosides based on a 3p-hydroxyholost-9(ll)-ene aglycone and those containing a 3P-hydroxyholost-7-ene skeleton. Usually aglycones that have a A^'^^ double bond are characteristic of sea cucumbers belonging to the order Aspidochirota, while those with a A^ unsaturation were generally isolated from animals of the order Dendrochirotida. 3p-Hydroxyholost-9(ll)-ene aglycones Bivittoside C, Fig. (2), a hexaglycoside isolated from the sea cucumber Bohadschia bivittata [18] is the simplest triterpene glycoside with a A^'^^ double bond:
590
R—a
2 Bivittoside C [18] R = [3-0-Me-3)-Glc-(l->4)-Qui
Fig. (2). Structure of Bivittoside C
A number of glycosides containing aglycones of this series show a carbonyl group at C-16 (Structure 3, Fig. (3)). With exception of glycoside 3a, all have an additional A25 double bond in the side chain
3a Ds-Penaustroside D [19] R = [3-O-Me-Xyl3)-Glc4)]-[Qui2)]-Qui-(l->2)^»-0SO5N8hX54 3b Holotoxin Ai [20] R = [3-0-Me-Glc4)]-[3-O^Me-Glc-3>XyHl-^)-Qui-(l->2)]-Xyl; A" 3c Holotoxio A [21] R = [3-0-Me-Glc^l->3)-Gic-(l-^)]-[3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui2)]-Xyl; A^ 3d Holotoxin Bi [20] R = [Glc3)-X)1-(l-^)-Qai-(l->2)]-Xyl; A^ 3e Holotoxin B [21] R = [G^c3)-G^c-(l->4>Qui-(1^2)^Xyi; A" 3f Neothynidioside [22] R = 3-0-Me-Glc-(l-y3)-Xyl-4)-Qui-2)^W)S03Na-Xyl; A^' 3g Psolusoside A [23] R = 6-OS03Na-3-^-Me-Glo3)-€-OS03Na-Glc4)-Qui-(l->2)-Xyl; A^' 3h Qadoloside A [24] R = 3-0-Me-Glc-(l-^3)-Xyi-(1^4)-Qui-(l->2)-Xyi; A" 3i Cladoloside B [24] R = [Glc-(l->4)]-[3-0-Me-Glc-2)]-Xyi; A" 3j Ds-Penaustroside C [19] R = [3-0-Me-Xyi4)]-[<^2)]-Qiu-2)-4-OSOjNa-Xyl; A^' 3kHemoiedemoside A [25] R = 3-0-Me-Glc-(1^3)-6-OS03NarGlc-(l-^)-Qiii-(l->2)-4-OS03NarXyl; A" 31 Hemoiedemoside B [25] R = 6-OS03Na-3-0-Me-Glc3)-6-OS03Na.Glc<1^4)-Qiii-{1^2)-4-OS03Na-Xyi; A^^ 3m Caudinoside A [26] R = 3-<9-Me-Glc-(l-^3)-Glo(l-^)-Qui-2)-Xyl; A^
Fig. (3). Structure of 3p-hydro3Qiiolost-9(l l)-en-16-one aglycone based glycosides
Another structural feature is the presence of a 12a-hydroxyl group in the aglycone, Fig. (4):
591
HO O
4a Bivittoside A [18] R = Qui-(l-^2)-Xyl 4b Bivittoside B [18] R = [3-0-Me-Glc4)]-[3-0-Me-Glc3)-Glc-4)-Qui-(l->2)]-X}d 4d Pervicoside C [27] R = 3-(9-Me-Gl4)-Qiji4-OS03Na-Xy! 4e Pervicoside B [27] R = 3-0-Me-Gio{\- •3)-Glc-(l->4)-Qui-(l->2)-4-OS03Na-Xyi; A^
Fig. (4). Structure of 3p,12a-dihydro3Qiiolost-9(l l)-ene aglyoone based glycosides
Some glycosides contain two hydroxyl groups at positions 12a and 17a of the holostanol skeketon, Fig. (5):
HO O
5a Echinoside B [28] R = Qui-(1^2)-4-OS03Na.Xyl; R' = H 5b Echinoside A [28] R = 3-0-Me-Glc3>Glo4>Qiii-(l->2>4-OS03Na-Xyl; R* = H 5c 22-Acetoxy-echinoside A [29] R = 3-0-Me-Glc3)-Glc<1^4)-Qui-(l->2)^M3S03Na-Xyl; R* = OAc 5d Holothurin A, [30] R = 3-<9-Me-Glc-(l-^3)-Glc-4)-Qtti-(l->2>4-0S03Na-Xyl; R* = C« 5e 24-Dehydroechinoside B [31] R = Qui-(l->2)^K)SOjNa-XyI; R* = H; A^ 5f 24-Dehydroechiiioside A [31] R = 3-0-MeGlc<1^3)-Gd-(l->4)-Qui-2)-4-0S03Na.Xyl; R^ = H; A^ 5g 22-Hydroxy-24-dehydroechiiioside A [29] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-2)-4-0S03NarX>i; R' = OH;A^
Fig. (5), Stiucture of 3p,12a, 17a-trihydrox54iolost-9(l l)-«ie aglycone based glycosides
Glycosides 5c, 5d and 5g together with glycosides 6, Fig. (6) and 7, Fig. (7) are characterized by additional acetoxy or hydroxy groups in the side chain.
592
OH
R
O'
6 24(5)-hydroxy-25-dehydroechinoside A [29] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-(l-^2)-4-OS03Na^Xyl Fig. (6). Structure of a sul&ted tetraglycoside isolatedfixMnthe sea oxccashei Actinopygaflammea
HO O^
^O
OAc
7a Holothurinosidc B [32] R = [3-0-Me-Glc-(l->3)-CHc-(l->4)-Qui-2)]-[CHc-(l->4)]-Xyl; R ' = OH; A^ 7b Pervicoside A (Neothyosidc A) [27] R = 3-C>-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l->2)-4-OS03Na-Xyl; R^ = H 7c Neothyoside B [33] R = Qui-(l->2)-4-OS03Na-Xyl; R ' = H Fig. (7). Structure of 25-acetoxi-3p,12a-dihydroxjiiolost-9(l l)-€ne aglycone based glycosides
Holothurins A (8a) and B (8b) isolated from the sea cucumber Holothuria leucospilota [34] as well as Desholothurin A (8d), and Holothurinosides A (8c), C (8e) and D (8f), Fig. (8)fromHolothuria forskali [32] are the only examples of glycosides containing the side chaininafiiranform. Compounds 3a, 3g-31 and 7c are the only A^'^ ^-glycosides isolated from sea cucumbers belonging to the order Dendrochirotida. In general, 3|3-hydroxyholost-9(ll)-ene based aglycones were characterized in holothurins isolated from animals of the order Aspidochirota.
593
8a Holothurm B [34] R = Qui-(1^2)-4-OS03Na-Xyl; R ' = OH 8b Holothurm A [34] R = 3-0-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l-^2)-4-OS03Na-X)4; R^ = OH 8c Holothurinoside A [32] R = [Glc-(1^4)]-[3-0-Me-Glc-(l-^3)-Glc-(l->4)-Qiii-(l->2)]-Xyi; R^ = OH 8d Desholothurm A [32] R = 3-0-Me-CHc-(l->3)-Glc-(l->4)-Qui-(l->2)-Xyl; R ' = OH 8e Holothurinoside C [32] R = 3-0-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l->2)-Xyi; R ' = H 8f Holothurinoside D [32] R = Qui-(1^2)-Xyi; R ' = H Fig. (8). Structures of glycosides isolatedfromtbe sea cucumbers Holothuria leucospilota and Holothuria forskalii
3P-HydroxyhoIost-7-ene aglycones Frondoside B (9a), Cucumariosides A2-4 (9b) and A7-3 (9c), Fig. (9) as well as several triterpene glycosides isolated from the sea cucumbers Stichopus chloronotus (lOa-lOh) and Thelenota ananas (lOi, lOj), Fig. (10) contain the simple 3P-hydroxyholost-7-ene as the aglycone. An additional acetoxyl group in the side chain is present in compounds 10alOj.
9a Frondoside B [35] R = [3-0-Me-Glc-(l-^3)-6-OS03Na-Glc-(l->4)]-pCyl-<1^2)]-Qui-(l->2)-4-OS03Na.Xyl; A'; A^ 9b Cucumarioside K2-A [36] R = [3-0-Me-Glo3)-Glc-(l->4)]-pCyl-(l->2)]-Qui-3)-6-OS03Na-Glc-(l-^)]-[Xyl-(l-^2)]-Qui-(l->2)-40S03Na-Xyl;A';A^ Fig. (9). Structures of glycosides isolatedfromthe sea cucumbers Cuctanariafrondosa and Cucumaria japonica
594
Glycosides lOa-lOj were isolated from Stichopus chloronotus and Thelenota ananas, two sea cucumbers belonging to the order Aspidochirota [37].
lOa Stichloroside C, (Stichoposide C) [37] R = [3-0-Me-Glo3)-Glc-(l->4)]-[3-0-Me-ac3)-Xyi-(l-»4)Qum-(l-^2)]-Xyi 10b Stichloroside B, (Stichoposide D) [37] R = [3-0-Me-Glc-(l->3)-Glc-(l-^)]-[3-0-Me-Glc-3)-Glc-(1^4)]-[3-0-Me-Glc-<1^3)-Glc-(1^4)-X5d-(l-^2)l-Xyl lOd Stichoposide A [37] R = Qui-(l->2)-4-OS03Na-Xyi lOe Stidioposide B [37] R = (ao2)-Xyl lOf Stichloroside C2 [37] R = [3-0-Me-Glc-(l->3)-Glc4)]-[3-<9-Me-ac-(1^3)-Xyl-(l->4)-Qui-(l->2)]-X>4; A^' lOg Stichloroside B2 [37] R = [3-0-Me-Glc-(l->3)-Glc-(l->4)]-[3-0-Me-Glo3)-Xyi-(l-M)-Glc-(l-^2)]-Xyi; A^^ lOh Stichloroside A2 [37] R = [3-0-Me-Glc-{l->3)-Glc-<1^4)]-[3-0-Me-Glc-(l->3)-CHc-2)]-Xyl; A^ lOi Thelenotoside A [37] R = 3-0-Me-Glc-(l->3)-Xyi-(l-^)-Qui-(1^2)-Xyl lOj Thelenotoside B [37] R = 3-<9-Me-Glc-(l->3)-X>i-(l->4)-Glc-(l->2)-Xyl Fig. (10). Structures o f glycosides isolated fixjm the sea cucunibers Stichopus chloronotus and Thelenota ananas
3p-Hydroxyholost-7-ene aglycones with a carbonyl group at C-16 have been isolated exclusively from the sea cucumber Cucumaria japonica, Fig. (11).
R—a
l l a Cucumarioside A2-3 [36] R = [3-0-Me-Glc-(l-^3)-Glc-2)-Xyi
595 l i b Cucumarioside Ar-2 [36] R = [6-OS03Na-3-0-Me-Glc-(l->3)-6-OS03Na-Glc-(1^4)]-[X>d-(l->2)]-Qui-(l->2)4-OS03N2hXyl l i e Cucumarioside Ao-3 [38] R = [3-0-Me-Glc-(l->3)-Xyi-2)]-Qui-(l-^2)-4-OS03Na.X)d; A" U d Cucumarioside A,-2 [38] R = [6-OAc^c-(l->3)-Glc-(l->4)]-pCyi-(l->2)]-Qui-(l->2H-OS03Na-Xyl; A^' l i e Cucumarioside A2-2 [36] R = [3-0-Me-CHc-(l->3)-Glc-(l-^)]-pCyi-(l->2)]-Qui-(1^2)-XyI; A" l l f Cucumarioside A7-I [36] R = [6-OS03Na-3-0-Me-Glc-(l-^3)-6-OS03Na-Glc-(l->4)]-[X>d-(l->2)]-Qui-(l->2)4-OS03Na-Xyl;A^' l l g Cucumarioside A3 [39] R = [3-(9-Me-ac4)]-[X54-(l->2)]-Qui-(l->2)-4-OS03NaXyi;A" l l h Cucumarioside A6-2 [39] R = [6-OS03Na-3-0-Me-Gac-(l->3)-Glc-(l->4)]-pCyi-(l->2)]-Qui-(l->2)-4-OS03NaXyl;A" Hi Cucumarioside A4-2 [36] R = [GIc-(l->3)-ac-<1^4)]-pCyi-(l-^2)]-Qui-(l-^2)-4-OS03Na-Xyl; A^ Fig. (11). Structures of glycosides isolated from the sea cucumber
Cucumariajaponica
Another structural feature that has been found only in this series of aglycones is the presence of an acetoxyl group at C-16. Glycosides with a p-configuration for this group are shown in Fig. (12). o^ ^o
R—a
12a Frondoside A [40] R = [3-<9-Me-Glc-(l-^3)-Xyl-(l-^)]-PCyl-(l->2)]-Qui-(l->2)-4-OS03Na-Xyl 12b Frondoside Ai [41] R = 3-0-Me-Glc-(l->3)-X>i-(l->4)-Qui-(l->2)-4-OS03Na-Xyl 12c Liouvilloside B [42] R = 6-OS03Na-3-0-Me-Glc-(l->3)-6-OSOjNa-Glc-(l->4)-Qui-(l->2)-4-OS03Na-Xyi 12d Cucumarioside Ao-2 [38] R = [3-0-Me-Glc-(l-^3)-Xyi-(l-^)]-pCyl-(l-^2)]-Qui-(l-^2)-4-OS03Na-Xyl; A^^ 12e Neothyonidioside C [43] R = 6-OS03Na-3-6>-Me-Glc-(1^3)-X5d-(l->4)-Qui-(l->2)-4-OS03Na-Xyi; A^ 12f Cucumarioside G, [44] R = 3-0-Me-Xyi-(l->3)-GJc-(l->4)-Qui-(l->2)-4-OS03N»-Xy!; A^ 12g Liouvilloside A [42] R = 6-OS03Na-3-0-Me-Glc-3)-6-OS03Na-Glc-(1^4)-Qui2)-4-OS03Na-Xyi; A^ 12h Cucumarioside C^ [45] R = [3-0-Me-Xyl-(l->3)-Glc4)]-[Xyi-(l->2)]-Qui-(l->2)-Xyi; HE, A^'* 12i Cucumarioside H [46] R = 3-0-Me-Xyl-3)-<31c4)-Qui-(l->2)-4-OS03Na-X>4; 22£'; A^ 12k Cucumarioside Cj [45] R = [3-0-Me-X)d-(l->3)-Glc-2)]-Qui-(l->2)-Xyl; 22Z; A^ 121 Cucumarioside G3 [47] R = 3-0-Me-ac-(l-^3)-Cac4)-Qui-(l-^2)-4-OS03Na.Xyl; 22Z; A^ Fig. (12). structure of 16p-acetoj^-3p4iydroxjiiolost-7-eoe aglycone based glycosides
Some of the glycosides containing a 16p-acetoxy group also present an allylic hydroxyl group at C-25, Fig. (13).
596
OH
13a Cucumarioside G4 [47] R = 3-(9-Me-X)4-(l->3)-Glc-{l->4)-Qui-(1^2)^U)S03Na.Xyl 13b Eximisoside A [48] R = 3-0-Me-Glc-(l-^3)-Xyl-(l->4)-Glc-(l->2)-X5d 13c Caldgeroside E [49] R = [6-OS03Na.3-0-Me-Glc3)-a(Kl->4)]-[Glc-(l-^2)]-Qui-{l->2)-4-OS03Na-Xyl Fig. (13). Structure of 16p-acetoxy-3p,25-dihydrox>iiolosta-7,22-diene aglyccaie based glycosides
Four glycosides isolated from the sea cucumber Cucumaria lefevrei [50] are the only examples of holothurins with a 16a-acetoxy group in their aglycones, Fig. (14). Lefevreiosides A2 (14b), B (14c) and C (14d) show the same monosulfated tetrasaccharide chain and differ in the degree of unsaturation or the position of the double bond in their side chains. Lefevreioside Ai (14a) is the desulfated analog of glycoside 14b.
14a Lefevreioside A, [50] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-(l-^2)-Xyl 14b Lefevreioside A2 [50] R = 3-0-Me-Glc-(l-^3)-Cac-(l->4)-Qui-2)-4-OS03Na-X>d 14c Lefevreioside B [50] R = 3-0-Me-Glc-(l-^3)-Glc-(l->4)-Qiii-(l->2)-4-OS03Na-Xyi; A^ 14d Lefevreioside C [50] R = 3-0-Me-Glc-(l->3)-G!c-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; A^^ Fig. (14). Structures o f glycosides isolatedfixatnthe sea cucumber Cucumaria
lefevrei
Several triterpene glycosides isolated from the sea cucumbers Cucumaria echinata and Pentamera calcigera contain a carbonyl group at C-23 in the side chain, Fig. (15). This structural feature is absent in 3phydroxyholost-9(ll)-ene aglycones.
597
15a Cucumechinoside C [51] R = 3-(9-Me-Glc-(l->3)-6-OS03Na-ac-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; R^ = H 15b Cucumedimoside F [51] R = 6-OS03Na-3-0-Me-Glc-(l->3)-6-OS03Na^c-(l->4)-Qui-(1^2)-4-OS03Na-Xyl; R' = H 15c Caldgeroside Cj [52] R = [3-0-Me-XyKl->3)-Glc-(l->4)]-[CHc-(l->2)]-Qui-(l->2)-4-OSOjNa-Xyl; R^ = H 15d Caldgeroside D2 [49] R = [3-C>-Me-XyH1^3)-6-OS03Na.Glc-(l->4)]4Glc2)]-Qm-3)-6-OS03Na-Glc-(1^4)-Qui-(l->2)-4-OS03Na-Xyi; R* = O 15f Cucumedimoside B [51] R = 3-O-Me-Glc-(l->3)-2-OSO3N2hXyi-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; R' = O 15g Cucumechinoside D [51] R = 6-OS03Na-3-0-Me-3)-6-OS03NarGlc4K^<1^2)-4-OS03Na-Xjd; R^ = O 15h Cucumediinoside E [51] R = 6-OS03Na-3-0-Me-Glc3)-2-OS03Na-X>d4-OS03Na-X>d; R^ = O 15i Cucumarioside Ao-1 [38] R= [3-O-Me-Glc-(l->3)-X>i-(l-^)]-[Xyi-2)]-Qui-(l->2)-4-OS03Na-Xyi; R^ = P-OAc Fig. (15). Structures of glycosides isolated fix>tn the sea cucumbers Cucumaria echinata and Pentamera calcigera
Recently, we have isolated an antifungal holothurin from the sea cucumber Psolus patagonicus [53]. Patagonicoside A (16), Fig. (16) is the &st example of a 3p-hydroxyholost-7-ene aglycone substituted with 12a- and 17a-hydroxy groups. H%o<^^-
16 Patagonicoside A [53] R = 3-6>-Me-Glc-(l->3)-6-OS03NarGlc-4)-Qui-(l->2)-4-OS03N»-Xyl Fig. (16). Structure of patagonicoside A, an antifungal oligoglycoside isolated fixxn the sea cucumber Psolus patagonicus
Non-holostane aglycones
598
Recently, some examples of holothurins having uncommon nonholostane aglycones have appeared in the literature. These glycosides have been isolated from seven species of sea cucimibers belonging to the order Dendrochirota. All are sulfated compounds, the majority monosulfated at the glucose or xylose units. Five glycosides contain aglycones with an 18(16)-lactone and a A^unsaturation, Fig. (17) and (18). OAc
17 Psolusoside B [54] R = [6-OSO,Na-Gl(Kl->4)]-[Glc-(l->4)-Glo(l-^2)]-X>i Fig. (17). Structure of Psolusoside B, isolatedfixmithe sea cucumber Psoltds fabricii
^N^^ •iiiH
18a Cucumarioside G^ [55] R = 3-0-Me-Xyl-(l-^3)-Glc(l->4)-Qui-(l-^2)-4-OS03Na.Xyl 18b Caldgeroside B [52] R= [3-0-Me-X>i-(l->3)-Glc(l->4)]-[Qw-2)]-Qiii-(l-^2)-4-OS03Na-Xyi 18c Caldgeroside C, [52]R = [3-0-Me-X>d3)-CHc(l->4)]-[Glc-(l-^2)]-Qui-(l->2)-4-OS03Na.X>1 18d Caldgeroside D, [49] R = [3-0-Me-Xyl-(l->3)-6-OS03Na-Glc(l-^)]-[CHcji-(l->2)-4-OS03Na-X^ Fig. (18). structures of noo4iolostane glycosides isolated fixxn the sea cucumbers Et^ntacta fraudatrix and Pentamera cahigera
Avilov et al. [56,57] reported three holothurins that are devoid of a lactonefiinctionand have a shortened side chain. Kurilosides A (19a) and
599
C (19b) contain a 9(ll)-double bond aglycone moiety and 16a-acetoxy group, Fig. (19).
OAc
R-
19« Kuriloside A [56] R = [3-O.Me-Glc-(1^3)-6-OS03N»4)-Qiii-(l->2)]-Xyl 19b Kuriloside C [56] R = [3-(9-Me-Glc-(l->3)-6-OS03Na^c-(l->4)]-[Qui-2)]-Xyl Fig. (19). Structures of ^yootsides isolatedfixxnthe sea cucumber Duasmodactyla kurilensis
Koreoside A (20) isolated from Cucumaria koraiensis is one of the two examples of non-holostane glycosides with three sulfete groups in the oligosaccharide chain, Fig. (20). COCH, IIH
20 Koreoside A [57] R = [6-OS03Na-3-C>-Me-3)-6-OS03Na-Glc-(l->4)]-PCyl-(l->2)]-Qui-(l->2)-4OSO^a-Xyi Fig. (20). Glycoside isolatedfixxntbe sea cucumber Cucumaria koraiensis
Ds-Penaustrosides A (21a) and B (21b), as well as Frondoside C (21c), also lack the lactone function and have an additional hydroxyl group at C20, Fig. (21).
600
21a Ds-Penaustroside A [19] R = [3-0-Me-Xyi<1^3)-Gic-(l->4)]-[Qui-(l-^2)]-Qui-(1^2)-4-OS03Na-Xyl; R^ = H 21b Ds-Penaustroside B [19] R = [3-0-Me-Xyi-<1^3)4Slc-(1^4)]-[Qui-(l->2)]-Qui-(l->2)-4-OS03Na-X>4; R^ = H; 21c Frondoside C [58] R = [3-0-Me-Xyi<1^3)-ac-(l->4)]-[Qiii-(l->2)]-Qui-2)-4-OS03Na-Xyl; R' = OAc; A^ Fig. (21). Structures of tiOQ4K>lostane glyoosides isolated from the sea cucumbers Pentacta australis and Cucumariafrondosa
Most of sea cucumber triterpene glycosides are tetra- or pentaglycosides. The few disaccharides that have been isolated show a Qui-(1^2)-4-OS03Na-Xyl chain attached to C-3 of the triterpenoid aglycone [28, 31, 33, 34, 37]. Bivittoside A (4a) and Holothurinoside D (8f) show no sulfate group while Stichoposide B (lOe) is the only example of a disaccharide with a glucose unit attached to C-2 of the xylose unit. Some hexasaccharides have been isolated from sea cucumbers of the order Aspidochirota: Stichopus japonica [21], Stichopus chloronotus [37], Parastichopus californius [20] and Bohadschia bivittata [18]. They are non-sulfated glycosides with a linear 3-0-Me-Glc-(l->3)Glc-(l->4)-Xyl chain and a branching of a linear trisaccharide at C-2 of the xylose unit. The only example with a glucose unit instead of the terminal 3-0-Me-glucose is Holotoxin Bi (3d). Most tetrasaccharides show a linear chain with the most common 3-0In some Me-Glc-(l->3)-Glc-(l->4)-Qui-(l->2)-Xyl structure. tetrasaccharides the glucose imit is replaced by a xylose [22, 24, 37, 38, 40, 43, 51] while Cucumariosides Gi (12f) and G4 (13a) show a terminal 3-0-Me-xylose unit. Thelenotoside B (lOj) and Eximioside A (13b) show a different tetrasaccharide chain: 3-0-Me-Glc-(l-^3)-Xyl-(l->4)-Glc(1~>2)-Xyl with no quinovose unit. Non-holostane triterpenoids, such as Psolusoside B (17), Kuriloside C (19b) and Bivittoside B (4b) are the only examples of tetrasaccharides with a non-linear chain. Most tetrasaccharides are sulfated at C-4 of the xylose unit. Additional sulfete
601
groups at C-6 of the 3-0-Me-glucose unit and at C-6 of the glucose unit have been found in trisulfeted tetraglycosides. Pentaglycosides isolated from sea cucumbers show a variety of carbohydrate chains, Fig. (22). Most glycosides contain chains I-IV. Chain IV is typical for glycosides isolated from the sea cucumber Pentamera calcigera: Calcigerosides Ci (18c), C2 (15c), Di (18d), D2 (15d) and E (13c). Cucumarioside Ai-2 (lid) is the only example of a triterpene glycoside containing an acetate group at C-6 of the terminal glucose unit (chain XII). Pentasaccharide chains with glucose as the terminal sugar are uncommon and were found in a few glycosides, such as Cucumarioside A4-2 (Hi) (chain VII), Cladoloside B (3i) (chain X) and Holothurinoside A (8c) (chain XT). [3-^-M&<51o4)]-pfyHl->2)]<^iiXyKl-^4)]-pfyl-(l-^2)]<^ii-(l-^2)]-Xyl^ycoi» [3-0-Me-XyKl-^3)-Glo4)HGl(Kl->2)]<^-(l->2)]-Xyl-aglyconea^^^ [3^-Me-Xyl-3)-Glc4)HQui-2)]<^ii2)]-Xyl-aglyooiie(rV0 [3-O.I^Xyl-(l-^3Kjlo2)]-Xyl-aglycQne(^ [3^-Me-Glc-(l-^3>Glc4)]4Glo4)]<^ii-(l-^2)]-Xyl^ycoDe(^ [GlcKl->3Hjlo4)]-pCyKl-^2)]<^ii-2)]-Xyl-aglycone(VII) [3-aMe-XyHl->3)-C]ao<1^4)HQui2)].Xyl-aglycc3oe(Vm^ [3^-Me<jlc^l->3)-Glc4K^ii-(l-^2)HGl<>(l-^4)]- Xyl^ycone (IX) [Glo4)H3-a.M©3)-XyKl->4)-Qui-2)]- Xyl-agjycone (X) [Glo4)]43-^-Me-Gl(Kl-->3)-Glo4)-Qui-3)-CHo4)]-pfyl-(1^2)]<^ii2)]-Xyl-aglycone(XII) Fig. (22). Pentaglycoside chains in holotfaurins
Most of the pentasaccharide chains are monosulfated at C-4 of the xylose unit linked to the aglycone. Only a few disulfeted or trisulfated pentaglycosides with additional sulfete groups at C-6 of the 3-O-Meglucose and glucose units have been isolated [35, 36, 39, 42, 49, 57]. STRUCTURAL ELUCIDATION Sea cucumber triterpene glycosides are quite fragile molecules. Acidic hydrolysis of intact holothurins results in the production of artifacts of the original aglycones due to migration of double bonds and dehydration reactions [59, 28]. Aqueous acid hydrolysis of glycosides containing a 25(26)-double bond in the aglycone side chain has led to the formation of artificial 25-hydroxy-genines [21, 60]. To overcome these diflSculties upland ^^C-NMR spectroscopy have been extensively used to determine the
602
Structure of the native aglycones as well as the glycosidic linkages in the oligosaccharide chain without degradation of the glycosides. Besides, the development of soft ionization methods, such as fest atom bombardment (FAB) [61] allowed the mass spectrometric analysis of polar thermally labile molecules of masses of up to a few thousand Daltons, in particular for samples which exist as preformed ions in solution. FAB-MS in positive- and negative-ion modes has been applied to obtain information on the molecular weight of underivatized glycosides of starfish and sea cucumbers on the basis of quasi-molecular ions [M+H]^, [M+Na]^and [M-H]' and [M-Na]", respectively, together with usefiil information on the saccharide sequence [2]. Nuclear magnetic resonance (NMR) has proved to be a very usefiil tool for structural elucidation of natural products. Recent progress in the development of two-dimensional ^H- and ^^C-NMR techniques has contributed to the unambiguously assignment of proton and carbon chemical shifts, in particular in complex molecules. The more used techniques include direct correlations through homonuclear (COSY, TOCSY, ROESY, NOESY) [62-65] and heteronuclear (HMQC, HMBC) [66, 67] couplings. ^H-NMR spectra of triterpene glycosides are complicated due to extensive interproton coupling. The first complete holothurin structures published in the literature [59, 68] reported only some characteristic proton signals, such as those due to methyl groups of the triterpenoid skeleton (I9-CH3, 2I.CH3, 26.CH3, 27-CH3, 3O-CH3, 3I-CH3, 32-CH3), olefinic protons at C-7, C-11 or those present in the side chain, and doublets ascribable to the anomeric protons of the oligosaccharide moiety. Originally, structural elucidation of holothurins was based mainly on ^^C-NMR data, acid hydrolysis and enzymatic and degradation reactions. In the last ten years bidimensional NMR experiments have allowed the assignment of all proton and carbon resonances of the aglycone and the oligosaccharide chain [32, 35, 39, 40, 48, 52, 57]. NOESY experiments [69] on the intact glycosides have been usefiil in determining the relative stereochemistry of all chiral centers of the aglycone. Recently, we have successfiilly assigned all proton and carbon resonances of a new aglycone in the disulfated tetraglycoside patagonicoside A (16) by a combination of ^H-^H COSY, COLOC, HETCOR and NOESY experiments [53]. Fig. (22) shows the NOESY correlations of the aglycone moiety of Patagonicoside A. Correlations of
603
H-3 with H-1', H-la, H-5a and H-31 confirmed the ^-configuration at C3. Of particular interest is the p-configuration of H-9 in 3(3hydroxyholost-7-ene based aglycones instead of the characteristic 9aconfiguration in natural steroids and triterpenoids. This proton showed a characteristic broad doublet at 6 3.02 ppm (in CD3OD) and a strong NOE correlation with H-19 and H-12p. This last correlation revealed the aconfiguration of the hydroxyl group at C-12. Correlations between H-12 and H-21 evidenced the a-configuration of the hydroxyl group at C-17 and consequently the S configuration at C-20. In this way we were able to confirm the stereochemistry assigned previously to these carbons by Kitagawa et al. [27, 28] only on the basis of solvent-induced shifts in the ^H-NMR spectra of the corresponding sapogenols obtained by hydrolysis of the native saponin.
Fig. (22). NOESY correlations of the agiyoone moiety of Patagooicoide A
Triterpene glycosides of sea cucumbers show characteristic signals due to the aglycone and the sugar moieties in their ^^C-NMR spectra. The chemical shifts of aglycone carbons of representative holothurins containing holostane aglycones are shown in Table 1. Characteristic resonances for C-3 (5 ca, 88-91 ppm) and C-20 (6 ca. 83-88 ppm) are observed. Both signals are easily distinguished by DEPT analysis [70]. The presence of a signal at 6 ca. 175-180 ppm is typical for a lactone carbonyl group ascribable to C-18. The two series of aglycones differing in the position of the double bond in the triterpenoid skeleton can be readily distinguished by the chemical shifts of their olefinic carbons. Holothurins containing a 3p-hydroxyholost-9(ll)-ene based aglycone such as glycoside 3k show characteristic resonances for a trisubstituted
604
9(ll).double bond at 5 151.0 ppm (s, C-9) and 111.0 ppm (d, C-11). The presence of an allylic hydroxyl group at C-12a shifts the resonance of C11 to 6 ca. 116 ppm (glycosides 4e, 5b, 6 and 8e). Besides, aglycones with a trisubstituted 7(8)-double bond, as in glycosides 9b, llf, 12a, 13c, 15c and 16, show typical resonances at 6 ca. 120-121 ppm (d, C-7) and 143-148 ppm (s,C-8). Table 1. ^^C NMR chemical shifts of holostane aglycones
c
3k'
4e^
5b ^
6'
8e^
9b*
llf
12a'
13c*
15c*
16^
1
36.1
36.8
36.4
36.1
36.4
36.0
36.8
36.0
35.8
36.0
37.3
2
26.8
27.3
27.0
27.1
27.3
26.9
27.7
26.8
26.6
26.8
27.8
3 4
88.4
89.1
88.5
88.8
88.8
88.9
90.0
89.2
88.9
88.7
90.8
39.5
40.2
39.9
40.1
40.1
39.5
40.4
39.5
39.2
39.4
40.4
5
52.7
53.2
52.7
52.8
52.8
47.9
49.5
48.0
47.7
47.8
50.2
6
20.9
21.4
21.2
21-3
21.2
23.2
24.2
23.3
23.0
23.2
24.0
7
28.3
29.0
28.2
28.4
28.7
119.8
122.7
120.4
120.2
119.8
121.4
8
38.6
40.4
40.8
41.0
40.8
146.6
144.8
145.8
143.2
146.5
148.4
9
151.0
153.6
154.0
154.1
153.5
47.2
48.2
47.2
47.3
47.2
46.1
10
39.7
39.8
39.6
39.8
40.1
35.4
36.7
35.5
35.2
35.4
36.4
11
111.0
116.4
115.6
115.7
116.1
22.8
23.3
22.6
22.3
22.7
35.9
12
31.9
68.5
71.3
71.5
71.5
30.2
30.7
31.4
31.0
30.0
73.6
13 14 15 16 17 18 19
55.7 41.9 51.9 213.4
64.4 46.8 24.3 37.3 47.2 177.5
58.5 46.3 27.0
57.9 46.7 53.0 215.3 64.6 180.3
83.2
86.9
87.4
83.6
23.9 84.1
25.0 84.9
54.2 180.1 23.7
57.5 51.2 34.0 25.5 53.4 179.8 23.9
20
59.5 47.5 43.6 75.4 54.5 180.6 24.0 85.9
59.1 46.9 43.4 74.9
20.0
63.7 46.2 23.6 38.4 47.6 177.5 22.0
58.6 51.2 24.4 34.2 53.0 180.2
18.3 84.7
57.8 46.5 36.5 36.8 89.5 174.8 20.1
84.9
82.5
60.2 52.0 35.7 36.5 90.7 178.5 24.4 88.0
21
26.7
26.4
23.0
23.3
18.7
25.9
27.2
28.4
28.7
27.1
23.0
22
38.3
39.6
36.5
35.2
80.2
39.2
39.2
39.1
41.7
51.8
39.2
23
22.2
23.4
22.2
30.7
28.7
22.9
23.2
22.8
143.2
207.5
23.0
24
37.9
124.5
38.8
75.4
36.7
123.3
38.8
39.6
120.5
52.0
40.7
25
145.4
132.1
28.2
150.0
81.2
131.8
146.5
28.1
70.0
24.3
29.0
26
110.4
25.7
22.6
110.6
28.7
25.5
111.4
22.4
29.6
22.3
23.0
27
22.1
17.7
22.6
18.2
28.0
17.8
23.2
22.9
29.8
22.3
22.9
30
16.4
16.8
16.7
16.8
16.7
17.3
18.4
17.5
17.2
17.3
29.3
31
27.8
28.3
28.0
28.2
27.2
28.6
29.8
28.8
28.1
28.6
17.7
22.7
22.7
20.1
30.8
32.8
32.4
32.0
30.6
31.2
171.0
171.0
21.5
21.1
32
61.2 176.1 21.9
20.5
22.1
35.8 89.1 174.7
AcO "InPynfi^j-DzO/lnPy-^/s/InCDaOD
605
The position of additional double bonds in the side chains can be determined from the carbon resonances of the olefinic carbons. A terminal isopropenyl group in the side chain shows characteristic signals for the olefinic carbons at 6 ca. 123-124 ppm (C-24) and 132 ppm (C-25) as well as for the methyl groups attached to C-25 at 6 ca. 25 ppm (C-26) and 18 ppm (C-27) (glycosides 4e and 9b). The presence of these two methyl vinyl groups is easily confirmed by the downfield shift of the methyl singlets in the ^H-NMR spectrum with respect to the corresponding doublets in a saturated chain. Liouvilloside A (12g), a virucidal trisulfated triterpene glycoside isolated from the Antarctic sea cucumber Staurocucumis liouvillei shows two singlets at 6 1.54 ppm (H26) and 1.64 ppm (H-27), while Liouvilloside B (12c), the saturated analog, shows two nearly overlapped doublets (J = 6.6 Hz) at 5 0.83 and 0.84 ppm [42]. Aglycones with a A^^-double bond (3k, 6 and llf) are characterized by olefinic carbon resonances at 5 ca. 145-150 ppm (C-25, s) and 110-111 ppm (C-26, t). This disubstituted terminal double bond shows a diagnostic multiplet for H-26 at 6 4.75 ppm (2H) and a vinyl methyl signal at 5 1.68 ppm (s, H-27) in the ^H-NMR spectrum [25]. Holothurins that contain a carbonyl group at C-16 (3k and llf) show a ketone carbonyl signal at 5 ca. 213-215 ppm. Aglycones substituted with hydroxyl groups at C-12 and C-17 with a-configurations (5b, 6,16) show a signal at 5 89-90 ppm due to the quaternary C-7. This carbon signal can be readily distinguished from the C-3 signal by a DEPT experiment. Holost-7-ene aglycones containing an acetate group at C-16 (12a, 13c) are characterized by the presence of a singlet at 6 2.0 ppm (CH3CO2) in their ^H-NMR spectra as well as signals at 6 ca. 171 and 21 ppm for the carbonyl and methyl groups of the acetate group. Recently, we have deduced the position of the acetoxyl group at C-16 in Liouvilloside A (12g) from the chemical shift of the H-16 signal (5 5.63 ppm) and its correlation with H-17, H-15a and H-15P in the ^H-^H COSY spectrum. The 16p-configuration was assigned by a NOESY experiment and by coupling constant analysis for the C-16 proton with the C-17a and C-15 protons. Calculated coupling constant values of 8.9 (Ji5a,i6a), 7.4 (yi5p,i6a) and 8.9 Hz (Ji6a,\ia) for the most stable conformation of 16(5acetoxyholosta-7,24-dien-3P-ol obtained by molecular mechanics were coincident with experimental and reported values [40] and differed
606
considerably from those calculated for the 16a-isoiner (4.1, 6.9 and 1.2 Hz, respectively) [42]. C-NMR data for non-holostane triterpenoid aglycones in intact glycosides are shown in Table 2. 1 -2
Table 2. ^^C-NMR chemical shifts of non-holostane aglycones Carbon
17*
18b**
Ds-19a''
20**
21a*'
1
35.0
35.6
36.5
35.6
36.6
2
26.1
26.6
27.2
27.0
27.3
3
87.8
88.7
88.6
89.1
88.9
4
38.4
39.2
39.5
39.4
39.4
5
46.9
47.2
53.0
48.5
6
22.4
23.1
21.4
23.4
53.2 21.6
7
146.5
122.4
28.6
122.5
28.5
8
121.7
147.3
41.5
147.7
41.7
149.0
49.3
149.0
9
45.2
46.3
10
34.8
35.3
40.0
35.7
40.0
11
20.9
21.6
114.0
22.4
115.3
12
19.3
19.9
35.8
33.6
38.1
13
53.9
56.7
46.4
45.1
45.2
14
45.0
46.0
46.8
53.2
47.6
15
43.7
43.6
42.7
33.6
33.9
16 17 18 19 20
78.3 59.4 180.2 23.5 82.9
81.0
75.9
59.1 181.9 23.7 139.9
21 22
23.0 38.7
23.0 113.8
66.0 16.7 22.4 206.5 30.9
22.5 62.1 24.9 24.7 211.5
22.7 53.3 16.6 23.0 74.2 26.3 45.7
23
20.7
22.6
24
37.1
40.1
25
144.5
28.3
26
110.1
22.5
27
21.7
30
16.6
30.7
22.6 17.1
28.2
17.5
17.0
31
28.1
28.5
17.4
29.0
28.2
32
33.0
33.9
19.6
30.5
19.1
CH3CO
169.0
170.2 21.0
21.5 CH3CO "InDMSa<4 *InPy^5-D20(4:l) ' Desdfeted analog of 19a
607
Aglycones with a 7(8)-double bond, as structures 17, 18 and 20, show typical olefinic carbon resonances at 5 146-147 ppm (C-8) and ca. 122 ppm (C-7), while those containing a A^^^^-trisubstituted double bond (19a, 19b, 21a, 21b and 21c) are characterized by signals at 5 149.0 ppm (C-9) and 114-115 ppm (C-11). Psolusoside B (17), the &st reported structure of a non-holostane glycoside with an 18(16)-lactone as well as holothurins 18a-18d show signals at 6 ca. 180-182 ppm (C-18, s) and 7881 ppm (C-16, d). These signals are easily distinguished from the chemical shifts of C-18 (5 ca. 178-180 ppm) and C-20 (5 ca. 83-88 ppm) in a A^-holostane aglycone with an 18(20)-lactone. Characterization of the oligosaccharide chain of hotothurins requires: a) the identification of each monosaccharide and its anomeric configuration in the oligosaccharide chain, b) the interglycosidic linkages and the sequence, c) the position of sulfate groups, and finally the site of attachment of the oligosaccharide chain to the triterpene aglycone. The identification of the monosaccharide composition is easily accomplished by acid hydrolysis of the intact holothurin, derivatization of each monosaccharide and fiirther analysis by GC and comparison with standards [42]. FAB-MS is a very usefixl technique for the determination of the sequence of monosaccharides in the carbohydrate chain of a glycoside. As shown in Fig. (24) for Liouvilloside A (12g), cleavages can occur on both sides of the glycosidic linkages with proton transfer. These cleavages give characteristic fragments that correspond to the sequential losses of each monosaccharide. In sulfated holothurins, fragment ion peaks due to the loss of SOsNa are diagnostic for the presence of sulfate groups in the glycosides. For example, Liouvilloside A (12g) showed fragment ion peaks at m/z 1355 [M - SOsNa + H + Na]^ 1253 [M 2S03Na + H + Nal^and 1151 [M - 3S03Na + 3H + Na]^ corresponding to the sequential losses of three sulfate groups. Although FAB-MS gives information on the sequence of monosaccharides in the oligosaccharide moiety, it is not possible to determine the location of the interglycosidic linkages by this method. NMR has been the method of choice for complete characterization of the oligosaccharide chain.
608
• 1179(+H+Na)
945(-Hf>fe) ^534(4Na)
'915(+HfNa) -898(+Na) -768(+Na)
i-518(4Na)
3-O-Me-Glc- -o- -GlcH-O- -Qd|--o4~Xyl4"0--|-Aglycon » ' I * I \ .SOsNa OSOsNa i OSOsNa ' —••lieiC-HfNa)
-752(+Na)
Fig. (24). Positive FAB-MSfragpaentationof Liouvilloside A
The ^H-NMR spectra of holothurins show complex and overlapping signals for hydroxymethine and hydroxymethylene protons of sugar residues in the downfield region at 8 3.0-5.0 ppm. * Nevertheless, the anomeric signals usually appear as almost separated doublets at 6 4.7-5.3 ppm in C5D5N:D20 (5:1) with Vi,2 of ca.7.7 Hz and are indicative of the P-anomers of the pyranose sugars with gluco and galacto configurations [72]. Another characteristic signal in the ^H-NMR spectra of holothurins is the methyl doublet resonance of the 6-deoxyhexose quinovose at 6 ca. 1.6 ppm in CsDsNrDaO (5:1). The resonance of the methyl carbon of quinovose is observed at 5 ca. 18 ppm. Glycosides containing a methoxyl group attached to C-3 of a glucose or a xylose unit show a typical singlet at 5 3.6 ppm in their ^H-NMR spectra as well as the corresponding carbon resonance at 5 ca. 60 ppm. Comparison of C-NMR data of the oligosaccharide chain of holothurins with those of reference methyl glycosides has been the method of choice to determine the interglycosidic linkages [73]. Terminal sugar residues exhibit remarkable resemblance of their C-NMR data with those of their respective methyl glycosides. Internal sugar moieties show deviations in their carbon resonances with respect to the corresponding methyl glycosides due to glycosidation. Measurement of the relaxation times (Ti) of the sugar units aided in some cases in the assignment of carbon resonances of the oligosaccharide chain [28, 60]. Permethylation of the intact non sulfated glycosides or the desulfated derivatives of sulfeted holothurins followed by GC-MS analysis of the
609
partially methylated alditol acetates [21, 28, 31] has also been used in order to determine the interglycosidic linkages. Table 3 shows the ^^C-NMR data for the sugar units of sea cucumber glycosides containing different pentasaccharide chains. Table 3. "C-NMR data for the sugar moieties of holotliurins with pentaglycosidic chains Carbon
12a*
Sc**
9a^
Des-lSb**'
r
104.5
103.5
105.7
104.6
81.6
83.3
81.7
83.0
2' 3'
75.3
75.7
75.3
76.9
4'
76.3
77.9
75.8
69.5
5'
64.2
64.1
64.2
65.9
1"
102.2
105.5
102.2
103.0
2"
82.6
75.8
83.9
83.4
3"
75.2
76.3
74.7
4"
85.3
87.1
75.1 85.5
5"
71.2
87.2 71.7
70.6
71.0
6"
18.0
18.0
17.5
17.9
1'"
104.7
104.9
104.3
103.9
2'"
73.6
73.7
73.4
73.5
3'"
86.2
87.8
86.4
86.5
4'"
68.9
69.8
69.7
69.0
5'"
66.0
77.3
74.9
77.1
62.1
67.6
61.5
6'" 1"" 2"" 3"" 4"" 5"" 6"" OMe
104.5 74.7 87.0 70.6 77.6 61.9 60.9
105.6 74.9 87.7 70.5 78.3
104.9 74.7 87.5 78.0 70.4
105.1 74.1 86.6 69.6 66.3
62.5 60.7
61.8 60.7
60.6
1'""
105.4
105.3
105.9
105.2
05SJ99
74.9
74.3
75.2
75.8
3'""
76.6
78.7
76.6
76.2
4»9'"
70.2
71.7
69.7
75.6
C9J»>9
66.6
78.1
64.2
73.4
6"'^^
62.2
17.9
•lnPy^3-D20(5:l) **InPy^5 ' Py-rfj-DzO (8:2) *toesulfeted analog of 18b "InPy^5P20(4:l)
610
One common structural feature is the presence of a xylose unit attached to C-3 of the aglycone and substituted at C-2' with a quinovose unit. As shown in Table 2, carbons involved in the interglycosidic linkages show chemical shifts at 6 ca. 82-88 ppm, shifted downfield from those expected for the corresponding methyl glycopyranosides. Glycosides containing a terminal glucose (12a, 8c, 9a) or xylose (Des18b) substituted with a methoxyl group at C-3 show an additional signal at 5 ca. 86-88 ppm due to the substitution at this carbon. Frondosides A (12a) and B (9a) and the desulfated derivative of Calcigeroside B (Des18b) present a branched 2,4-disubstituted quinovose residue with a xylose unit attached to C-2" in 9a and 12a and a quinovose unit in Des-18b. This substitution pattern is deduced from the downfield shifts of C-2" and C-4" of the 2,4-disubstituted quinovose in comparison with those carbon resonances in a terminal quinovose vmit (Des-18b). On the other hand, Holothurinoside A (8c) with a 2,4-disubstituted xylose unit attached at C-3 of the aglycone shows signals at 6 83.3 ppm (C-2') and 77.9 ppm (C-4') for the carbon atoms involved in the glycosidic bonds. As shown in Table 3, due to the proximity of carbon resonances of the different sugar units in the oligosaccharide chain, it is diflBcult to assign unambiguously each signal on the only basis of comparison with published data, sometimes performed in different solvents or solvent mixtures. Recent application of two-dimensional NMR techniques (^H-^H COSY, relay COSY, HETCOR, COLOC, HMBC and HMQC) to the structural elucidation of holothurins [32, 35, 39, 40, 48, 52, 57] has allowed the unambiguous assignment of all ^H and ^^C resonances of the oligosaccharide chain. The NOESY spectrum of Patagonicoside A clearly showed the correlations between the protons (Fig. (23)) of the oligosaccharide chain. These correlations confirmed the iaterglycosidic linkages deduced previouslyfi-omanalysis of ^H-^H COSY and HETCOR spectra as well as the site of attachment of the sul&ted xylose unit to the C-3 of the aglycone [53].
611
Fig. (23). NOESY oorrelatiocis of the oligosaccharide moiety ofpatagonicoide A
Another common structural feature in holothurins is the presence of one, two or three sulfate units attached to the sugar residues of the oligosaccharide chain. The location of these groups has been determined by comparison of ^^C-NMR data of the native glycosides and the corresponding desulfated derivatives. Desulfetion of the native holothurins is easily achieved by hydrolysis in a mixture of pyridine and dioxane at 120X and further purification of the desulfated derivatives by HPLC [53]. Those holothurins containing an acetoxyl group at C-16, as Liouvilloside A (12g), are desulfeted by acid hydrolysis in HCl-MeOH in order to prevent hydrolysis of the acetate group [42]. Table 4 shows the ^H- and ^^C-NMR data for two glycosides, Patagonicoside A (16) and Hemoiedemoside A (3k), containing the same disulfeted tetrasaccharide chain and the trisulfated Liouvilloside A (12g), that differsfi^om16 and 3k in the presence of an additional sulfete group at C-6 of the terminal 3-0-Me-glucose unit. The three glycosides differ in their aglycone structures. As observed in Table 4 the esterified carbons with a sulfete group show downfield shifts of ca, 4-6 ppm with respect to their desulfeted derivatives, while upfield shifts of ca. 2-3 ppm are observed for the vicinal carbons. The chemical shifts of these carbons vary with the solvent used for performing the spectra. For example, the xylose unit with a sulfate group at C-4', common to all sulfeted holothurins, shows a characteristic signal for C-4' at 5 77.1 ppm in CD3OD, while the same carbon resonance is shifted downfield to 5 75.8 and 74.4 ppm in CsDsN.DaO (5:1) and DMSO-e/6, respectively. Sulfate groups at C'6 of glucose or a 3-0-Me-glucose residue show typical signals for C-6 at 5 65.6-68.5 ppm in these deuterated solvents.
612
Table 4. *H- and "C-IVMR data for the Sugar Moieties of Patagonicoside A (16), Hemoiedemoside A (3k) and LiouviUoside A (12g).
16
c r T 3' 4' 5'
3k
12g
Sc*-'
SH'(«/mHz)
5c ^'^
5H"(./inHz)
Sc^-^
SH'(>/mHz)
105.6 82.7
4.46 d (7.9) 3.55 m 3.78 m 4.23 m 3.37 m;
104.9 82.4
4.69 d (7.1) 3.69 m 4.27 dd (9, 8.7) 5,11m 3.72 m
104.2 82.0 14.1 (+2) 74.4 (-4.8) 63.2 (+2.3)
4.32 d (7.3) 3.36 m 3.54 m 3.97 m 3.19 m
103.8 74.9 74.2 86.2
4.49 d (8) 3.10 3.32 m 3.03
70.4 17.4
3.34 m 1.25 d (5.3)
103.1 72.6 85.9
4.40 d (7.8) 3.25 m 3.49 m
68.7 74.2 (+2.1) 65.9 (-4.9)
3.20 m 3.53 m 3.78 m, 4.04 dd
75.1 (+2.3) 77.1 (-6) 63.8 (+2.6)
74.8 (+2.6) 75.8 (-5.5) 63.9 (+2.3)
4.17 m
1" 2" 3" 4"
104.8 76.3 75.6
5" 6"
115
r"
4.75 m 4.92d/7.7)
4.61 d (7.6) 3.36 m 3.55 m 3.23 m
104.6 75.5 75.6 87.8
18.0
3.49 m 1.35 d (6.1)
71.3 17.8
3.88 dd 3.97 m 3.44 dd (8.7, 8.9) 3.66 m 1.63 d (6.1)
873
104.8
4.45 (6.9)
104.6
4.76 d (7.7)
ij'i'tt
74.3
3'"
87A
74.3 86.5
4»»»
10.2 75.2 (+2.7) 68.5 (-6.1)
3.41m 3.60 m 3.42 m 3.69 m 4.12 m;
3.95 4.25 3.79 4.21 4.68 m, 5.14 dd (2,10.7)
5'" 6'"
69.9 74.7 (+2.7) 67.5 (-5.7)
4.38 m 1»»9
105.2 75.4 87.6 71.1
4.57 d (7.9) 3.31m 3.10 m 3.34 m
104.9 74.5 87.4 70.3
78.1 62.5
OCH3
61.1
3.32 m 3.65 m; 3.85 bd (10.5) 3.62 s
113
6""
2"" 3"" 4"" «9»»
61.8 60.6
5.29 d (7.8) 3.96 m 3.71m 4.02 dd (8.9, 9.3) 3.95 m 4.19 m, 4.43 dd (2,11.9) 3.85 s
(18,10) 103.8 73.6 85.8 69.3
4.47 d (7.9) 3.15 m 3.00
75.1 (+1.8) 65.6 (-4.6)
3.34 m 3.83m,4.04dd (18,10) 3.49 s
60.1
3.21m
' Reoofxied at 125 MHz in Me^hanoW4; ^ Italics = interglycx)sidic positions, bold = sul&te positions; (Ac = 6c - ScdesDtfated andog); *" Rfiootded at 500 MHz in Metbanol-
The determination of the position of sulfate groups in holothurins is important in order to establish structure-activity correlations. Recently, we have evaluated the antifungal activity of di- and trisulfated glycosides and their semi-synthetic desulfated analogs against the phytopathogenic fimgus Cladosporium cucumerinum [25, 53]. We have found that Hemoiedemosides A (3k) and B (31) and Patagonicoside A (16) wrere more active than their desulfated analogs. On comparing the antifungal
613
activities of the disulfated glycosides 3k and 16, Hemoiedemoside A resulted more active. Both glycosides present the same oligosaccharide chain and differ in the aglycone structure. On the other hand, Hemoiedemoside B (31) differing from 3k in the presence of a third sulfate group at C-6 of the terminal 3-0-methylglucose residue is less active than 31. These results suggest that both the aglycone structure and the presence and number of sulfete groups at the oligosaccharide chain play an important role in the antifungal activity of holothurins. ACKNOWLEDGEMENTS The authors gratefiilly acknowledge grants from the International Foundation for Science, Stockhohn, Sweden, the Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, CONICET, ANPCyT and the Universidad de Buenos Aires. H. D. Chludil thanks FOMEC-UBA for a fellowship. M. S. M. and A. M. S. are Research Members of the National Research Council of Argentina (CONICET).
REFERENCES [1]
Stonik, V.A.; Elyakov, G.B. In Bioorganic Marine Chemistry; Scheuer P.J., Ed.; Springer-Verlag: Berlin, 1988; Vol. 2, pp. 43-86. [2] Minale, L.; Riccio, R.; ZoUo, F. In Progress in the Chemistry of Organic Natural Products; Herz, W.; Kirby, G.W.; MOOTC, R.E.; Steglich, W.; Tamm CR, Eds.; Springer-Verlag: New York, 1993; Vol. 62, pp. 65-308. [3] Minale, L.; Riccio, R.; Zollo, F. In Bioactive Natural Products - Studies in Natural Products Chemistry; Atta-iff-Rahman., Ed.; Elsevier Science Publishers: Amsterdam, 1995; Vol. 75, pp. 43-110. [4] D'Auria, M.V.; Minale, L.; Riccio, R.; Chem. Rev., 1993, 93, 1839-1895. [5] lorizzi, M.; De Marino, S.; Zollo, F.; Curr. Org Chem., 2001, 5, 951-973. [6] Verbist, J.F. In Echinoderm Studies; Jangoux, M.; Lawrence, J.M., Eds.; A.A. Balkema: Rotterdam, 1993; Vol. 4, pp. 111-186. [7] Chludil KD.; Seldes, A.M.; Maier, M.S. In Research Advances in Lipids; Mohan, R.M., Ed.; Global Research Network: India, 2002; in press. [8] Roccatagliata, A.J.; Maier, M.S.; Seldes, A.M.; Pujol, C.A.; Damonte, E.B.; J. Nat Prod., 1996, 59, 887-889. [9] Roccatagliata, A.J.; Maier, M.S.; Seldes, A.M.; J. Nat. Prod., 1998, 61, 370-374. [10] Riccio, R.; D'Auria, M.V.; Minale, L.; J. Org Chem., 1986, 51, 533-536.
614
[11]
[12]
[13] [14] [15] [16]
[17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]
Kalinin, V.I.; Avilov, S.A.; Stonik, V.A. In Saponins in Food, Feedstuffs and Medicinal Plants; Oleszek, W.; Marston, A., Eds.; Kluwer Academic Publishers: The Netherlands, 2000; pp. 155-162. Kalinin, V.I.; Anisimov, M M ; Prokofieva, N.G.; Avilov, S.A.; Afiyatullov, Sh.Sh.; Stonik, V.A. In Echinoderm Studies; Jangoux, M ; Lawrence, J.M, Eds.; A.A. Balkema: Rotterdam, 1996; Vol. 5, pp. 139-181. Kalinin, V.I.; J, theor, Biol, 2000, 206, 151-168. Stonik, V.A.; Kalinin, V.I.; Avilov, S.A.; J. Nat. Toxins, 1999, S, 235-248. Kobayashi, M.; Okamoto, Y.; Kitagawa, I.; Chem. Pharm. Bull, 1991, 39, 28672877. Kalinin, V.I.; Volkova, O.V.; Likhatskaya, G.N.; Prokofieva, N.G.; Agafonova, I.G.; Anisimov, M M ; Kalinovsky, A.I.; Avilov, S.A.; Stonik, V.A.; J. Nat Toxins, 1992,1, 31-37. Makarieva, T.N.; Stonik, V.A.; Kapustina, I.I.; Boguslavsky, V.M; Dmitrenoik, A.S.; Kalinin, V.I.; Cordeiro, ML.; Djerassi, C ; Steroids, 1993, 58, 508-517. Kitagawa, I.; Kobayashi, M ; Hori, M.; Kyogoku, Y.; Chem. Pharm. Bull. 1989, 37, 61-67. Miyamoto, T.; Togawa, K.; Higuchi, R.; Komori, T.; Sasaki, T.; J. Nat. Prod, 1992; 55, 940-946. Maltsev, I.I. ; Stonik, V.A.; Kalinovsky, A.I.; Elyakov, G.B.; Comp. Biochem. Physiol. 1984, 78B,A2\. Kitagawa, I.; Yamanaka, H ; Kobayashi, M ; Nishino, T.; Yosioka, I.; Sugawara, T.; Chem. Pharm. Bull. 1978, 26, 3722-3731. Zurita, M.B.; Ahond, A.; Poupat, C ; Potier, P; Menou, J.L.; J. Nat. Prod, 1986; 49, 809-813. Kalinin, V.L.; Kalinovsky, A.I.; Stonik, V.A.; Khim. Prirodn. Soedin, 1985, 2, 212-217. Avilov, S.A.; Stonik, V.A.; Khim. Prirodn. Soedin, 1988, 5, 764-766. Chludil, H.D.; Muniain, C.C; Seldes, A M ; Maier, M.S.; J. Nat. Prod., 2002; in press. Kalinin, V.I.; Malutin, A.N.; Stonik, V.A.; Khim. Prirodn. Soedin, 1986, 3, 378379. Kitagawa, L; Kobayashi, M ; Son, B.W.; Suzuki, S.; Kyogoku, Y.; Chem. Pharm. Bull. 1989, 37, 1230-1234. Kitagawa, I.; Kobayashi, M.; Inamoto, T.; Fuchida, M ; Kyogoku, Y.; Chem. Pharm. Bull. 1985, 33, 5214-5224. Bhatnagar, S.; Dudouet, B.; Ahond, A ; Poupat, C ; Thoison, O.; Clastres, A ; Laurent, D.; Potier, P.; Bull. Soc. Chim. France, 1985,1, 124-129. Oleynikova, G.; Kuznetsova, T.; Ivanova, N.; Kalinovsky, A.; Rovnikh, N.; Elyakov, G.; Khim. Prirodn. Soedin, 1982, 4,464. Kitagawa, I.; Kobayashi, M ; Kyc^oku, Y.; Chem. Pharm. Bull. 1982, 30, 20452050. Rodriguez, J.; Castro, R ; Riguera, R ; Tetrahedron, 1991, 47,4753-4762. Encamacion, R ; Murillo, J.; Nielsen, J.; Christophersen, C ; Acta Chem. Scand. 1996, 50, 848-849.
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[34] [35] [36] [37]
[38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54]
Kitagawa, I.; Nishino, T; Kobayashi, M; Kyc^oku, Y.; Chem. Pharm. Bull 1981,29,1951-1956. Findlay, J.A.; Yayley, M; Radios, L.; J. Nat Prod, 1992, 55, 93-101. EJrozdova, O..A.; Avilov, S.A.; Kalinovsky, A.I.; Stonik, V.A.; Mlgrom, Y.; Rashkes Y.; Khim. Prirodn, Soedin, 1993, 5, 369-31A, a) Kitagawa, I.; Kobayashi, M.; Inamoto, T.; Yasuzawa, T.; Kyogoku; Y., Chem. Pharm, Bull. 1981, 29, 2387-2391. b) Stonik, V.A; Maltsev, I.I.; Kalinovsky, A.I.; Elyakov, G.B.; Khim. Prirodn. Soedin, 1982,2, 200-204. c) Sharipov, V.F.; Chumak, A.D.; Stonik, V.A.; Elyakov, G.B.; Khim. Prirodn. Soedin, 1981, 2, 181-184. d) Stonik, V.A.; Maltsev, I.I. ; Kalinovsky, A.I.; Konde, K.; Elyakov, G.B.; Khim. Prirodn. Soedin, 1981, 2, 194-199. Drozdova, O..A.; Avilov, S.A.; Kalinovsky, A.I.; Stonik, V.A.; Mlgrom, Y.; Rashkes Y.; Khim. Prirodn. Soedin, 1993,2, 242-248. Drozdova, O.A.; Avilov, S.A.; Kalinin, V.I.; Kalinovsky, A.L; Stonik, V.A.; Riguera, R; Jimenez, C; Liebigs Ann./Recueil 1997, 2351-2356. Girard, M.; Bflanger, J.; ApSimon, J.; Gameau, F.; Harvey, C; Brisson, J.; Can. J. Chem.; 1990, 68, 11-18. Avilov, S.A.; Kalinin, V.I.; I>rozdova, O.A.; Kalinovsky, A.I.; Stonik, V.A.; Gudimova, E.N.; Chem. Nat. Comp., 1993,29,216-218. Maier, MS.; Roccatagliata, A.J.; Kuriss, A.; Chludil, HD.; Seldes, A.M.; Pujol, C.A.; Damonte, E.B.; J. Nat. Prod., 2001, 64^ 732-736. Avilov, S.A.; Kalinovsky, A.I.; Stonik, V.A.; Chem. Nat. Comp. 1990,26,42-45. Afiyatullov, S.S.; Tishchenko, L.Y.; Stonik, V.A.; Kalinovsky, A.I.; Elyakov, G.B.; Khim. Prirodn. Soedin, 1985, 2, 244-248. Afiyatullov, Sh.Sh.; Kalinovsky, A.I.; Stonik, V.A.; Khim. Prirodn. Soedin, 1987, 6, 831-837. Kalinin, V.I.; Afiyatullov, Sh.Sh.; Kalinovsky, A.I.; Khim. Prirodn. Soedin, 1988, 2,221-226. Kalinin, V.I.; Avilov, S.A.; Kalinovsky, A.I.; Stonik, V.A.; Mlgrom, Y.M. Rashkes, Y.N.; Khim. Prirodn. Soedin, 1992, 6, 691-694.. Kalinin, V.I.; Avilov, S.A.; Kalinina, E.Y; Korolkova, O.G.; Kalinovsky, A.I. Stonik, V.A.; Riguera, R.; Jimenez, C; J. Nat. Prod., 1997, 60, 817-819. Avilov, S.A.; Antonov, A.S.; I>rozdova, O.A.; Kalinin, V.I.; Kalinovsl^^, A.I. Riguera, R; Lenis, L.A.; Jimenez, C; J. Nat. Prod., 2000, 63, 1349-1355. Rodriguez, J.; Riguera, R; J. Chem. Res., 1989, 2620-2636. Myamoto, T.; Togawa, K.; Hguchi, R; Komori, T.; Sasaki, T.; Liebigs Ann. Chem., 1990,453-460. Avilov, S.A.; Antonov, A.S.; Drozdova, O.A.; Kalinin, V.I.; Kalinovsky, A.L; Stonik, V.A.; Riguera, R; Lenis, L.A.; Jimenez, C; J. Nat. Prod, 2000, 63, 6571. Murray, A.R; Muniain, C.C; Seldes, A.M.; Maier, M.S.; Tetrahedron, 2001,57, 9563-9568. Kalinin, V.I.; Kalinovsky, A.I.; Stonik, V.A.; Dmitrenok, RS.; Elkin, I.N.; Chem. Nat. Comp., 1989, 25, 311-317.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 28 © 2003 Elsevier Science B.V. All rights reserved.
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SULFUR-CONTAINING NATURAL PRODUCTS FROM MARINE INVERTEBRATES MICHELER.PRINSEP Department of Chemistry, University ofWaikato, Private Bag 3105, Hamilton, New Zealand. ABSTRACT: An overview of sulfur-containing natural products isolated from the marine invertebrate phyla that are commonly studied by natural products chemists, is provided. The material is arranged by phyla and sulfated compounds are included, except for the Echinodermata where sulfated saponins and steroids are specifically excluded. A total of 638 compounds and 530 references are recorded. The review covers the published literature up until the end of 2001. References to reported syntheses and comments on biological activities of metabolites are included.
INTRODUCTION Marine invertebrates such as sponges, bryozoans and tunicates are well known sources of a wide variety of secondary metabolites or natural products. Many of these compounds possess novel structures with unprecedented ring systems or unusual combinations of functional groups. There is a high incidence of biological activity among the secondary metabolites of marine invertebrates, which is not surprising, given the environment that the organisms live in. Marine invertebrates are either completely sessile, or if able to move, such as is the case for example, for nudibranchs (sea slugs) and ophistobranchs (sea hares), they do so only very slowly. Most marine invertebrates lack physical protection in the form of spines, a sting or a shell, so would seem an ideal meal for a passing predator. However, these invertebrates flourish and the fact that they do so, is thought to be due to chemical protection from predation, overgrowth and infection that their secondary metabolites convey. For this reason, many research groups worldwide investigate the natural products of marine invertebrates, primarily concentrating on their biological activity. A myriad of structural types are found amongst the secondary metabolites of marine invertebrates. Halogenation is typical of marine natural products, which is not unexpected, given that seawater contains
618
very high concentrations of chloride, bromide and iodide ions (559 mM, 0.86 niM and 0.45 |iM respectively) [1]. Sulfur is the fourth most common element in seawater after chlorine, sodium and magnesium and the sulfate anion is the second most abundant after chloride [2]. There are many excellent reviews available on marine natural products in general. The annual reviews by Faulkner [3-20] cover all secondary metabolites from the major marine phyla. Very few reviews however, deal solely with sulfur-containing marine metabolites. The first of these to be published arranged compounds according to structural types [21]. Another review covers sulfated marine compounds only and these are arranged by phylum [2]. The most recent review deals with marine sulfur-containing natural products excluding sulfates and metabolites are organised according to the sulfur functional groups that they contain [22]. This review will discuss all sulfur-containing natural products from the main marine invertebrate phyla studied by natural product chemists: Bryozoa, Chordata, Cnidaria, Mollusca, Porifera and a selection of those from the Echinodermata. The vast majority of sulfurcontaining metabolites that have been isolated from echinoderms are sterol sulfates and saponins and this review will not include coverage of these but will only deal with other types of sulfur-containing metabolites from echinoderms. For discussions of the sterol sulfates and saponins of echinoderms, the reader is referred to other sources [2,3-20]. The current review concentrates on isolation and biological activity of sulfurcontaining metabolites and covers the literature up to the end of 2001. As pointed out by Christophersen [21], the distinction between primary and secondary metabolites is somewhat blurred, therefore, some compounds that could perhaps be classified most readily as primary metabolites are included, but macromolecules are specifically excluded. Every endeavour has been made to be comprehensive, but inevitably some metabolites may have been overlooked. Metabolites are organised by phylum and within phyla, compounds are arranged in families, or by structural type.
Bryozoans A bryozoan or moss animal is a sedentary colony of minute filterfeeding individuals called zooids [23]. They are widely distributed
619
throughout the marine environment. There are about 4,000 living species and over 10,000 as preserved fossils. Apart from a very fev^ freshw^ater species, all living bryozoans are marine dwelling [24]. The zooids that comprise a bryozoan colony each consist of a body wall (the box or tube) and a polypide. The polypide is made up of a simple U-shaped gut and an apparatus of tentacles called a lophophore [25]. The inner faces of the tentacles are covered with cilia that beat to generate a current of water towards the mouth. The supportive outer body wall is often reinforced by either chitin or calcium carbonate or both. Zooids can be specialised and take on specific roles within a colony, such as feeding, brood or funicular (transport or communication) zooids [25]. Contact with the bryozoan Alcyonidium gelatinosum gives rise to "Dogger Bank itch", an allergic contact dermatitis. The causative agent is (2-hydroxyethyl)dimethylsulfoxonium ion (1). Synthesis of 1 was achieved by base-catalysed condensation of trimethylsulfoxonium chloride and formaldehyde [26].
OH X"
The sulfur-containing p-carboline alkaloid, l-ethyl-4-methylsulfone-p-carboline (2) was isolated from the New Zealand bryozoan Cribricellina cribraria, along with several other p-carboline alkaloids. l-Ethyl-4-methyl-sulfone-P-carboline (2) exhibited only modest antimicrobial activity, especially compared to some of the other alkaloids isolated, which were both cytotoxic and antimicrobial [27].
Two sulfur-containing isoquinoline alkaloids, 2-methyl-6,7di(methylthio)-2//-isoquinoline-3,5,8-trione (3) and 2-methyl-6methylthio-2H-isoquinoline-3,5,8-trione (4) were isolated from a
620
Tasmanian collection of the Australian bryozoan Biflustra perfragilis. The crystal structure of compound 3 was determined and it exhibited activity in the brine shrimp assay and against cultures of marine bacteria but was inactive against human bacterial pathogens [28]. Simultaneously, 7-amino-2-methyl-6-methylthio-"2//-isoquinoline-3,5,8trione (5) and compound 3 were reported from a South Australian collection of the same bryozoan by another research group and named perfragilins A and B respectively. The bryozoan was referred to as Membranipora perfragilis but is in fact the same species as B. perfragilis [28]. Both perfragilins showed activity against the P388 murine leukaemia cell line, with perfragilin B (3) being about one order of magnitude more active than perfragilin A (5) [29]. Single crystal Xray structures of perfragilins A (5) and B (3) were determined [30] and perfragilin B (3) was later synthesised [31]. These compounds are all structurally very similar to mimosamycin, which has been isolated from a terrestrial bacterium and from two marine sponges [28]. This suggests that the perfragilins are of bacterial origin.
Me-S
The Japanese fouling bryozoan, Dakaira subovoidea was the source of two 6//-anthra[l,9-Z?c]thiophene derivatives (6) and (7). The structure of compound 6 was determined by X-ray crystallography [32]. Both compounds were found to act as antioxidants and were patented for use as hypolipemics, but no other biological activity was reported [33]. Compound 7 has also been synthesised [34]. Our own studies of the bryozoan Watersipora subtorquata [35] led to the isolation of the closely related anthraquinone 5,7-dihydroxy-6-oxo-6//-anthra[l,9-Z?c] thiophene-l-carboxyhc acid (8), which was identified by nuclear magnetic resonance (NMR) and mass spectral analysis [36]. Compound 8 exhibited significant cytotoxicity against the African green monkey kidney cell line, BSC-1 [36].
621
6 R = CH2OH 7 R = C02Me 8 R = CO2H
Two ceramide 1-sulfates (9-10) have been obtained from the Japanese bryozoan Watersipora cucullata. These compounds are potent deoxyribonucleic acid (DNA) topoisomerase I inhibitors [37].
HO3SO,
C9H19 HO3SO.
C7H 7"15
Three new alkaloids, euthyroideones A, B and C (11-13) were isolated from the New Zealand bryozoan Euthyroides episcopalis [38]. All three compounds contain the unique heterocyclic pyrido(4,3-h)-l,4benzothiazine skeleton. The structure of euthyroideone A (11) was determined by X-ray crystallography, NMR spectroscopy and mass spectrometry. Euthyroideone B (12) exhibited modest cytotoxicity against the BSC-1 cell line [38].
11
12
13
Tunicates (Ascidians) Tunicates or ascidians belong to the phylum Chordata. Ascidiacea is a class of the subphylum Urochordata (Tunicata) and members of this
622
class are often referred to as tunicates or sea squirts, because their body is covered with a sack or tunic and many species expel water through a siphon when disturbed [39]. There are approximately 2,000 living species of tunicate and of these, ascidians are the most abundant. They may be solitary or colonial and are sessile, filter-feeding organisms [39]. Biologically active metabolites are quite conmionly found in ascidians and many of these compounds are derived from amino acids. Ascidiacyclamide (14), a cytotoxic, cyclic peptide, was isolated from an unidentified species of ascidian [40]. The absolute configuration was determined and the structure confirmed by total synthesis [41]. An Xray crystal structure was carried out [42] and further X-ray crystallographic studies determined the conformation of the molecule in the solid- and solution-states [43].
VN H
V
o=< \
y/^
NH
HN
\
14
Lissoclinum species of tunicates produce a range of cyclic peptides, which contain thiazole, thiazoline and oxazoline rings. Most belong to the general families patellamides (octapeptides), lissoclinamides (heptapeptides) or bistratamides (hexapeptides) [22]. The heptapeptide ulicyclamide (15) and the octapeptide ulithiacyclamide (16) were the first representatives of a series of cyclic peptides to be isolated from Lissoclinum patella. Their structures were elucidated by interpretation of spectral data [44]. A revised structure was later put forward for ulicyclamide (15) as a result of a detailed analysis of the fast atom bombardment (FAB) mass spectrum. The same paper reported the isolation of two more polar cyclic peptides and another, which was present as a minor component. These heptapeptides were called lissoclinamides 1-3 (17-19) [45]. An unidentified tunicate from the Great Barrier Reef contained ulithiacyclamide (16) and ascidiacyclamide (14) [46]. Two syntheses of ulithiacyclamide (16)
623
have been reported [47,48]. Ulicyclamide (15) was later also synthesised in high yield by solid phase synthesis [49]. The conformational properties of ulithiacyclamide (16) were probed using NMR spectroscopy and molecular mechanics calculations [50]. Ulithiacyclamide B (20) was isolated from L. patella from Pohnpei and was cytotoxic against the human oral carcinoma cell line, KB [51]. o
R
'•\. K'\ O
Ph O 15
\ ^
O
16 R = CH2CHMe2 20 R = CH2Ph
Y
^
17
18 Rj = Me, R2 = H 19Ri=H,R2 = Me
Three cyclic octapeptides, patellamides A-C (21-23) were isolated from L. patella and cytotoxicity data for these compounds and for ulicyclamide (15) and ulithiacyclamide (16) against L1210 murine leukaemia cells and the human acute lymphoblastic leukaemia (ALL) cell line CEM were reported [52]. The structures of the patellamides were later reassigned on the basis of synthetic studies. The proposed structures of patellamides B (22) and C (23) were synthesised and the products were shown to differ from the natural products. This led to new structures being proposed [53,54]. Separate syntheses of
624
patellamide A (21) [55], patellamide B (22) [56] and of patellamides B and C (22-23) [57] produced compounds that were identical to the natural products.
yr^
oK
21
o=(
y-T
22 R = CH2CHMe2 23 R = CH(Me)Et
The spectral data of the patellamides was also reasssigned and the new assignments used for elucidation of the structures of three new metabolites, the octapeptide prepatellamide B formate (24) and the heptapeptides, prelissoclinamide 2 (25), and preulicyclamide (26) [58]. The molecular conformation of patellamide A (21) was determined by X-ray crystallography [59,60] and the solution conformations of patellamides B (22) and C (23) were determined by NMR spectroscopy and molecular dynamics [61]. The octapeptide preulithiacyclamide (27) is a potent inhibitor of Macrophage Scavenger Receptor and was isolated from L. patella from Palau along with other known cyclic peptides [62].
\=M
H
^U
>=0
o\"r<:/25
625
HO-'Z \ (Qy-NH
HN H
"
'
HN f
"^
S
N .NH
"^ H " N ' ^ O
PK 26
27
Patellazoles A-C (28-30) are further cytotoxic, thiazole-containing macrolides isolated from L. patella [63,64]. Structural elucidation of patellazoles B and C (29-30) relied on interpretation of data that did not permit stereochemical assignments.
2o R} — H, R.2 — H 29 Ri = H, R2 = OH 30 Ri = OH, R2 = OH
Three cytotoxic peptides, patellamide D (31) and hssoclinamides 4-5 (32-33) were isolated from a Great Barrier Reef specimen of L. patella and identified by interpretation of spectral data. The peptides were found within the obligate algal symbiont of the genus Prochloron [65]. Another study of the same Australian L. patella reported lissoclinamide 6 (34), in addition to Hssoclinamides 4-5 (32-33) and patellamide D (31). The structure of patellamide D (31) was obtained by X-ray crystallography and its conformation compared with those obtained by molecular modelling [66]. Patellamide D (31) has been reported to be a
626
resistance-modifying agent in the multidrug resistant CEM/VLBIOO human leukaemic cell line [67]. The structure of lissoclinamide 5 (33) was revised as a result of total synthesis [68] and a total synthesis of lissoclinamide 4 (32) was described [69]. Later, total syntheses of lissoclinamides 4 (32) and 5 (33) allowed the revision of their configurations [70].
^6^^.\ ^ a \
^'^\\ P h / s ^ - /
32 X = thiazoline
31
^P^
34
33 X = thiazole
Lissoclinamides 7 (35) and 8 (36) were isolated from a Great Barrier Reef specimen of L. patella. Structure-activity studies showed that lissoclinamide 7 (35) with two thiazoline rings, was the most effective in vitro cytotoxin of those tested [71]. The stereochemistry of lissoclinamide 7 (35) was determined by synthesis using Burgess reagent to form oxazoline and thiazoline rings without scrambling adjacent chiral centres [72]. Lissoclinamides 9 (37) and 10 (38) were isolated from an Indonesian L. patella sample [73] while prepatellamide A (39) was isolated as a minor component of another Indonesian specimen of L. patella [74].
: o Y f^N^O
^
HN
JL N /
O-l""
VN
HN
ph-^ 35 X = thiazoline 36 X = thiazole
"^^Q
H
37
°
/
627
38
39
A further cytotoxic, octapeptide, patellamide E (40), was isolated from L patella from Singapore and the structure was elucidated by chemical and spectral methods [75]. Patellamide F (41) was isolated from L patella from north-western Australia and was also cytotoxic. The structure and absolute stereochemistry of patellamide F (41) were established by chemical and spectroscopic methods. Patellamide B (22), ulithiacyclamide (16) and lissoclinamide 3 (19) were also isolated from the same sample [76]. The octapeptides, patellamide G (42) and ulithiacyclamides E-G (43-45) were isolated from L. patella from Pohnpei, along with known series members [77].
I
•^
NH
f~f
O N ^
OH O
HN
H VP
40 Rj = CH(Me)Et, R2 = Me 41 Rj = CHMe2, R2 = H
^
;
NH
fll
HN
H Yp 42
628
- - O OH
f ^
OH O
"V^N^S O^IH H J > "" /^^ H
HN\ S^NYJ>^. Ph-^
O
OH
(^
^
"S^N^S o^L H JJ A) ^^Jl^N^^^
"""VN
J^
p^/^ O
43
O
f ^
^ ^ ^ N \ ^S
oCj fi T """VN
H HN\
S ^ N ^ ^ p ^ ^ O OH
44
45
Two cyclic hexapeptides, bistratamides A (46) and B (47) were isolated from L bistratum. They were found in the obligate algal symbiont Prochloron [78]. L. bistratum from the Philippines contained bistratamides C (48) and D (49), with bistratamide D (49) being a central nervous system depressant [79]. The structure of bistratamide C (48) was confirmed by total synthesis using enantiomerically pure oxazole and thiazole amino acids [80], while bistratamide D (49) was synthesised using a convergent strategy and enantiomerically pure oxazole, thiazole and oxazoHne segments [81].
r^NH
46 X = thiazoline
HN^O
48
^--f^NH
HN"^0
49
47 X = thiazole
L. patella from Fiji contained patellins 1-5 (50-54) [82] and earlier, solution- and solid-state conformational studies were carried out on patellin 2 (51), and the structure was determined by X-ray analysis [83]. A Lissoclinum sp. from the Great Barrier Reef yielded patellins 3 (52), 5 (54) and 6 (55) and the heptapeptide trunkamide A (56) [82]. Compounds 50-56 were all identified by interpretation of spectral data and through use of Marfey's method to determine the absolute stereochemistry of the constituent amino acids [82]. A total synthesis of the proposed structure of trunkamide A (56) revealed that the structure
629
of the natural product should be reinvestigated [84] and the structure was indeed later revised as the result of a total synthesis [85]. Another total synthesis was later reported [86].
50
51
Solution- and solid-state conformations of tawicyclamides A (57) and B (58), proline-containing cyclic peptides from a Philippines specimen of L. patella, were determined by spectroscopic and X-ray analyses respectively [87]. Patellins 1-6 (50-55), trunkamide A (56) and the tawicyclamides A-B (57-58) all lack the oxazoline ring present in most other cyclic peptides isolated from the Lissodinum genus [22].
0 - ^ N -
52 R = CH2CHMe2
54
53 R = CHMe2
55
56
""
630
57 R = CH2Ph 58 R = CH2CHMe2
A number of cyclic peptides have been isolated from the tunicate Didemnum molle. The structure of mollamide (59), a cytotoxic heptapeptide isolated from Didemnum molle from the Great Barrier Reef, was determined by X-ray crystallography and chemical degradation [88]. Mollamide has also been synthesised [89]. Cyclodidemnamide (60) is a weakly cytotoxic, heptapeptide from D, molle from the Philippines [90]. Total synthesis of the proposed structure of cyclodidemnamide gave a product with different spectral data to those of the natural product, which is thought to be a stereoisomer [91]. The stereochemistry of one of the two valine residues in cyclodidemnamide was revised from L-valine to D-valine as a result of the total synthesis of both isomers [92] and the configuration was also reassigned as a result of total synthesis [93]. The hexapeptides, comoramides A (61) and B (62), were isolated from D. molle from Mayotte lagoon in the Comoros Islands, while the heptapeptides, mayotamides A (63) and B (64) were isolated from a separate collection of D. molle from the Comoros Islands [94].
r~^ k^O 0=(
O
"
N=r
>-NH HN-< \ P - -
O 59
^^ Ph 60
631
°y:A: ^"xrx VrT 61
62
63 R = Me 64R = H
A Didemnum sp. from Palau was the source of didemnaketal C (65), which contains a sulfonic acid group [95]. The in vitro anti-human immuodeficiency virus (anti-HTV) activity of D. molle from Pohnpei is associated with the sulfated mannose polysaccharide kakelokelose (66) [96].
o o o" o Me02C>,,^s!>s^AA,^^.->^^
-^Ov.^ OSOjNa HO-^JUQ;^
OSOsNa
NaOsSO-^^"^"^^
66
Lamellarin T-V and Y sulfates (67-70) were isolated from an unidentified ascidian from the Arabian Sea coast of India [97]. Four additional lamellarin sulfates, the 20-sulfates of lamellarins B, C and L and lamellarin G 8-sulfate (71-74) were isolated from Didemnum chartaceum from the Great Barrier Reef [98]. Unusually long relaxation times were observed for certain signals in the ^H NMR spectra of these compounds. Lamellarin a 20-sulfate (75) was isolated from an unidentified ascidian from India and was an inhibitor of human immunodeficiency virus type 1 (HTV-l) integrase [99].
632
.O^^O
R,0. R7O
OH
R.O
o R i OMe
OR4
OMeORs
67 Ri Ell = Me, R2 = OMe, R3 = H
71 ^5, R^ = SOsNa, R2 = Me, R3 = H, R4 = Me, R5 = Me, R^ = OMe,
68 Ri = Me, R2 = H, R = H
72 RJ = SOaNa, R2 = Me, R3 = H, R4 = Me, R5 = Me, Re = OMe
69 Ri = Me, R2 = OMe, R3= OH
73 R^ = S03Na, R2 = Me, R3 = Me, R4 = H, R5 = H, R^ = H
70 Ri = H, R2 = H, R3= H
74 Ri = Me, R2 = H, R3 = Me, R4 = H, R5 = S03Na, Re = H 75 Ri = S03Na, R2 = Me, R3 = H, R4 = Me, R5 = Me, Rg = H
Didemnum rodriguesi from New Caledonia contained the unusual peptidyl alkaloid caledonin (76), that formed a complex with Zn^"^ and Cu^ ions between thiol and primary amine groups [100]. The minalemines D-F (77-79) are peptide guanidine derivatives isolated from a Caribbean collection of D. rodriguesi and contain a sulfamic acid group [101]. The stereochemistry of cyclodidemniserinol trisulfate (80) from a Palauan specimen of Didemnum guttatum was partially determined [102]. f^
N
o NH2
. N O "
H2N
H N^ NH
H
»03?
9
O R
f "
NH
H O
'N H
77 R = C7H15 78R = C8Hn 79R = C9Hi9
OSOaNa NHS03Na
NaOSOj^ 80
U
NH2 ^
633
The structure of the cytotoxic metabolite dendrodoine (81) from the tunicate Dendrodoa grossularia was determined by X-ray analysis [103] and it was later synthesised by a convergent route [104].
cxA:^ V
N
.Me
Me
81
The eudistomins are p-carboline alkaloids isolated from Eudistoma olivaceum. Eudistomins C, E, F, K and L (82-86) all contain a novel oxathiazepine ring [105]. It was later proposed that the stereochemistry of eudistomins C, E, F, K and L (82-86) should be revised on the basis of a nuclear Overhauser enhancement difference spectroscopy (NOEDS) study of eudistomin K (85) from Ritterella sigillinoides [106]. Eudistomin K sulfoxide (87), an antiviral agent from /?. sigillinoides [107] was synthesised from eudistomin K (85), and the structure and absolute configuration of compound 85 were determined by X-ray analysis [108]. R. sigillinoides also contains debromoeudistomin K (88), in addition to known eudistomins [109]. A^(10)-Methyleudistomin E (89) was isolated from E, olivaceum from the Caribbean [110].
82 Ri = H, R2 = OH, R3 = Br, R4 = H
87
89
83 R, = Br, R2 = OH, R3 = H, R4 = H 84 Ri = H, R2 = OH, R3 = Br, R4 = C2H3O2 85 R, = H, R2 = H, R3 = Br, R4 = H 86 Ri = H, R2 = Br, R3 = H, R4 = H 88 Rj = H, R2 = H, R3 = H, R4 = H
Syntheses of both (-)-eudistomin F (84) [111] and of (-)-eudistomin L (86) [112] have been reported, while (-)-eudistomins C, E, F, K and L (82-86) were later also synthesised from the corresponding A^hydroxytryptamines and D-cysteinal [113].
634
Eudistomidin C (90) was one of three antileukaemic p-carboline alkaloids isolated from an Okinawan sample of Eudistoma glaucus. It was identified by spectral methods and synthesis of a derivative [114]. Eudistomidins E (91) and F (92) were isolated from E, glaucus from Okinawa and identified by spectroscopic techniques [115]. Eudistomidins C (90) and F (92) contain a methyl sulfide group while eudistomidin E (91) contains a methyl sulfoxide. 14Methyleudistomidin C, (93) was isolated from Eudistoma gilboverde along with known eudistomins [116]. 14-Methyleudistomidin C (93) exhibited potent cytotoxic activity with IC50 values less than 1 |Lig/mL against four human tumour cell lines.
SMe
Citorellamine (94) is an indole disulfide dihydrochloride from the Fijian tunicate Polycitorella mariae. It exhibits potent antimicrobial and insecticidal activity in addition to cytotoxicity [117]. The structure was later revised and syntheses of the proposed and true structures carried out [118].
N H 94
'NH k / S f .2HC1
A Didemnum sp. from Rota in the northern Mariana Islands contained four new p-carboline alkaloids, didemnolines A-D (95-98), together with known metabolites [119]. The didemnolines are characterised by substitution at N9 as opposed to CI. A straightforward synthesis of the didemnolines was reported [120].
635
MeS^N
1
Me
H
95 R = Br
97 R = Br
96R = H
98 R = H
Two antineoplastic 24-membered macrolide sulfates, iejimalides C (99) and D (100) were isolated from Eudistoma cf. rigida and identified by interpretation of spectral data [121]. o Me0s.^x^^x::^>s^^^^0^.Av^'<^^-^j^A^NHCH0
»X
0S03Na
The structures of two thiazoles (101-102) from Aplidium pliciferum were elucidated by spectral interpretation and confirmed by synthesis [122]. A New Zealand species of Aplidium contained a cytotoxic and antimicrobial 1,2,3-trithiane derivative (103), which on standing at room temperature for one month in neutral solution, gave 2-vanilloyl imidazole [123], previously reported as a metabolite of A. pliciferum [122]. Hysistozoa fasmeriana from New Zealand gave the new (-)enantiomer of rran5^-5-hydroxy-4-(4'-hydroxy-3'-methoxyphenyl)-4-(2"imidazolyl)-1,2,3-trithiane (103). A second collection of the same ascidian yielded compound 103 and two dithiane alkaloids, fasmerianamines A (104) and B (105). The structures were determined by spectral studies. Both enantiomers of the trithiane 103 exhibited identical biological activities in a range of assays, including modest cytotoxic and antimicrobial properties, while the fasmerianamines (104105) were inactive [124].
636
S
J U
MeO.
OMe 103
104 R = OMe 105 R = OH
Lissoclinotoxin A (106), the antimicrobial constituent of L perforatum from France, was reported to be a 1,2,3-trithiane derivative by analysis of spectral data [125]. The structure of lissoclinotoxin A was later revised to the corresponding pentasulfide, based on unpublished mass spectral data of a derivative and lissoclinotoxin B (107), another pentasulfide, was identified by spectral data interpretation [126]. A Lissoclinum sp. from the Great Barrier Reef, Australia contained lissoclinotoxin A (106), lissoclinotoxins C (108) and D (109) and Hssoclins A (110) and B (111) [127]. Lissoclinotoxin C (108) is a dithiomethyl compound while lissoclinotoxin D (109) is dimeric. Diplamine (112), a further cytotoxic methyl sulfide-containing pyridoacridine, was isolated from the tunicate Diplosoma sp. [128] and later, a total synthesis in 21 steps was reported [129]. Lissoclin disulfoxide (113) inhibits interleukin-8 receptors and is a dimeric alkaloid from a South African species of Lissoclinum [130]. The proposed structure is a symmetrical head-to-head dimer but the spectral data does not eliminate a symmetrical head-to-tail possibility [17]. NH2 OMe
OMe
S-S^
HOY^'SMe
OMe
HO^iyS-S,
S'S NH2 106
107
NH2 108
NHo 109
637
OMeO
MeS
T
N ^^
MeS^ y
NHR 110 R = COCH2CHMe2
OMe
^S" ^
NMe2
"SMe NMe2
113
111 R = CO(Me)C=CHMe (E) 111 R = COMe
Varacin (114), a cytotoxic compound closely related to lissoclinotoxin A (106), was isolated from a Fijian sample of L. vareau and a benzopentathiepin structure was proposed on the basis of spectral data [131]. Two total syntheses of varacin (114) have been carried out [132,133] and later, further syntheses were described [134-135]. N,Ndimethyl-5-(methylthio)varacin (115) and the corresponding trithiane (116) were obtained from L. japonicum from Palau and 3,4desmethylvaracin (117) was isolated from a Eudistoma sp. from Pohnpei [136]. An inseparable mixture of 5-(methylthio)varacin (118) and the corresponding trithiane (119) was obtained from a Pohnpeian Lissoclinum sp. [136]. Three additional antimicrobial polysulfides of the varacin family (120-122) were isolated from Polycitor sp., collected by dredging in the Sea of Japan [137]. RoQ
NR4
NR2
114 Ri = Me, R2 = Me, R3 = H, R4 = H2
116 Rj = SMe, R2 = Me2
115 Rj = Me, R2 = Me, R3 = SMe, R4 = Me2
119 Rj = SMe, R2 = Hj
117 Ri = H, R2 = H, R3 = H, R4 = H2
120 R, = H, R2 = H2
118 Ri = Me, R2 = Me, R3 = SMe, R4 = H2
OMe o
's
121
638
The methyl sulfide-containing alkaloids, varamines A (123) and B (124) were isolated from L. vareau. Their structures were determined by interpretation of NMR spectral data and by comparison with related alkaloids. The varamines were cytotoxic towards L1210 murine leukaemia cells with IC50 values of 0.03 and 0.05 |J.g/mL, respectively [138]. The varamines (123-124), Hssoclins (110-111) and diplamine (112) all contain a methyl sulfide group linked to a pyridoacridine ring system [22].
MeS" OMe 123 R = Me 124 R = H
The shermilamines are a group of alkaloids containing a thiazinone ring attached to a pyridoacridine ring system. The structure of shermilamine A (125), an orange pigment from a species of Trididemnum from Guam was determined by X-ray analysis [139]. Shermilamine B (126) was later isolated from the same Trididemnum extract and from a Eudistoma sp. [140]. Shermilamine B (126) was also isolated from the Red Sea tunicate Eudistoma and the structure was elucidated on the basis of spectroscopic data [141].
126
Dehydrokuanoniamine B (127) and shermilamine C (128) were isolated from a Cystodytes sp. from Fiji. Their structures were determined by analysis of spectroscopic data. These compounds displayed dose-dependent inhibition of proliferation in human colon tumour cells in vitro [142]. Shermilamines D (129) and E (130) were
639
isolated from Cystodytes violatinctus from the Comoros Islands, along with tintamine (131), which has a novel heterocyclic skeleton [143]. Cycloshermilamine D (132) was also isolated from C violatinctus from the Comoros Islands in extremely low yield and is another pyridoacridine alkaloid with a novel heterocyclic ring system [144].
129R = NMe2 130R = N(O)Me2
OH
0;5s^N.
NMe2
132
The pyridoacridine alkaloids, kuanoniamines A-D (133-136) were isolated from an unidentified Micronesian tunicate and its nudibranch predator, Chelynotus semperL The structures were established by extensive NMR spectral analysis. Cytotoxicity against KB cells ranged from IC50 values >10 |Lig/mL for kuanoniamine B (134), 5 [Xg/mL for kuanoniamine D (136), to 1 |Lig/mL for kuanoniamine A (133) [145]. Kuanoniamine A (133) has also been synthesised [146,147].
133
640
NHR 134 R = COCH2CHMe2 135 R = COEt 136 R = Ac
Polycarpamines A-E (137-141) are unusual sulfur-containing antifungal agents from Polycarpa auzata from the Philippines. The structures were elucidated by interpretation of spectral data [148]. Polycarpine (142), a cytotoxic, dimeric, disulfide alkaloid, the corresponding dihydrochloride (143) and two sulfur-containing related monomers (144-145) were isolated from Polycarpa clavata from Western Australia [149]. Polycarpine (142) was also isolated with two monomers (144,146) from P. aurata from Chuuk [150] and later it was synthesised in three steps from p-methoxyphenacyl bromide [151]. NMeo
NMe2
Y\S
MeO.
MCO^'YTC^ MeSS
R OMe
NMe7
MeO.
HO,
MeSS
MeOS
R
137 R = H
138 R = O
140 R = COMe
139 R = S
OMe
141
NH2 OMe
01U'
MeO
Me^^^Y
NH2
c
Me
VN
jr^'^" ..o^^
142
144 R = OMe
143 = .2HC1
145 R = OH
146
The in vivo antitumour activity of extracts of the tunicate Ecteinascidia turbinata was noted in the late 1960s [152] but the active metabolites were only isolated and identified much later by two research groups. These complex alkaloids were termed the ecteinascidins and are
641
abbreviated as Et with a number representing the value of the highest mass ion observed in the positive ion FAB mass spectrum. The Harbor Branch group [153] identified two compounds that were identical to ecteinascidins 729 (147) and 743 (148), identified at Illinois where compounds 745 (149), 759A (150), 759B (151) and 770 (152) were also reported [154]. The stereochemical representations at the 11,13 bridgehead differ between the two groups. Ecteinascidins 759A (150) and 759B (151) were tentatively assigned as A^-oxides of ecteinascidin 743 (148) [154]. X-ray crystal structures of the N12-formyl derivative of ecteinascidin 729 and of the natural N12-oxide (153) of ecteinascidin 743 (apparently different from compounds 150 and 151) were determined [155]. An enantioselective total synthesis of ecteinascidin 743 (148), which entered phase I clinical trials as an anticancer agent, has been reported [156] and synthesis of 148 from the fermentation product cyanosafracin B can provide sufficient quantity for clinical trials [157]. OMe HO^ Js^^Me
OMe H0,^Js^Me
MeO. J ^
147 Ri = H, R2 = OH
-NH
153
148 Rj = Me, R2 = OH 149Ri=Me, R2 = H 150 Ri = Me, R2 = OH, TV-oxide 151 Ri = Me, R2 = OH, N-oxide 152Ri=Me,R2 = CN
Ecteinascidins 597 (154), 583 (155), 594 (158) and 596 (158) are putative biosynthetic precursors of ecteinascidins and were isolated from £". turbinata from the Caribbean [158]. A recent review on the chemistry and pharmacology of the ecteinascidins has been published [159].
642
OMe
OMe
OMe
MeO
Four simple sulfates (158-161) were identified as antimicrobial constituents of Halocynthia roretzi from Japan [160]. Sodium (or potassium) 2,6-dimethylheptyl sulfate (161) was also found in Polycitor adriaticus from Croatia [161]. The absolute configuration of 2,6dimethylheptyl sulfate (161), which has also been found in other Mediterranean ascidians, has been determined using Mosher's method [162].
The Mediterranean ascidian Halocynthia papillosa contained two cytotoxic sulfates, 6-methylheptyl sulfate (162) and (F)-oct-5-enyl sulfate (163) [163]. ^OSOjNa
^OSOjNa 162
163
Ascidia mentula from the Mediterranean was the source of two antiproliferative alkyl sulfates, sodium salts of 3,7,11,15tetramethylhexadecane-l,19-diyl disulfate (164) and heneicosane-1,21diyl disulfate (165) respectively [164]. Microcosmus vulgaris, also
643
collected in the Mediterranean, was the source of the sodium (or potassium) salt of (3Z)-4,8-dimethylnon-3-en-l-yl sulfate (166) [165]. ^OSOgNa OSO.Na 164 NaO^; 165
166
The Mediterranean tunicate Sidnyum turbinatum contained four alkyl sulfates, 1-heptadecanyl sulfate (167), 1-octadecanyl sulfate (168), sodium (25)-2,6,10,14-tetramethylpentadeca-l,18-diyl sulfate (169) and 1-hexyl sulfate (170). The structures were determined by spectroscopic and chemical methods. All exhibited antiproliferative activity in vitro against the murine fibrosarcoma cell line, WEHI 164 [166]. NaO.SO
NaOiSO 168
167 ^0S03Na NaOaSO
169
Na03S0 170
The structure of polyclinal (171), an aromatic sulfate from a Califomian specimen of Polyclinum planum, was determined by X-ray crystallography [167]. OH ^CHO ^OSOsNa OH 171
644
Uoamines A (172) and B (173) are piperidine alkaloids, isolated from Aplidium uouo from Maui, Hawaii. They differ only in the geometry of the 3-thiomethylacrylate ester group [168]. OH
A^A^ O
SMe
H 172
173
Tasmanian collections of Clavelina cylindrica yielded the alkaloids cylindricines F (174) and G (175), the first thiocyanates isolated from an ascidian [169]. Cylindricines H-J (176-178) were later isolated from the same species [170].
,0Ac
,0Ac SCN
NCS"
RH2C
174R = (CH2)4Me
176 R = SCN
175 R = (CH2)2Me
177 R = NCS
A Micronesian ascidian, Nephteis fasicularis, was the source of fasicularin (179), a tricyclic, thiocyanate-containing alkaloid that was active in a DNA damaging assay [171]. The structure was confirmed by total synthesis [172]. C^Hv NCS.
The virenamides A-C (180-182), thiazole-containing cytotoxic linear peptides, were isolated from the colonial ascidian Diplosoma virens collected on the Great Barrier Reef, Australia. Their structures were
645
deduced from NMR spectral data and confirmed using Marfey's procedure [173]. Virenamides D (183) and E (184) were also obtained from D. virens from the Great Barrier Reef [174] and virenamide B (181) has been synthesised [175].
^ . \
,,
180
181 R = CHMej 182 R = CHjPh
183
184
An enediyne antitumour antibiotic, namenamicin (185) was isolated from Polysyncraton lithostrotum from Fiji [176].
NHC02Me
HO-^'
wo
o^
Y
OH
HN.,
185
646
Cnidaria (Coelenterates) The Cnidaria comprise about 8,000 living species and include jellyfish, corals, soft corals or gorgonians, sea anemones and hydrozoans. They are the lowest members of the animal kingdom with cells organised into specialised organs [177]. Cnidarians have a single internal cavity, which acts as a stomach and a single opening above, which is encircled by tentacles and through which food enters and waste escapes [178]. Some Cnidaria are solitary and consist of a single polyp such as sea anemones and others are colonial such as corals but all Cnidaria are radially symmetrical [178]. Many have nematocysts or stinging cells but these organisms are less likely to contain secondary metabolites for use in chemical defence, as they are not really required. Terpenoids are very commonly isolated from this phylum but very few sulfur-containing compounds have been found in Cnidarians. The marine hydroid Tridentata marginata contained the aromatic alkaloids tridentatols A-C (186-188). Tridentatol A (186) inhibited feeding by the planehead filefish. The structure of tridentatol C (188) was elucidated by a single crystal X-ray diffraction study [179]. ^QXJ
SMe
^QXJ
NySMe
kjk ^
SMe 186
1 >-SMe
187
188
^
A zoanthid from the Indian Coast, Zoanthus sp., contained the sulfated sphingolipid hariamide (189) [180]. o C9H19
Two new ultraviolet (UV) absorbing compounds, palythrinethreonine-sulfate (190) and palythrine-serine-sulfate (191) were isolated from the reef-building coral Stylophora pistillata [181].
647
jj
R
OMe
OH yC HO ^OSOjH 190R=Me 191 R = H
The sea anemone Anthopleura elegantissima was the source of the sulfonic acid-containing compound mycosporine-taurine (192) [182], o HOH2CJ X
^
SOH
H 192
Molluscs The phylum MoUusca comprises approximately 100,000 species, making it one of the largest animal phyla [177]. The name mollusc means "softbodied". Molluscs are non-segmented, have a head with tentacles and move by crawling on a foot. For bivalves such as mussels and oysters, the foot is a digging tool and for cephalopods such as squid and octopuses, it is formed into tentacles. The outer body covering is termed the mantle and usually secretes a shell to protect the body [183]. The shell-less molluscs such as the carnivorous nudibranchs (sea slugs) and herbivorous ophistobranchs (sea hares), are well known sources of bioactive secondary metabolites but in many cases the mollusc itself does not produce the compound but sequesters it from its diet. Similarly, filter-feeding bivalves have been the sources of large toxic compounds but the actual producers of these compounds are thought to be microorganisms. Adenichrome is an Fe (Ill)-containing pigment from bronchial heart of Octopus vulgaris. It consists of a mixture of closely related peptides derived from glycine and the isomeric amino acids adenochromines A, B and C (193-195) [184,185].
648
.CO2H
193 Ri= ^-S^
NH
. R,= f - S
R4N^N
194Ri = H.
R2= ^-S^ =(
^pNH2
h
R3 = H
R4 = H or Me .CO2H R3= ^ NH2 R4N^N
R4 = H or Me 195 R
NH9
R4N^N
R4 = H or Me CO2H
ro^H /-<
R2 = H,
NH2
R4 = H or Me
.CO2H
=( R4Nv^N
.CO2H
R3=
^-S
NH,
,C02H NH2
R4N.^N
R4 = H or Me
R4 = H or Me
The brominated alkaloid neoaplaminone sulfate (196) was isolated from the sea hare Aplysia kurodai and its structure was determined by spectral and chemical methods [186]. A. kurodai obtains most, if not all of its metabolites from the red algae on which it feeds [10]. OMe
Me2N
196
The antineoplastic, cyclic peptide dolastatin 3 (197) was isolated from the sea hare Dolabella auricularia in small quantities [187]. It bears much structural resemblance to the cyclic peptides of tunicates. Synthetic attempts indicated that the original published structure was incorrect [188]. Three reports of research directed towards synthesis of possible components of dolastatin 3 (197) failed to help with the correct structure [189-191]. Reisolation of 197 allowed the determination of the correct sequence of amino acids in this cyclic pentapeptide and the new structure was confirmed by synthesis [192]. Synthesis of dolastatin 3 (197) and the corresponding 12/? diastereoisomer permitted study of the solution
649
conformations by NMR and circular dichroism (CD) spectroscopy and by molecular forcefield calculations [193,194]. CONH9
The pentapeptide dolastatin 10 (198) was isolated from D. auricularia and the structure was proposed on the basis of spectral studies [195]. Dolastatin 10 (198) is a very potent antineoplastic agent. The absolute configuration proposed from spectral analysis was confirmed by synthesis [196] and dolastatin 10 (198) has subsequently been synthesised by several other research groups [197-199].
•X"vA
Me, A . , N . ^
^.j^N-
Me O yK^ Me OMeO
M^
198
D. auricularia from Japan contained dolabellin (199), a cytotoxic bisthiazole. The structure was elucidated by spectral data examination and chemical degradation [200]. D. auricularia also contained the cyclic hexapeptide dolastatin E (200) [201], the stereochemistry of which was determined by chemical degradation and total synthesis [202]. A further cytotoxic hexapeptide, dolastatin I (201) was isolated from D. auricularia from Japan [203] and the stereostructure was confirmed by enantioselective synthesis [204]. Dolastatin 18 (202) was isolated as a very minor cytotoxic component of D. auricularia from Papua New Guinea [205].
650
CI CI
OH OH
199
H
NT
H
^
1
-V- "-^^ yN
"
'NH
N
O
r^
Ph
HN'^^0
201
202
The ichthyotoxic diacylglycerol umbraculumin C (203) was isolated from the Mediterranean ophistobranch Umbraculum mediterraneum [206]. The stereochemistry of compound 203 was determined by chemical interconversion and total synthesis [207].
The nudibranch Glossodoris quadricolor which feeds on the sponge Latrunculia magnifica, contained the sponge metabolite, latrunculin B (204) [208], while the nudibranch Chromodoris elisabethina from Guam and Enewetak in the Marshall Islands contained the icthyotoxic latrunculin A (205) [209]. Both of these compounds were previously isolated from the Red Sea sponge, L. magnifica [210,211]. Latrunculin A (205) was also found in the nudibranch Chromodoris lochi and the sponge, Spongia mycofijiensis [212].
651
p? -^r? ^p i^
X"
X"
HN'A
204
205
Extracts of the digestive gland of the nudibranch Doris verrucosa contained the unusual xyloside, 9-(5-deoxy-5-methylthio-P-Dxylofuranosyl)adenine (206), identified by comparison with synthetic material [213]. NH2
OH
206
The Australian nudibranch, Cerastoma brevicaudatum contained (methylthio)furodysinin (207) and dithiofurodysinin disulfide (208) [214], derivatives of thiofurodysinin acetate (209) that had been isolated earlier from a sponge [215]. Compounds 207 and 208 are also assumed to be of sponge origin.
Specimens of the nudibranchs Phyllidia pustulosa and P. varicosa from the Philippines contained 4a-isothiocyanogorgon-ll-ene (210), in addition to known compounds [216].
652
SCN 210
Keenamide A (211) is a thiazoline-containing cytotoxic, cyclic hexapeptide that was isolated from the mollusc Pleurobranchus forskalii, which is known to feed on ascidians [217].
O 211
Tyrindoxyl sulfate (212) was originally isolated from the gastropod Murex truncatus and has been used as a source of the dye Tyrian purple since antiquity. It has also been identified in several other species of marine molluscs [2]. pSOgNa ^^^SMe ^
ax 212
The kidney of the giant clam Tridacna maxima yielded an arseniccontaining sugar sulfate (213), the structure of which was determined by X-ray crystallography [218]. Me 0=As.
HO OH 213
653
Yessotoxin (214) is a polyether from the scallop Patinopecten yessoensis and has been implicated in diarrhetic shellfish poisoning (DSP). The structure and partial stereochemistry of yessotoxin were deduced from spectral data [219]. The relative stereochemistry of yessotoxin and the structures of two new analogues, 45-hydroxyyessotoxin (215) and 45,46,47-trinoryessotoxin (216) were also established [220]. The absolute stereochemistry of yessotoxin (214) was determined by NMR spectroscopy using a chiral anisotropic reagent [221]. The absolute configuration at C45 in 45-hydroxyyessotoxin (215), isolated from P. yessoensis, was determined by the use of a modified Mosher's method [222].
214 Ri = SOgNa, R2 =
\
215Ri = S03Na,R2=
V
OH 216Ri = S03Na,R2= \'^^^''^tx'^ 220Ri = H,R2=
V
222Ri = S03Na,R2 =
Two analogues of yessotoxin, homoyessotoxin (217) and 45hydroxyhomoyessotoxin (218) were isolated from the digestive glands of mussels from the Adriatic Sea. Their structures were determined by NMR spectroscopy and mass spectrometry [223]. Adriatoxin (219), a further yessotoxin analogue, was isolated from the digestive glands of the mussel
654
Mytilus galloprovincialis from the Adriatic coast of Italy [224]. 1Desulfoyessotoxin (220) was isolated from the digestive glands of Norwegian Af. edulis [225].
HO.SO' HO3SO' 217 R = H 218 R = OH
OSOaNa
219
Carboxyhomoyessotoxin (221) was isolated from the digestive glands of mussels from the northern Adriatic Sea [226]. Two further analogues of yessotoxin, carboxyyessotoxin (222) and 42,43,44,45,46,47,55heptanor-41-oxohomoyessotoxin (223) were isolated. Carboxyyessotoxin (222) was isolated from DSP-infested M. galloprovincialis from the Italian Coast of the Adriatic Sea [227], while 42,43,44,45,46,47,55-heptanor-41oxohomoyessotoxin (223) was also isolated from digestive glands of mussels from the northern Adriatic. The structure of compound 223 was determined by NMR spectroscopy and mass spectrometry [228].
655
CO2H
NaOgSO'
111
NaO^SO* NaO.SO' 223
A chlorosulfolipid (224) has been isolated from the hepatopancreas of mussels from the northern Adriatic Sea. The structural determination, including the absolute stereochemistry, was carried out by extensive NMR spectral analysis and through molecular mechanics and dynamics calculations [229].
224R = S03H
656
The cockle, Austrovenus stutchburyi from New Zealand contained brevetoxin Bi (225) [230] and the greenshell mussel, Pema canaliculus contained brevetoxin B3 (226) [231]. A further brevetoxin analogue, brevetoxin B2 (227) was isolated from the hepatopancreas of P. canaliculus [232], while the major toxin in neurological shellfish poisoning (NSP) associated with P, canaliculus was identified as brevetoxin B4 (228) [233].
NaOaSO
CO2H
H '-'iV
^ H
226 R = COC13H27, COC15H31
HQ
q )-C02H
227
657
The cytostolic phospholipase A2 inhibitor tauropinnaic acid (229) was isolated from the Okinawan bivalve Pinna muricata (pen shell). The structure and stereochemistry at all but one centre were elucidated by interpretation of spectroscopic data [234].
The genus Conus comprises approximately five hundred species of predatory cone snails and is therefore, one of the largest, if not the largest, single genus of marine animals alive. Each species of snail produces a unique venom with between 50 and 200 components. These sulfur-rich peptides or conotoxins are neuropharmacologically active and modulate ion channel function [235]. Any attempt to deal with these toxins within this review would not be feasible and the reader is referred to other excellent reviews on the subject [235,236].
Sponges (Porifera) The phylum Porifera comprises about 5000 living species [177]. Porifera or "pore-bearers" are filter-feeding organisms, which consist of a main body cavity perforated by holes. They are considered to be the most primitive of marine invertebrates, with littie internal organisation [178]. The supporting skeleton of a sponge consists of spicules composed of calcium carbonate or silica or of silica spicules and spongin fibres [183]. Sponges in general, and in particular those without the physical protection of spicules, produce secondary metabolites that are thought to provide protection against predation, overgrowth or fouling. Many sponges contain symbiotic
658
microorganisms and thus there is usually some uncertainty as to the true source of any isolated metabolites. [4]. Isothiocyanates are one of the most common classes of sulfurcontaining marine metabolites and usually occur as terpenes. The majority of terpene isothiocyanates isolated are from the sponge orders, Axinellida (Acanthella and Axinella genera), Halichondrida {Cymbastela, Phakellia, Axinyssa (= Trachyopsis) and Halichondria genera) and Lithistida {Theonella genus) [22]. Terpene isothiocyanates are often accompanied by the corresponding isocyanides and formamides. Biosynthetic studies involving incorporation of ^^C labelled precursors including 2-isothiocyanopupukeanane (230) into a Hymeniacidon sponge, revealed that the isocyano group was the precursor of the formamido and isothiocyano groups, the reverse reactions did not take place and the sponge did not utilise formate in the isocyano biosynthesis [237].
"K4N^^CS
230
The sponge Axinella cannabina was the source of the sesquiterpenoid, axisothiocyanate 1 (231) [238]. Later, the structures of axisothiocyanate 2 (232) [239] and axisothiocyanate 3 (233) from A. cannabina were determined from chemical and spectral data. The latter has a novel spiro[4,5]decane skeleton [240]. Further study of A. cannabina resulted in the isolation of axisothiocyanate 4 (234) [241]. The absolute configurations of axisothiocyanate 1 (231) and axisothiocyanate 4 (234) were determined from X-ray crystallography and from CD measurements [242]. Synthesis of racemic axisothiocyanate 4 (234) has been accomplished [243]. Axisothiocyanate 2 (232) was synthesised in a stereospecific manner from aromadendrene. The report also stated that some sesquiterpene isothiocyanates can cause allergic reactions with headaches, nose itching and rashes [244]. Two additional isothiocyanates (235-236) were isolated from A. cannabina [245]. A third isothiocyanate (237) was later isolated from the same extract and axisothiocyanates 1-3 (231-233) were reported to be cytotoxic in vitro [246]. Another sesquiterpene isothiocyanate (238) was isolated as a minor metabolite of A. cannabina [241],
659
A Halichondria species of sponge from Oahu, Hawaii contained a complex mixture of sesqui- and diterpenoids. The sesquiterpenoids belonged to the a-amorphene (zizanene) series and included an isothiocyanate (239) [248]. H
SCN 239
A sesquiterpene isothiocyanate (240) was isolated from the Califomian nudibranch Cadlina luteomarginata but was presumed to be concentrated from the Axinella species of sponge which constitutes much of its diet [249]. Famesyl isothiocyanate (241) was isolated from Pseudaxinyssa pitys [250], while theonellin isothiocyanate (242) was isolated from the sponge Theonella cf. swinhoei [251].
NCS
SCN 242
Two further sesquiterpene isothiocyanates were isolated from A. cannabina as minor metabolites. The first (243) is based on the epieudesmane skeleton while the next (244) is related to alloaromadendrene [252]. A third isothiocyanate (245) which is a c/^-eudesmane derivative
660
was found in A. cannabina, while the same isothiocyanate was found in higher yield from Acanthella acuta [253]. Three isothiocyanate compounds (246-248) were isolated as minor metabolites of A. acuta. Their structures were elucidated by spectral data interpretation [254]. NCS
NCS 244
NCS 245
A species of Halichondria from the Marshall Islands contained an isothiocyanate (249) [255] and an isothiocyanate based on the guai-6-ene skeleton (250) was isolated from an unidentified sponge from Japanese waters [256]. Acanthella pulcherrima from Australia contained two isothiocyanates (251-252), in addition to known sesquiterpenes [257]. NCS
. NCS
SCN
249
The first sesquiterpene thiocyanate to be isolated from a marine sponge was (15*,45*,65*,7/?*)-4-thiocyanato-9-cadinene (253) from Trachyopsis aplysinoides. The structures of this compound and of an isothiocyanate with a new carbon skeleton (254), were determined by Xray analysis and two additional isothiocyanates (255-256) were identified [258]. Isothiocyanate 254 was synthesised using an oxidative radical cyclisation reaction as a key step [259].
661
SCN
NCS
^ < &
•Mx; 253
5&-
r
SCNJ
/
00
i H ""
>
256
255
254
The structure of 5-isothiocyanatopupukeanane (257), a sesquiterpene isothiocyanate from an Axinyssa species from Guam, was determined by X-ray analysis [260]. Two isomeric sesquiterpene thiocyanates, 2thiocyanatoneopupukeanane (258) and 4-thiocyanatoneopupukeanane (259) were isolated from an unidentified sponge from Pohnpei and from Phycopsis terpnis from Okinawa [261]. A sample of Axinyssa (= Trachyopsis) aplysinoides from Palau yielded a rare thiocyanate, 2thiocyanatopupukeanane (260), while two specimens from Pohnpei yielded 13-isothiocyanatocubebane (261), 1-isothiocyanatoaromadendrane (262) and 2-thiocyanatoneopupukeanane (258) [262]. This last compound had previously been assigned different stereochemistry at C2 [261]. (-)-4Thiocyanatoneopupukeanane has been synthesised in an enantiospecific manner (259) [263]. Both enantiomers of 2-thiocyanatoneopupukeanane (258) have been synthesised from (/?)-carvone [264].
SCN\
NCS
,
. H '
258 Ri = SCN, R2 = H ^^'
259Ri=H,R2 = SCN
^60
261
262
A sesquiterpene isothiocyanate, halipanicine (263) has been isolated from an Okinawan specimen of Halichondria panicea [265]. The relative stereochemistry of halipanicine (263) was established by synthesis [266] and later, a total synthesis was achieved [267].
SCN
CO w 263
662
Three new antiparasitic sesquiterpene isothiocyanates, 4isothiocyanato-9-amorphene (264), 10-isothiocyanato-4,6-amorphadiene (265) and 10-isothiocyanato-5-amorphen-4-ol (266) were isolated from a Fijian specimen of Axinyssa fenestratus. The compounds were identified by spectral data interpretation [268]. Two isomeric isothiocyanates (267268) were isolated from Acanthella klethra from the Great Barrier Reef and their structures were determined by X-ray crystallography and spectral data examination [269]. . .Ncs SCN»
A sesquiterpene thiocyanate, cavemothiocyanate (269) was isolated from Acanthella cf. cavernosa and the structure was elucidated on the basis of spectral data. The nudibranch Phyllidia ocellata also contained cavemothiocyanate [270]. Acanthene B (270) is a sesquiterpene isothiocyanate isolated from a British Columbian Acanthella sp. [271]. The sesquiterpene thiol, T-cadinthiol (271) was isolated from Cymbastela hooperi from Kelso Reef on the Great Barrier Reef [272]. A sesquiterpene isothiocyanate that displayed modest in vitro antimalarial activity, (-)-9-isothiocyanatopupukeanane (272) was isolated from an Axinyssa sp. from the Great Barrier Reef [273]. Great Barrier Reef samples of A. cavernosa contained lO-isothiocyanatocadin-4-ene (273) [274]. H\^SH
269
^^^
270
271
^^^^f^
272
HTNCS
273
Two isothiocyanates, epipolasins A and B and the corresponding (3phenylethylamine adducts, epipolasinthioureas A (274) and B (275) were isolated from the sponge, Epipolasis kushimotoensis. The structures of the epipolasins were determined by chemical degradation to known compounds [275]. The structure of epipolasin A is identical to that
663
previously assigned to a metabolite of the nudibranch Cadlina luteomarginata (240) [249] and the physical data is also similar except for the sign and magnitude of the optical rotation. The structure of epipolasin B is identical to that previously assigned to axisothiocyanate 2 (232) [239]. Synthesis of (-)-(10/?)-10-isothiocyanoaromadendrane indicated that it was the enantiomer of epipolasin B, previously isolated from E, kushimotoensis [276]. .-'^"S"'^
w)
CQ ^K!^
^
240 R = NCS
232 R = NCS
274 R = NHC(S)NHCH2CH2Ph
275 R = NHC(S)NHCH2CH2Ph
The structure of a diterpenoid isothiocyanate (276) extracted from a Halichondria sponge, was determined from chemical and spectral data [277].
NCS 276
Kalihinols G (277) and H (278) were trace components of a species of Acanthella from Guam and kalihinol X (279) was isolated from a Fijian species of Acanthella, All inhibited growth of Bacillus subtilis. Staphylococcus aureus and Candida albicans [278]. 10-Epi-isokalihinol H (280) and 15-isothiocyanato-l-epi-kalihinene (281) were isolated from Acanthella cavernosa from the Seychelles [279]. A Japanese specimen of A. cavernosa contained a sesquiterpene isothiocyanate (282) and lOPformamido-5P-isothiocyanatokalihinol A (283). Structures were assigned by spectral data interpretation [280]. Phakellia pulcherrima from the Philippines contained the minor diterpene isothiocyanates kalihinol L (284), 10-isothiocyanatokalihinol G (285), 10-epi-kalihinol H (286) and 10-isothiocyanatokalihinol C (287) [281]. 10-Epi-kalihinol I (288) and 5,10-bisisothiocyanatokalihinol G (289) were isolated from an Acanthella sp. from Okinawa [282].
664
HOJ
HOj
1 HJ
1 HJ THJ[>
C N ^
A^^
NCS
H CI
279
277 Ri = NC, R2 = NCS
280
281
278 Ri = NCS, R2 = NC
NCS
H\.NHCHO
^K
- ^
282
283
SCN ^yJC P
SCN V C
CN V Q
CI
NCS
NCS
NCS
NCS
HO-
289
A Japanese sponge of the Adociidae family contained 10isothiocyanatobiflora-4,15-diene (290), which was identified by spectral analysis [283]. A. cavernosa from Fiji yielded a diterpene isothiocyanate (291) [284]. NHCHO
NCS
NCS 291
665
Cymbastela hooperi from the Great Barrier Reef contained four diterpene isothiocyanates (292-295) amongst other diterpenes [285]. An amphilectene isonitrile (296) was isolated from a Caribbean Cribochalina sp. [286]. NCS
NCS
A series of eighteen long chain, aUphatic a,a)-bis-isothiocyanates (297-314) and three a-isothiocyano-c?-formyl analogues (315-317) was isolated from a Fijian species of Pseudaxinyssa [287]. The major constituents (297), (305) and (315) all have the same length of aliphatic chain (CI8). Unlike terpenoid isothiocyanates, this series was not accompanied by the corresponding isocyanides or formamides. SCNr ^ ( C H 2 ) n / ^ ^ I NCS
SCN-(CH2)n
297n=14 301n = l l
3 0 5 n = 1 6 310n = 13
315 n = 15
298n = 8
302n = 12
306n = 9
316 n = 9
299n = 9
303n=13
3 0 7 n = 1 0 312n = 15
300n=10
304n=14
308n=ll
313n=17
309n=12
314n=18
NCS
311n=14
SCN
(CH2)n-CH0
317n=16
Dysidea herbacea contains linear polychlorinated peptides with a thiazole residue. The metabolites can be divided into the dysidenin, the isodysidenin and the dysideathiazole series of compounds [22]. Dysidenin (318) was isolated from D. herbacea from Cooktown, Australia without stereochemical assignments [288]. The structure of isodysidenin (319), isolated from a sample of D, herbacea from Papua New Guinea, was determined by X-ray diffraction analysis [289]. It was proposed that the two compounds differ in stereochemistry at C5 [290]. The absolute configurations were later revised [291].
666
Me
^H JL
Me
Q H
318 R = Me 325 R = H
*^
-^H J ^
Q H '^
319 R = Me, X = CI, Y = CI 322 R = H, X = CI, Y = CI 323R = H,X = C1,Y = H 324R = H,X = H,Y = C1
Two thioacetates, thiofurodysin acetate (320) and thiofurodysinin acetate (209) were isolated from a Dysidea species from Sydney, Australia. They were converted by treatment with Raney nickel to a mixture containing furodysin and furodysinin respectively [214]. These were the first thiol acetates isolated from natural sources. The absolute configurations of (-)-(6/?,ll/?)-thiofurodysinin acetate (209), (-)-(6/?,ll/?)furodysinin disulfide (208) and (+)-(6/?,ll/?)-methoxythiofurodysinin acetate lactone (321), isolated from a Fijian specimen of D. herbacea were determined by chemical interconversion [292]. H
Acs
HA 320
^o
^""YT^^) ^""^^
OMe O
^ 209 R = SAc
321
208 R = -S-S- (dimer)
A collection of D. herbacea from near Bowen, Australia yielded 13demethylisodysidenin (322), 11 -monodechloro-13-demethylisodysidenin (323) and 9-monodechloro-l3-demethylisodysidenin (324), all derivatives of isodysidenin, and a dysidenin derivative, 13-demethyldysidenin (325). 13-Demethylisodysidenin (322) and 13-demethyldysidenin (325) were epimeric at C5 by direct comparison of the dechlorinated derivatives of each [293]. Syntheses of both (+)-13-demethyldysidenin (325) and (-)-13demethylisodysidenin (322) have been described [294]. The results of this synthetic study imply that absolute configurations at C2 and C7 in all of the natural materials are 5, opposite to those assigned by X-ray crystallography to isodysidenin (319). In an Australian specimen of D, herbacea, 13-demethylisodysidenin (322) was found to be localised in cells of the cyanobacterium Oscillatoria spongeliae, while two sesquiterpenes were associated with the sponge cells [295].
667
Thiofurodysinin (326), a furanosesquiterpene from Dysidea avara from Australia, was the first report of a sesquiterpene mercaptan from a sponge [296]. HS^V^r^^x 326
A Palauan species of Dysidea contained 15-acetylthioxyfurodysinin lactone (327), that binds to human leukotriene B4 (LTB4) receptor. The structure was determined by spectral data analysis and confirmed by synthesis involving photo-oxidation of 15-acetylthioxyfurodysinin (328), which co-occurs with it in the sponge [297,298]. An Australian species of Euryspongia also contained 15-acetylthioxyfurodysin (329) and 15acetylthioxyfurodysinin (328) [299]. A sample of Z). herbacea from the Great Barrier Reef contained (-)-neodysidenin (330) and the absolute configuration was determined by capillary electrophoresis of Marfey's derivatives [300]. H OH AcS''"V**'"'^f'^^^^>^Q
Acs
H/ 327
H
329
OH
W
NH
330
The dysideathiazoles A-E (331-335) are a series of polychlorinated amino acid derivatives from Pacific Island collections of D. herbacea. The structures were determined by X-ray analyses and the absolute configurations were determined by X-ray crystallography of a brominated derivative [301]. Herbamide A (336), a chlorinated amide was isolated from a Papua New Guinean sample of D. herbacea as a minor component [302]. D. herbacea from the southern Great Barrier Reef contained a thiazole (337) amongst other known metabolites [303]. A Dysidea sp.
668
from Okinawa contained the benzothiazole S1319 (338), as a Padrenoreceptor agonist [304]. XCI2C.
CCI2Y
331 R = H, X = CI, Y = CI
336
332 R = Me, X = CI, Y = CI 333R=:Me,X = Cl,Y = H 334 R = H, X = CI, Y = H 335 R = Me, X = H, Y = H Me ^ C ^ N ^ , CHCI2
NFP 337
NHMe
y
338
Dysidea avara from the Solomon Islands contained the melemeleones A (339) and B (340), which were identified by spectroscopic analyses [305]. They consist of a sesquiterpene linked to a quinone with an attached taurine residue [22].
SOaH
340
Dysidea sp. from Bararin Island in the Philippines, has yielded the dysideaprolines A-F (341-346), proline-derived analogues of dysidenin (318). The barbaleucamides A (347) and B (348), which are structural analogues of the cyanobacterial metabolite barbamide, were also isolated. The structures were elucidated by NMR spectroscopic analysis. It is most probable that all of these compounds are derived from a symbiotic cyanobacterium found in close association with the Dysidea sp. [306].
669
OMe
^^A, X2HC' " ^ ^N R2
Cl^C
R .^N,^-^CCl3
O
341 Ri = H, R2 = Me, X = CI, R3 = CHCI2
347 R = H
342 Ri = Me, R2 = Me, X = CI, R3 = CHCI2
348 R = Me
343 Ri = H, R2 = H, X = CI, R3 = CHCI2 344 Ri = H, R2 = Me, X = H, R3 = CHCI2 345 Ri = H, R2 = Me, X = CI, R3 = CH3
346 Ri = H, R2 = Me, X = CI, R3 = CH2CI
The burrowing sponge Siphonodictyon coralliphagum and other species of the same genus, contain a series of sesquiterpene hydroquinones. Siphonodictyal D (349), and siphonodictyols G (350) and H (351) occur as sodium sulfates and the structure of siphonodictyal D (349) was determined by X-ray crystallography [307]. A deep water collection of S. coralliphagum contained bis(sulfato)cyclosiphonodictyol A (352) which inhibits binding of LTB4 to human neutrophils [308]. Na03S0,^^^CH0
349
OH
350
Na03S0^^
Na03S0-f3-0S03Na "CH2OH
351
Na03S0
670
Agelas nakamurai from Japan produced the sesquiterpene sulfone, agelasidine A (353), which possessed antispasmodic activity. The structure was deduced from spectral data [309]. A simple synthesis of agelasidine A (353) utilised a hetero-Claisen rearrangement [310]. A biomimetic synthesis of 353 was also reported [311] and another synthesis of agelasidine A (353) was carried out in three steps from famesol [312].
353
Two diterpene derivatives of hypotaurocyamine, agelasidines B (354) and C (355) were also isolated from Agelas nakamurai The structures were determined by interpretation of spectral data. Both are antimicrobial, inhibit smooth muscle contraction and enzyme activity of Na"*'/K"^transporting adenosine triphosphate (ATP)ase [313]. Agelasidine C (355) has been synthesised [314]. (-)-Agelasidine C (356) and (-)-agelasidine D (357) were isolated from the Caribbean sea sponge Agelas clathrodes. The structures were confirmed by interpretation of the spectral data and by comparison of this data with those of the known antipode (+)-agelasidine C (355) [315]. NH
o \ 354
355
O'^O
356
H .N>^NH2 NH
NH
357
Suvanine (358), an acetyl cholinesterase inhibitor was first isolated from species of Ircinia [316] and then later from a Coscinoderma species from Fiji and Palau when the structure was revised [317].
671
Me,
+ NHo
Me
NHo
A Califomian sponge of the Halichondriidae family contained a sulfated sesterterpene hydroquinone and five sulfated sesterterpenes. The structures of the halisulfates 1-5 (359-363) were determined by interpretation of spectral data and a structure was proposed for halisulfate 6 (364). The halisulfates are antimicrobial and antiinflanmiatory [318]. The absolute configuration of halisulfate 3 (361), which was also isolated from Ircinia sp. from the Philippines, has been determined by application of the chiral amide method and by chemical degradation techniques [319]. Halisulfate 7 (365) is a sesterterpene sulfate from a Coscinoderma sp. from Yap, Micronesia [320].
359
360 NaOgSO-
CH20S03Na-0 361
362
672
NaOaSO'
Bioassay directed isolation of serine protease inhibitors from Coscinoderma mathewsi yielded the 1-methylherbipoline salts (366-367) of known sesterterpenes halisulfate-1 (359) and suvanine (358) [321]. Coscinosulfate 1 (368), a sesquiterpene sulfate, was isolated from a New Caledonian collection of C mathewsi. It displayed significant activity as an inhibitor of the protein phosphatase Cdc25 [322]. A total synthesis starting from (+)-sclareolide was described [323].
OSOa
^O
Me
367
OS03Na
Sulfircin (369), an antifungal sesterterpene sulfate was isolated as the N,A^-dimethylguanidinium salt from a deepwater Ircinia species and its structure was determined by X-ray analysis [324]. Two sesterterpene sulfates, hipposulfates A (370) and B (371), were isolated from Hippospongia cf. metachromia from Okinawa and their structures were elucidated by interpretation of spectroscopic data. Both compounds possess an enolsulfate functionality [325].
673
oso
NaO^SO^
370R = H 371 R = OH
Akaterpin (372) is an inhibitor of phosphatidylinositol-specific phospholipase C from a Callyspongia sp. [326]. The relative stereochemistry of the ring junction in the upper decalin moiety of akaterpin was shown to be cis by synthesis of model compounds [327]. NaOsSO-f
VoSOgNa
Four unstable sulfate esters (373-376) of known furanosesterterpenes were obtained from Ircinia variabilis and from /. oros from the northern Adriatic Sea [328]. The 22-(9-sulfates of palinurin (377) and fasiculatin (378) were isolated from /. variabilis and from /. fasiculata respectively. Both were toxic to brine shrimp [329]. Ircinianin sulfate (379) was isolated from /. (Psammocinia) wistarii from the Great Barrier Reef as a very unstable metabolite [330]. PSO3K
674
379
Adociasulfates 1-6 (380-385) were isolated from a Haliclona (aka Adocia) sp. from Palau and were all inhibitors of kinesin motor proteins [331]. Adociasulfate 2 (381) had earlier been shown to inhibit the activity of the motor protein kinesin by interference with its binding to microtubules [332]. An Adocia sp. from the Great Barrier Reef contained adociasulfates 1 (380), 7 (386) and 8 (387), which inhibit vacuolar YCATPase [333]. Adociasulfates 5 (384) and 9 (388) were obtained from Adocia aculeata from the Great Barrier Reef [334]. The structure of adociasulfate 1 (380) was confirmed by an enantioselective total synthesis [335]. Adociasulfate 10 (389) from Haliclona sp. from Palau also inhibits the kinesin motor proteins [336].
NaOaSO-ZT^OSOsNa
HO^: 380Ri = SO3Na,R2 = SO3Na
381R = S03Na
384 Ri = SOsNa, R2 = H
385 R = H
386 Ri = H, R2 = SOsNa
382
675
H0-/^-0S03Na
OSOaNa
387
383
NaOaSO.
COoH
OSOaNa
389
388
Shaagrockols B (390) and C (391) from the Red Sea sponge Toxiclona toxius, are antifungal hexaprenylhydroquinone disulfates and were identified by spectral data interpretation [337]. Toxicols A (392), C (393) and toxiusol (394) are hexaprenoid hydroquinones that were also isolated from Toxiclona toxius. The structures were determined by spectral data examination [338].
390
OSOaNa
GSOsNa
0S03Na
OSO^Na
676
OSOsNa
OSOgNa
392 R = SOgNa
394
393 R = H
A hexaprenyl-hydroquinone sulfate (395) was identified as an H^/K"^ATPase inhibitor from a Japanese species of Dysidea [339]. Sarcotragus spinulosus from deep water contained the Na"^/K"^-ATPase inhibitors sarcochromenol sulfates A-C (396-398) and sarcohydroquinone sulfates A-C (399-401) [340]. The structures were determined by spectral data analysis of the natural products and of derivatives.
.rd"*^"
NaOaSO'
396 n = 5 397 n = 6 398 n = 7
OSOaNa 399 R = H, n = 6 400R = SO3Na,n = 7 401 R = H, n = 8
A heptaprenylhydroquinone derivative (402) was isolated from an Indian sample of Ircinia fasciculata [341]. Ircinia spinulosa from the Adriatic contained three sulfated 2-prenylhydroquinones (403-405) that are toxic to brine shrimp [342]. An Ircinia sp. from New Caledonia contained a sulfated 2-prenylhydroquinone (406) and a sulfated furanoterpene (407) [343]. An Australian Sarcotragus sp. contained octaprenylhydroquinone sulfate (408) and nonaprenylhydroquinone sulfate (409) as inhibitors of al,3-fucosyltransferase VII [344].
677
OSO^Na
402
ry^^^yiry^
0S03Na OSO^Na
407
The yellow pigment halenaquinol sulfate (410) has been isolated from the Okinawan sponge Xestospongia sapra and is a pentacyclic hydroquinone [345]. The absolute stereochemistry was determined to be 65 by comparing the CD spectrum of a derivative with a theoretically calculated spectrum [346]. Theoretical calculation of CD spectra of halenaquinol sulfate (410) isolated from X exigua and X sapra determined that the absolute stereostructure was 12b5' [347]. The pentacyclic compound, xestoquinol sulfate (411) has been isolated from an Okinawan collection of X. sapra and its structure was elucidated on the basis of spectroscopic data and a chemical conversion [348]. NaOjSO. OSOaNa
Xestoquinolide B (412) was obtained from Xestospongia cf. carbonaria and the protein kinase activity of it and related compounds reported. The structure of this merosesquiterpenoid was elucidated by spectral data interpretation [349]. A Xestospongia species from the
678
Philippines contained the topoisomerase n inhibitors, secoadociaquinones A (413) and B (414) [350]. HN"^
0<^^
H 0 3 S ' ^
SO3H
0,s^.x<^^NH O
412X = NH,Y = S02
413
orX = S02,Y = NH
Species of Adocia from Truk contained adociaquinone A (415), the mildly cytotoxic adociaquinone B (416) and 3-ketoadociaquinone (417) as The absolute minor components. All were synthesised [351]. configurations of (+)-adociaquinones A (415) and B (416) were determined by total synthesis [352]. 02S^
.If! ° rxT ^0 415 R = H2
rV^o l y^o y^^-^
0
416
417 R = 0
A number of sulfur-containing cyclic peptides have been isolated from sponges and many of these are cytotoxic or exhibit other biological activities. Discodermins A-D (418-421) from Discodermia kiiensis are antimicrobial, cyclic tetradecapeptides which contain a sulfonic acid group [353,354]. The structure originally proposed for discodermin A [354] was later corrected by incorporation of L-proline instead of D-proline [353]. Four additional tetradecapeptides, discodermins E-H (422-425), were isolated from D. kiiensis from Japan and were cytotoxic and antimicrobial [355,356]. The amino acid sequence in discodermins A-D (418-421) was revised, reversing the positions of threonine and asparagine from their original assignment [355].
679
CONHj
o N>'
^ .
,-^5
^ . OH
NH NH9
1^
^N'
Me
"7
NH
H2NOC
N H O
^ 418 Rj = H, R2 = H, R3 = Me, R4 = Me, R5 = a 419 Ri = H, R2 = H, R3 = H, R4 = Me, R5 = a 420 Ri = H, R2 = H, R3 = Me, R4 = H, R5 = a 421 Ri = H, R2 = H, R3 = H, R4 = H, R5 = a 422 Rj = H, R2 = H, R3 = Me, R4 = Me, R5 = b 423 R, = H, R2 = H, R3 = Me, R4 = Et, R5 = a 424 Ri = Me, R2 = H, R3 = Me, R4 = Me, R5 = a 425 Ri = H, R2 = OH, R3 = Me, R4 = Me, R5 = a
A deepwater species of Discodermia contained the cytotoxic depsipeptide, polydiscamide A (426), which was identified by spectroscopic means, chemical degradation and derivatisation [357]. O
HzN^NH
OHC^J^IJ^ t H N'
HNv > ' "SOjNa NH
HNO'ITV ^ , ^ C O N H
426
680
Halicylindramides A-C (427-429) are antifungal and cytotoxic depsipeptides from Halichondria cylindrata from Japan [358]. Two additional peptides, halicylindramides D (430) and E (431) of which the former is antifungal and cytotoxic, were also isolated from a Japanese collection of//, cylindrata [359]. H V.
.
O
/
***, „ ' O
^ ^ ° / O rf^ H
Ph
CONH2 427 Ri = H, R2 = Me 428 Ri = Me, R2 = H 429 Ri = Me, R2 = Me j ^
O^^NHCHO
r S
?ONH2
Ph O H2N
N "
H2NOC
u ^NH O
-^
430
Br-f
V-,
O. NHCHO A-<
V-\
CONH2 CONH9 O .-\_Me
H2N ^
^
.pj^
N H 431
Microspinosamide (432), a cyclic peptide incorporating 13 amino acid residues, was isolated from an Indonesian collection of Sidonops
681
microspinosa. The structure of compound 432 was elucidated by NMR and mass spectral analyses, and by chemical degradation and derivatisation studies. This tridecapeptide is the first naturally occurring peptide to contain a P-hydroxy-p-bromophenylalanine residue. Microspinosamide (432) inhibited the cytopathic effect of HIV-1 infection in an in vitro colorimetric assay, with an EC50 value of approximately 0.2 |Lig/mL[360]. f^'^^NH
OHC.
.
j^ O /=<
O^
H ^ ^
0w,^^^N H 2
''^T ^
H ^
f^ H I
OH NH H9N O 432
Studies of Theonella sponges from Okinawa resulted in the isolation of the thiazole-containing cyclic peptides, keramamides F, G, H, J, K, M and N (433-439) [361-364]. Keramamides F (433) and K (437) were cytotoxic against KB and LI 210 cell lines and keramamide K (437) contained an unusual tryptophan residue. A different Theonella sp. from Okinawa yielded a theonellapeptolide congener (440), which contained a methylsulfinylacetyl group at the iV-terminus [365]. Synthesis of the proposed structure of keramamide J (436) indicated that the natural product structure needs to be re-examined [366]. O
O
OHC.
^
OH "
Q^
^ HN
433
KJ
O^X^O NH N. J w .OMe
OMe
682
OHC
^
^OMe
435Ri = Br,R2 = OH
437
436Ri = H,R2 = H
438 R = Me 439 R = Et
MeOS
V -
HN^C
NH
: sV-T0
^cyK^
440
Oriamide (441), a cytotoxic peptide containing the unusual amino acid, 4-propenoyl-2-tyrosylthiazole, was isolated from a Theonella sp. from South Africa [367]. Theonella cupola from Indonesia and from
683
Okinawa contained a cytotoxic, cyclic heptapeptide, cupolamide (442) [368]. NH
X ^
^ n
' HNy.0
I 0;:yJ--y'
HO>^A.„,
441
NH
-OSOaNa O
442
Phakellistatin 5 (443), a cytotoxic heptapeptide from Phakellia costata from Chuuk (Truk) contains a methyl sulfide group [369]. Solution- and solid-phase syntheses of phakellistatin 5 have been accomplished and the products were chemically identical but not biologically identical [370]. Hymenamide F (444) from an Okinawan Hymeniacidon sp., is another cyclic heptapeptide that contains a methyl sulfide group. Structural elucidation of hymenamide F (444) was carried out through spectral data analysis of the corresponding sulfoxide [371]. MeS^
H2N-C ^
H
. . . Ph 443
^, ^ MeS
V^' H
^^-^
444
The marine red alga Ceratodictyon spongiosum containing the symbiotic sponge Sigmadocia symbiotica, which was collected from Biaro Island, Indonesia, yielded two isomers of a bioactive thiazole-containing cyclic heptapeptide, d^,d5'-ceratospongamide (445) and trans.transceratospongamide (446). The structures of the ceratospongamides, which each consist of two L-phenylalanine residues, one (L-isoleucine)-L-
684
methyloxazoline residue, one L-proline residue, and one (Lproline)thiazole residue, were established through extensive NMR spectroscopy, as well as chemical degradation and chiral analysis. Cis.cis(445) and rran5',rran5-ceratospongamide (446) are stable conformational isomers of the two proline amide bonds. rran5,rrans-Ceratospongamide (446) exhibits potent inhibition of secreted phospholipase A2 (SPLA2) expression in a cell-based model for antiinflammatory activity (ED50 32 nM), whereas the cis.cis isomer (445) is inactive. Trans.transCeratospongamide (446) was also shown to inhibit the expression of a human-sPLA2 promoter-based reporter by 90% [372].
' > .
445
446
The cyclic hexapeptide, waiakeamide (447) was isolated from an Indonesian collection of Ircinia dendroides [373]. Haliclona nigra from Papua New Guinea contained two cyclic hexapeptides, haligramides A (448) and B (449). They showed an unusual activity pattern in the National Cancer Institute (NCI) 60 cell-line panel [374].
Ph"
U
V
l^ "U
Me^% 447
448 R = SMe 449 R = S(0)Me
685
The prianosins and the discorhabdins are sulfide-containing pyrroloiminoquinones. Prianosin A (450) is a potent antileukaemic agent isolated from the Okinawan sponge Prianos melanos. The structure was determined by X-ray analysis [375]. Prianosins B-D (451-453) are further sulfur-containing alkaloids from P. melanos with potent antineoplastic activity. The structures of the prianosins were elucidated by interpretation of spectral data [376]. Discorhabdins A (450), B (454) and D (453) are cytotoxic pigments from a New Zealand species of Latrunculia [377]. Discorhabdin D (453) was also found in an Okinawan species of Prianos [378]. The structure of discorhabdin A (450) was identical to that previously reported for prianosin A [375] and the other discorhabdins were identified through analysis of spectral data. The spectral data of prianosins C (452) and D (453) were identical to those of 2hydroxydiscorhabdin D and discorhabdin D (453) from Latrunculia brevis, so the structures were therefore revised [379]. Discorhabdin Q (455) was isolated from L. purpurea and from at least three Zyzzya species and is cytotoxic [380]. Two Antarctic sponges, Latrunculia sp. and Negombata sp. contained the antibacterial discorhabdin R (456) [381].
Like the prianosins and the discorhabdins, the batzellines and isobatzellines are sulfides with a pyrroloquinone skeleton [22]. A deepwater Batzella sponge contained the alkaloids, batzellines A (457) and B (458), which possess methyl sulfide groups. The structure of
686
batzelline A (457) was determined by X-ray analysis and that of batzelline B (458) was proposed on the basis of spectral data analysis and chemical conversion [382]. Further studies of the deep water Caribbean Batzella sponge resulted in the isolation of isobatzellines A (459), B (460) and D (461), which also contain methyl sulfide groups. The structures of the isobatzellines were proposed on the basis of spectroscopic analyses and the compounds exhibited cytotoxicity in vitro against P388 cells and moderate antifungal activity against Candida albicans [383]. BatzelHnes A (457) and B (458) and isobatzellines A (459) and B (460) have all been synthesised [384]. SMe
SMe
H,N
The Fijian sponge Zyzzya cf. marsailis (now Z fuliginosa) [13] contained the pyrroloiminoquinone alkaloid makaluvamine F (462). Discorhabdin A (450) was also found in the sponge [385]. Total synthesis of makaluvamine F (462) was achieved using hypervalent iodine(in)induced reactions [386,387]. Zyzzin (463) is a thiolactam from Zyzzya {Zyzza) massalis, that during purification, undergoes hydrolysis to the corresponding lactam [388]. o
X N
NH
N H
463
Dercitin (464) is a violet acridine alkaloid from a deepwater species of Dercitus with antitumour, antiviral and immunomodulatory properties in vitro [389] and with in vivo antitumour activity [390]. The structure was proposed on the basis of spectral properties. Cyclodercitin (465) was isolated as a minor alkaloid from a deepwater Dercitus species and three
687
dercitin related compounds, nordercitin (466), dercitamine (467) and dercitamide (135) were found in a deepwater species of Stelletta. The structures were determined by spectral data interpretation. All four compounds inhibited the proliferation of P388 cells in vitro, and all but cyclodercitin (465) exhibited immunosuppressant activity [391].
466 R = NMe2 467 R = NHMe 135 R = NHCOEt
The structure of the pyridoacridine alkaloid stellettamine (468) from a deepwater species of Stelletta, was unambiguously determined by X-ray analysis. This, together with long range ^H-^^C NMR coupling constants and metal-binding studies on kuanoniamine C (135), a metabolite of the tunicate Cystodytes [145], recognised to have identical data to that of dercitamide, led to structural revision of dercitin (464), cyclodercitin (465), nordercitin (466), dercitamine (467), and dercitamide (135) [392]. The structures of the kuanoniamines and dercitins were confirmed by total Sagitol (469) is a pyridoacridine alkaloid from synthesis [393]. Oceanapia sagittaria. It can be obtained from dercitin (464) by singlet oxygen oxidation, but its CD spectrum suggests that it is not entirely an artifact [394]. A paper reporting insecticidal activity and cytotoxicity for kuanoniamines C (135) and D (136) also reported the isolation of an additional pyridoacridine alkaloid, A^-deacetylkuanoniamine C (470) from Oceanapia sp. from Truk, Micronesia [395].
469
688
Corallistine (471) was isolated from Corallistes fulvodesmus from deep water off New Caledonia. The structure was elucidated by X-ray crystallographic analysis [396]. The unusual alkaloid neamphine (472) was isolated from Neamphius huxleyi from Papua New Guinea and its structure determined by X-ray crystallography [397].
MeS^
NH2
O
471
Me
472
The antibacterial and cytotoxic bicyclic alkaloid phloeodictine B (473) was isolated from a New Caledonian species of Phloeodictyon and is unusual as it contains a cyclic aminoketal functionality. The structure was proposed on the basis of spectral data [398]. A Phloeodictyon sp. from New Caledonia contained the cytotoxic and antibiotic alkaloids, phloeodictines Ci (474) and Ci (475). Their structures were elucidated by mass spectrometry and NMR spectroscopy [399]. H2N-H
^ \
\ NH
HN-< Ky
474 n = 3 475 n = 2
LatruncuHns A (205) and B (204) are powerful ichthyotoxins isolated from Latrunculia magnifica. The structure of latrunculin A (205) was determined by X-ray diffraction analysis to be a 16-membered macrolide attached to a 2-thiazolidinone moiety [210]. Latrunculin B (204) is a 14membered macrolide. The ichthyotoxicity of the latrunculins is probably due to their haemorrhage causing ability [211]. The structures of two minor metabolites of L. magnifica, latrunculins C (476) and D (477) were elucidated by analysis of spectral data [400]. Studies of the action of the latrunculins on mouse neuroblastoma and fibroblast cells show that they cause reversible changes in cell morphology [401]. Latrunculin B (204) has been synthesised by a highly convergent, stereocontroUed route [402].
689
Several reactions of latrunculin B (204) were carried out as part of a study on structure-activity relationships but the biological activities of the products were not reported [403]. Two quite different total syntheses of latrunculin A (205) were briefly communicated [404,405] and full experimental details were reported later [406,407].
.c^ 6,7-Epoxy-latrunculin A (478) and latrunculin M (479) were isolated from L magnifica. The structures were established by chemical and spectral methods [408]. Latrunculin A (205) was isolated from Fasciospongia rimosa collected in Okinawan waters and the absolute configuration was determined by X-ray analysis [409]. F. rimosa also contained the cytotoxic latrunculin S (480) and the structure was elucidated by NMR spectroscopy and chemical correlation with known congeners [410].
478
Mauritamide A (481), a taurine-containing metabolite, was isolated from the sponge Agelas mauritiana [411]. The taurine-containing alkaloids tauroacidins A (482) and B (483) were isolated from a Hymeniacidon sp. from Okinawa and are tyrosine kinase inhibitors [412]. An imidazole alkaloid, (9£')-clathridine 9-//-(2-sulfoethyl)-imine (484), a taurine derivative of clathridine, was isolated from the calcareous sponge
690
Leucetta microraphis [413]. Taurospongin A (485) is a sulfated acetylenic fatty acid derivative from Hippospongia sp. from Okinawa. It inhibits both DNA polymerase b and HIV reverse transcriptase [414]. B
O
481
B
^^
^O^MQ
482 R = Br (95/9/? = 6:4) 483R = H(95/9/?=l:l)
Me
N
O
^^
AcO
O.
H03S 484
Ct^H 16'^32
485
The pyrrole-imidazole alkaloid taurodispacamide A (486) has been isolated from the Mediterranean sponge Agelas oroides. The structure was established from spectroscopic data. Taurodispacamide A (486) exhibited antihistaminic activity when tested on guinea pig ileum [415].
SO3H
486
The Senegalese sponge Ptilocaulis spiculifer has been shown to contain dakaramine (487), a tyrosine derivative containing iodine and an alkyl sulfate (488) as a counterion of dakaramine [416]. I A ^ O v ^ x - v ^ NMe2
"V'^^^'^^^OSOsH 487
488
691
Microxine (489), a purine derivative was isolated from the Australian marine sponge Microxina sp. Microxine was found to weakly inhibit Cdc2 kinase activity [417]. .N^^O
NH
489
Three aldose reductase inhibitors (490-492) were isolated from Dictyodendrilla sp. from Japan and their structures were determined by Xray analysis and spectroscopic studies [418].
490 R = Na
492
491 R = H
Hyrtiomanzamine (493), a compound consisting of a 6-hydroxy-Pcarboline associated with a betaine unit, was isolated from Hyrtios erecta from the Red Sea and the structure elucidated by spectral data examination. Hyrtiomanzamine exhibited immunosuppressive activity in the B lymphocytes reaction assay [419].
MeS. N N. Me" ^ Me
493
692
The sponge pyridinebetaine B members of a new of Echinodathria.
Agelas dispar from the Bahamas contained (494) [420]. Echinoclathrines B-C (495-496) are class of pyridine alkaloids from an Okinawan species They exhibit weak immunosuppressive activity [421]. 1 +^ N I^SO-
1
^N-^(CH2)i2SR H
495 R = Ac
494
496 R = H
The sesterterpene pyridinium alkaloid spongidine D (497) was isolated from a Spongia sp. from Vanuatu as an antiinflammatory agent [422]. S0.H
/olfO' 497
The diketopiperazine cyc/c>-(L-proline-L-thioproline) (498) was isolated from Tedania ignis but a bacterial origin for the metabolite was suggested [423]. Cyd(7-(L-proline-L-methionine) (499) was isolated from Pseudomonas aeruginosa associated with the Antarctic sponge, Isodictya setifera. The structure was elucidated by spectroscopic methods and confirmed through synthesis [424]. o
o
498
499
o
693
There have been three reports of the same dimeric disulfide. It was first isolated from an unidentified sponge from Guam and the structure elucidated by analysis of spectral data. The (E,E) stereochemistry of the disulfide (500) was defined by comparing the ^^C NMR spectroscopic data with those of the (E,Z)Asomer (501) that was obtained as an unstable minor product [425]. Compound 500 was isolated from a species of Psammaplysilla and was called psammaplin A [426]. It was also isolated from Thorectopsamma xana, collected from the same location in Guam, together with a minor dimeric metabolite bisaprasin (502). Both compounds inhibited growth of Staphylococcus aureus and Bacillus subtilis [427]. Psammaplin A (bisprasin) (500) was later isolated from a Dysidea species of sponge and shown to act on Ca^^-induced Ca^"^ release channels of skeletal muscle [428].
500n= !(£,£) 501 n = 1 (E,Z) 502n = 2
Four minor metabolites, psammaplins B-D (503-505) and presammaplin A (506) were isolated from Psammaplysilla purpurea, in addition to psammaplin A (500). Psammaplin B (503) is a thiocyanate bromotyrosine derivative, while psammaplin C (502) is a sulfanamide. Psanmiaplin D (505) displayed antimicrobial activity and mild tyrosine kinase inhibition [429]. The psammaplins Ai (507) and A2 (508) and aplysinellins A (509) and B (510) were isolated from Aplysinella rhax from both Pohnpei and Palau. These compounds inhibit famesyl protein transferase and leucine aminopeptidase [430]. Another sample of A. rhax from the Great Barrier Reef, Australia contained psammaplin A 11'sulfate (511) and bisaprasin 11'-sulfate (512), both of which inhibited [ H]-l,3-dipropyl-8-cyclopentylxanthine binding to rat brain adenosine Ai receptors [431].
694
O
H
T
H
0
HO' 506
503 R = SCN 504 R = SO2NH2 505 R=
—S-S^^^^^
o N H
A
OMe
507 Ri = H, R2 = SO3', n = 1 508 Ri = SO3-, R2 = 503', n = 2 511 Ri = H, R2 = S03Na, n = 0
^^H
OH Br Br^
HO2C
695
34-Sulfatobastadin 13 (513) is an inhibitor of endothelin A receptor from lanthella sp. from the Great Barrier Reef [432]. Three new bastadin analogues including 15,34-0-disulfatobastadin 7 (514) and lO-Osulfatobastadin 3 (515) were isolated from lanthella basta from Exmouth Gulf, Western Australia. They showed moderate differential activity as sarcoplasmic reticulum-Ca^'*"-channel agonists of the skeletal muscle receptor-protein complex, RylR FKBP12 [433]. NOH
ON,5j^^Na03SO ^>Ss^Br
NOH NOH
0--V^Br 0S03Na
NaOjSO^ H(
OlP
v^
Br' NOH 514
NOH 515
lantherans A (516) and B (517) are dimeric tetrabrominated benzofuran derivatives that were isolated from an Australian lanthella species. The structures were determined by spectroscopic and chemical methods. lantheran A (516) includes a (Z,Z)-1,3-butadiene moiety, whereas iantheran B (517) is the geometric isomer possessing a (Z,£:)-1,3butadiene moiety. Both compounds were Na"^/K'^-ATPase inhibitors [434,435]. lanthesines C (518) and D (519) showed potent NaVK^-
696
ATPase activity and are additional dibromotyrosine derivatives from an Australian lanthella sp. [436].
516
517 R = S03Na
MeO
.SOgNa H
N^xv^O
OMe 518
Br,
i)H
MeOBr HN^^^^^Oyk
Hj^^S03Na
B,A:A^C02H 519
A two Sponge association of a thin crust of Haliclona sp. overlaying an unidentified choristid (probably not Jaspis) sponge contained two enol sulfates, presumed to be from the choristid sponge [437]. These enol sulfates were also found as the sodium salts jaspisin (520) [438] and isojaspisin (521) [439] and (£)- and (Z)-narains (522-523) [440] from
697
Japanese specimens of Jaspis sp. The jaspisins (520-521) inhibited hatching of sea urchin embryos and the narains (522-523) induced metamorphosis in ascidian larvae. Three 3,4-dihydroxystyrene sulfate dimers (524-526) were also isolated from the same Jaspis species [441]. H0^^^5yx%^OS03-R
H0.,^<^^^^^
520R = Na^
521R = Na^
522 R = Me2NC(NH2)NH2^
523 R = Me2NC(NH2)NH2''
"""ij "^
HO^^^XAcHO 524
"^
^
SIS
HOsSOv^O,
"^^^
526
Aplysillin A (527), with unknown double bond geometries was isolated from Aplysina fistularis and is a weak thrombin receptor antagonist [442].
Southern Australian Echinodictyum sp. contained the echinosulfonic acids A-C (528-530) and echinosulfone (531), which were all antibacterial [443].
698
528R = Et 529 R = Me 530 R = H
No Sterols with sulfate groups in the sidechain have been isolated from sponges to date. This contrasts with echinoderms. Many of the sterol sulfates isolated from echinoderms, especially from ophiuroids (brittle stars) contain sulfate groups in the sidechain [2]. Halistanol sulfate (532) from Halichondria cf. moorei is a tris-sodium sulfate salt, which possesses antimicrobial, hemolytic, and ichthyotoxic activity [444]. It was later isolated from two sponges of the genus Topsentia as the free sterol and found to inhibit pp60v-src protein tyrosine kinase activity [445]. The tris(2-aminoimidazolium) salt of halistanol sulfate (533) was isolated from Topsentia sp. from Japan and is also antimicrobial [446].
^K^^lX^X) I I I
RO3SO,
OSO:.Na 532
R= R: [ > - !NH2 ^ - - NT
ROsSO' 6SO3R 533
A Sterol disulfate (534) and a sterol trisulfate (535), both closely related to halistanol, have been found in a species of Halichondria [447] and in Trachyopsis halichondrioides [448] respectively.
699
.-kAJ
H4N"'03SO'
OSOsNa 534
535
A Japanese species of Epipolasis contained five sterol sulfates named halistanol sulfates A-E (536-540), which differ from the original halistanol sulfate (532) from Halichondria moorei [449]. Structures were elucidated by spectroscopic and chemical techniques. Halistanol sulfates F-H (541543) are three additional sterol sulfates from Pseudaxinyssa digitata that inhibit HIV in vitro [450].
>s.-4Ct/
Na03S04 3380
H • 0S03Na
b=
536 Ri = a, R2 = H 537 Ri = b, R2 = H
c=
538 Ri = c,R2 = H 539 Ri = d, R2 = H
d=
540 Ri = e, R2 = O H
The sterol sulfate, halistanol disulfate B (544) was isolated from a South African Pachastrella sp. The structure and stereochemistry of compound 544 were established mainly by interpretation of spectral data. Halistanol disulfate B (544) was active in the endothelin converting enzyme (ECE) assay at a micromolar concentration [451]. Three sterol trisulfates (545-547) have been isolated from the sponges Trachyopsis halichondrioides and Cymbastela coralliophila [452].
700
R Na03Sa
I
I J H
NaOjSO' OSOgNa 544
545 R = ^^.^.^-^^ 546 R = ^^Y^ 'VV,
547 R =
f
The structure of sokotrasterol sulfate (548), isolated from sponges of the family Halichondriidae was determined by X-ray analysis [453-455]. The steroid, 26-norsokotrasterol sulfate (549), was isolated from the marine sponge Trachyopsis halichondrioides and was identified by NMR spectroscopic analysis [456].
HOaSQ HO3SO' OSOjNa 548
Ibisterol sulfate (550) is a sulfated sterol from a deepwater Topsentia sp. that was cytoprotective against HIV-1 in the NCI primary screen [457].
0S03Na 550
701
A Topsentia sp. from Okinawa contained five antimicrobial 14-methyl sterol sulfates, topsentiasterol sulfates A-E (551-555) [458]. Ophirapstanol trisulfate (556) from deepwater Topsentia ophiraphidites showed inhibition in the guanosine diphosphate/G-protein RAS exchange assay [459].
Na03S04
I^T'TI''^
NaOjSO'' HO ^ OSOjNa 551 R =
^ "r"^^ '^ o V-OH 552 R = ^^t^^r=\ -OH HO^O
553 R =
o-^^o 554 R =
il \ O
555 R =
OSOaNa 556
702
An unusual 6a-sterol sulfate (557) was isolated from Dysideafragilis, from the Venetian lagoon and displayed cytotoxicity against two different tumour cell lines in vitro [460]. Tamosterone sulfates (558-559) are a C14 epimeric pair of polyhydroxylated sterols isolated from a new species of Oceanapia [461]. The Japanese marine sponge Epipolasis sp. contained the steroid polasterol B sulfate (560) along with the known compound halistanol sulfate (532). The structure of compound 560 was determined on the basis of spectroscopic evidence and a chemical conversion [462].
NaOjSQ
HO Y Y ^OH GSOsNa
OH OH 558 R = a-H 559 R = P-H
557
NaOaSO^i 4 y . ^ L i x ^ Na03S0' OSOjNa 560
An Acanthodendrilla sp. from Japan contained ten steroidal sulfates, acanthosterol sulfates A-J (561-570). Acanthosterol sulfates I (569) and J (570) showed antifungal activity against Saccharomyces cervisiae and its mutants [463]. Clathsterol (571), was isolated from the Red Sea sponge Clathria sp. The structure was established mainly by interpretation of spectral data and a chemical transformation. Clathsterol (571) was active against HIV-1 reverse transcriptase (RT) at a concentration of 10 |LiM [464]. Toxadocia zumi contains three sterol sulfates (572-574) that are antimicrobial, cytotoxic, ichthyotoxic and larvicidal [465].
703
RjO. R20^ 0S03Na 561
R,0,
^ T1 l Tii OSOaNa
OSOsNa 562Ri=H,R2 = Ac
564 Ri = H, R2 = Ac
563Ri = H,R2 = H
566 Ri = H, R2 = H
568Ri=Ac,R2 = H
569 Ri = Ac,R2 = H
NaOjSOi HI H NaO^SO' " ^ ^^
v^
OH' OAc
«^jdb
I JL J NaOaSO^^-^^^
0S03Na 565 Ri = H, R2 = Ac
571
572 R =
567 Rj = H, R2 = H
573 R =
570Ri=:Ac,R2 = H 574R= .
The sterol sulfates haplosamates A (575) and B (576) are inhibitors of HFV-l integrase from two Philippines Haplosclerid sponges and were reported to be the first naturally occurring sulfamates [466] but the structures were revised after re-examination of spectral data [467].
-O •bP02H0Me
NaOsSO' Y Y ^OR OH OH 575 R = H 576R = P03H2
704
A sterol sulfate, 3P,4P-dihydroxypregn-5-en-20-one 3-sulfate (577), was isolated from Stylopus australis from New Zealand and was the first known sterol sulfate with a 5-pregnene skeleton [468].
HO3SO'
The Pacific deepwater sponge Poecillastra laminaris contained annasterol sulfate (578), which had glucanase inhibitory activity [469].
'^OAc
NaOaSa 578
Polymastiamide A (579), an antimicrobial steroid with an unusual side chain modification involving an amide bond to a non-protein amino acid, was isolated from the Norwegian marine sponge Polymastia boletiformis. The structure of polymastiamide A (579) was elucidated by analysis of spectroscopic data and chemical interconversions [470]. Polymastiamides B-F (580-584), additional amino acid conjugates of steroids, were later isolated from the same sponge [471]. O
'CO2H NaOsSO'^ >
MeO 579
NaOjSa 580Ri = H,R2 = OMe 581Ri=Me,R2 = H
CO2H
705
O
NaOsSO^^^V""^"--^
CO2H
582 Ri = Me, R2 = OMe 583Ri = H,R2 = OMe 584Ri=Me,R2 = H
Echinoclasterol sulfate phenethylammonium salt (585), an antifungal and cytotoxic steroid, was isolated from the South Australian sponge Echinoclathria subhispida [472].
^x:!;55^^\^NH3
0380^
585
Three antiviral sterol disulfate orthoesters, orthoesterol disulfates A-C (586-588) were isolated from Petrosia weinbergi and their structures were determined by spectral data elucidation [473].
NaOaSa
I
I J H
NaOjSO'
586R= / .
587
R=c5^0C
588R:
c^^
706
Weinbersterol disulfates A (589) and B (590) are also antiviral metabolites from P. weinbergi [474].
589Ri = H,R2 = OH 590Ri = OH,R2 = H
Haliclostanone sulfate (591) is an unusual polyhydroxylated sterol sulfate from Haliclona sp. from Malaysia [475].
HO' Y ' y ^OH O l f OH 591
Crellastatin A (592) is the first of a series of cytotoxic bis-steroidal sulfates isolated from a Crella sp. from Vanuatu [476].
,0S03Na
592 Ri = OH, R2 = OH 593 Ri = H, R2 = OH 594 Rj = OH, R2 = H 595 Ri = H, R2 = H
707
Crellastatins B-M (593-604) are twelve additional cytotoxic, dimeric 4,4'-dimethylsterols from the same Crella sp. [477,478].
,0S03Na
596
.OSOaNa
597 R = OH 598 R = H
.OSOjNa
OSOjNa
600
708
^OSOjNa
^OH
r^^. 602
,0S03Na
OSO^Na
^OH OSOjNa
Benzylthiocrellidone (605) was isolated from Crella spinulata and the structure was confirmed by synthesis [479]. It is the first reported example of a natural product containing a dimedone unit [22].
709
Ph O
S^
OH
00 605
Pateamine (606), a potent cytotoxin containing a dilactone functionality, was isolated from a New Zealand species of Mycale and identified by analysis of spectral data [480]. Total synthesis of pateamine A (606) involved a P-lactam based macrocyclisation [481,482], while another total synthesis of pateamine employed a concise and convergent route [483].
A sulfated galactolipid, M-6 (607) was isolated from Phyllospongia foliascens. M-6 (607) consisted of an inseparable mixture of compounds with variations occurring in the carboxylic acid portion of the molecule. Compound 607 has resistant activity against complement fixation in serological reactions [484]. CH20S03"Na'"
OH 607
hOR ^O"^
R = (a:b=l:2) a =C0(CH2)6CH=CHC7Hi5 b = C0Ci5H3i
710
The cytotoxic polyether acanthifolicin (608), which is structurally very similar to okadaic acid, was isolated from Pandaros acanthifolium. It is unique in having an episulfide group on a long-chain polyether (C38) backbone. The structure and absolute configuration were determined by X-ray analysis [485]. Acanthifolicin (608) is thought to be a product of a microbial or microalgal symbiont of the sponge. Desulfurisation of acanthifolicin with a Zn/Cu couple yielded okadaic acid [486]. HQH
Mycothiazole (609) is a novel thiazole-containing lipid with anthelmintic properties isolated from Spongia mycofijiensis. The structure was established by analysis of spectral data [487]. A total synthesis of (-)mycothiazole (609) utilised a convergent strategy. The optical rotation of the product was the same sign as that of the natural material but significantly larger [488].
NHC02Me 609
The theonezolides are 37-membered macrocycles, consisting of fatty acid chains with attached functionalities such as a sulfate ester and a thiazole [22]. Theonezolide A (610) is a cytotoxic metabolite of Theonella sp. from Okinawa. The structure was reported without stereochemical details [489], The structures of theonezolides B (611) and C (612) from a Japanese Theonella sp. were determined by spectroscopic methods but without stereochemistry, except at one centre [490].
711
Toxadocials A-C (613-615) and toxadocic acid (616) are sulfated long chain alcohols isolated from Toxadocia cylindrica that inhibit thrombin. Their structures were determined by chemical and spectral means [491,492]. NaOgSO
GSOsNa
NaOsSO 613 R = CHO 616 R = CO2H
0S03Na
712
NaOsSO
OSOgNa
CHO
NaOaSO
OSO^Na
614 OHC NaOaSO NaOsSO
0S03Na
OSOaNa 615
Callyspongins A-B (617-618) are sulfated compounds from a Japanese sample of Callyspongia truncata. They inhibit fertilisation of starfish (Asterias amurensis) gametes [493].
"OSO^Na 617 R = SGjNa 618 R = H
Mycale sp. from Japan contained thiomycalolides A (619) and B (620) as minor metabolites. They are highly cytotoxic glutathione adducts of the known metabolites mycalolides A and B [494]. OHC
^o HO2C
NH
HO2C o'^-'^s^ o
I O
VQ OMe
619R = 0 620 R = H, 0C0CH(0Me)CH20Me
713
Penares sp. from Japan contained penarolide sulfates Ai (621) and A2 (622), which were a-glucosidase inhibitors [495]. O
•3"7 o c.H
C4H9
An Oceanapia sp. collected off the northern Rottnest Shelf, Australia, has yielded three novel dithiocyanates, thiocyanatins A-C (623-625). The structures were determined by spectroscopic analysis and confirmed by total synthesis. The thiocyanatins contain an unprecedented dithiocyanate functionality and an unusual 1,16-difunctionalised n-hexadecane carbon skeleton. They possess nematocidal activity [496]. NCS^
^SCN
SCN 624
623 "'SCN 625
Three sulfated ceramides, calyceramides A-C (626-628) were isolated as inhibitors of neuraminidase from the marine sponge Discodermia calyx. Their structures were determined by spectroscopic and chemical methods [497].
NaOjSO.
NaOjSi
714
NaOaSO.
Irciniasulfonic acid (629) was obtained from Ircinia sp. from Japanese waters. Spectroscopic and chemical analyses revealed it to consist of three different kinds of acids; common fatty acids, a novel unsaturated branched CIO fatty acid and an isethionic acid. Irciniasulfonic acid (629) reverses multidrug resistance in human carcinoma cells caused by overexpression of membrane glycoprotein [498].
R=
629
Bastaxanthins B, C, D, E, and F (630-634) are novel carotenoid sulfates from the marine sponge lanthella basta from the Great Barrier Reef, Australia [499]. The stereostructure of bastaxanthin C (631) was determined on the basis of infrared (IR), ^H and ^^C NMR, and CD spectra, and by chemical transformations [500]. Bastaxanthins were also isolated from /. flabelliformis from the Great Barrier Reef including bastaxanthin C (631) (major), B (630), D (632), and F (634) and bastaxanthin G (635) [501]. Bastaxanthin G (635) was not fully characterised but was the most polar of the carotenoids isolated and was tentatively described as a disulfate [501],
715
630 R = CH2OH
Na03S0'
631 R = CHO 633 R = CO2H
632 R = CH2OH
NaOgSa
634 R = CO2H
A sulfone (636) is a minor constituent of the Mediterranean sponge Anchinoe tenacior [502]. Sulfolane (637), a familiar industrial chemical, was isolated from a mixture of the sponge Batzella sp. and a Lissoclinum tunicate from Victoria, Australia [503]. It is possibly an absorbed compound rather than a natural product [12].
(!) 637
636
5-Thio-D-mannose (638), the first example of a naturally occurring 5thio-sugar has been isolated from Clathria pyramida [504] and was later synthesised in 12 steps from D-mannose [505]. HOpH
HOAOH
638
716
(2-Hydroxyethyl)dimethylsulfoxonium chloride (1), the causative agent of Dogger Bank Itch which has previously been isolated from the marine bryozoan Alcyonidium gelatinosum [26], has now been isolated as a cytotoxic component of the marine sponge Theonella aff. mirabilis [506].
Echinoderms The phylum Echinodermata comprises about 7000 living species [177]. Echinoderm means "spiny-skinned" and these organisms are characterised by the tube feet, which they use to move about. These have suction discs on the ends, which operate by an internal bulb pumping water in and out of the foot, causing expansion and contraction. The phylum is sub-divided into five classes; the asteroids (sea stars), the holothurians (sea cucumbers), the crinoids (sea lilies), the echinoids (sea urchins) and the ophiuroids (brittle stars) [178]. As stated in the introduction to this review, sulfated sterols and saponins, which comprise the majority of echinoderm metabolites containing sulfur, are not included here. The histidine derivatives, l-methyl-5-thiolhistidine (639) and its disulfide (640) were isolated from unfertilised eggs of the sea urchin Paracentrotus lividus [507] and from those of other echinoderms [508]. Their structures were revised after an unambiguous synthesis [509]. LOvothiol A disulfide (640) was also shown to be the egg release pheromone of the marine polychaete worm Platynereis dumerilii [510]. Me CO2H NNf--^NH2 /—< S-S NH2 H2N Y\ N^N-Me H02C^^-^N
HS
639
640 ^^
The ovothiols, a family of mercaptohistidine compounds, have been isolated from marine invertebrate eggs. Ovothiol B (641) from the scallop
717
Chlamys hastata, ovothiol C from the sea urchin Strongylocentrotus purpuratus (642) and ovothiol A from the starfish Evasterias troschelli were isolated from eggs of ovarian tissue [511,512]. The structure of ovothiol A is identical to that of l-methyl-5-thiolhistidine (639). CO2H
641 R = H 642 R = Me
The ovaries of the Japanese sea urchin Hemicentrotus pulcherrimus contained a bitter tasting amino acid, pulcherrimine (643) [513]. A sulfonoglycolipid isolated from the shell of the sea urchin Anthocidarias crassispina was a 96:4 mixture of r-0-palmitoyl-3'-0-(6-sulfo-a-Dquinovopyranosyl)glycerol (644) and the myristoyl counterpart (645) [514]. o
OH
^^ 643
OH^ 644R = Ci5H3i 645R = Ci3H27
Many starfish cause an escape response in usually sessile marine invertebrates [7]. The starfish Dermasterias imbricata causes the sea anemone Stomphia coccinea to release its basal disc from the substratum and swim away on contact. Bioassay-directed fractionation of the starfish extract led to the isolation of the compound found to elicit this response, the benzyltetrahydroisoquinoline alkaloid imbricatine (646). The structure of compound 646 was elucidated by spectral data interpretation. The amino acid residue in imbricatine is related to the thiol containing amino acids ovothiols A-C. Imbricatine (646) is active in both L1210 and P388
718
assays [515,516]. The structural elucidation and partial synthesis of imbricatine (646) were later reported fully [517].
Forbesin (647), a sulfated glycolipid and a disodium salt, eicosane1,16-disulfate (648) were isolated from the sea star Asterias forbesi, Forbesin was also isolated from A. vulgaris [518]. NaOsSO
647
HOMO
NaOaSO OSOgNa
648
The sea star Henricia laeviuscula contained the anthraquinone sodium isorhodoptilometrin-2'-sulfate (649) [519].
OSOjNa
719
The naphthopyrone, comantherin sulfate (650) was isolated from the crinoid Comantheria perplexa [520] and three naphthopyrones (651-653) were isolated from Comanthus parcivirrus timorensis. Both species were collected off Australia [521].
NaOsSO' 651 Ri = OH, R2 = H
650
652 Ri = OH, R2 = OMe 653Ri = OMe,R2 = OMe
The crinoid Comatula pectinata contained three anthraquinones (654656) [522] while two further anthraquinones (657-658) were isolated from the crinoid Ptilometra sp. l,8-Dihydroxy-3-propyl-9,10-anthraquinone-60-sodium sulfate (657) and l,8-dihydroxy-3-(r-hydroxypropyl)-9,10anthraquinone-6-O-sodium sulfate (658) have not previously been isolated from a natural source. l,8-Dihydroxy-3-(l-hydroxypropyl)-9,10anthraquinone-6-O-sodium sulfate (658) was cytotoxic [523]. OH O NaOgSO.
.OMe NaOsSO' OR2 0
ORi
0
654 Ri = H, R2 = H
657R = H
655 Rj = Me, R2 = H
658R = OH
656 Ri = Me, R2 = Me
The deepwater stalked crinoid Gymnocrinus richeri contained the gymnochromes C (659) and D (660) and isogymnochrome D (661). These compounds have a helical chirality and chiral atoms in the sidechains give rise to isomers [524].
720
HO
0
OH
fWS
Brv
HO" HO.
HO -%H
Wy^ OH 0 OH 659
riT
HOs ^B,6S03H
rl
\J
Br^
OH
0
if' ^ II ""^
HO"JLX
TI T11 TL
Br''
Brv
HO ,Br
O
OH
9SO3H
II
1
11^
^
JS ^
PSO3H 'Br
OH 0 OH 660
PSO3H OH O OH 661
The holothurian Cucumaria frondosa contains 2,6-dimethylnonane-lsodium sulfate (662) and 2,4,6-trimethyl-nonane-l-sodium sulfate (663). The structures were proposed without stereochemical detail [525].
LAjJx.'
OSOsNa
662 R = H 663 R = Me
The Japanese sea cucumber Cucumaria echinata contained a ganglioside CG-1 (664) with neuritogenic activity toward the rat pheochromocytoma PC-12 cell line [526]. Similar activity was reported for the ganglioside HPG-8 (665) isolated from the sea cucumber Holothuria pervicax from Japan [527]. 0SO3H HO^^^A^OH HN^3H ..OH HO' ^ *OHHO^ OH 664
721
OH H
loX>^2C0^0.
H03SO'
HO' ^ 'OHHO^ OH 665
A carotenoid sulfate, ophioxanthin (666), was isolated from the ophiuroid Ophioderma longicaudum from the Mediterranean Sea and shown to be 5,6,5',6-tetrahydro-P,P-carotene-3,4,3',4-tetraol 4,4'-disulfate [528]. The carotenoid, dehydroophioxanthin (667) was isolated from the ophiuroid Ophiocomina nigra off Spain and the structure was determined by spectral data analysis [529].
(3Z)-4,8-Dimethylnon-3-en-l-yl sodium sulfate (166), which is also found in the ascidian Microcosmus vulgaris [166], is a sulfated alkene that was isolated from the ophiuroid Ophiocoma echinata from Colombia [530].
722
ABBREVIATIONS ALL
= acute lymphoblastic leukaemia
Anti-fflV
= anti-human immunodeficiency virus
ATP
= adenosine triphosphate
CD
= circular dichroism
DNA
= deoxyribonucleic acid
DSP
= diarrhetic shellfish poisoning
EC50
= effective concentration needed to reduce cell growth by 50%
ECE
= endothelin converting enzyme
ED50
= effective dose needed to reduce cell growth by 50%
FAB
= fast atom bombardment
fflV-1
= human immunodeficiency virus type 1
IC50
= inhibitory concentration needed to reduce cell growth by 50%
IR
= infrared
mg
= milligram
mL
= millilitre
mM
= millimolar
[iM
= micromolar
nM
= nanomolar
NCI
= National Cancer Institute
NMR
= nuclear magnetic resonance
NOEDS
= nuclear Overhauser enhancement difference spectroscopy
723
NSP
= neurotoxic shellfish poisoning
RT
= reverse transcriptase
SPLA2
= secreted phospholipase A2
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753
SUBJECT INDEX (Vol. 28) Abamectin 434,406 eigmnst Hyalomma spp, 407 against Phytoseiulus persimilis 434 against Rhipicephalus spp. 407 against Tetranichus urticae 434 Absolute stereostructure 11 of broussonetine C 11 ofbroussonetineL 12 Aburatubolactam A 139 f^om Streptomycessp. 139 Aburatubolactam C 139 fvom Streptomycessp. 139 (2S)-Abyssinone II 17 as aromatase inhibitor 17 Acacia honey 386 aroma of 386 Acanthella 663 kalihinol G of 663 kalihinol H of 663 Acanthella cavernosa 663 10-^/?/-isokalihinolHfrom 663 15-isothiocyanato-1 -epi-kalihinene from 663 Acanthella klethra 662 isothiocyanates from 662 A canthella pulcherhma 660 isothiocyanates from 660 sesquiterpenes from 660 Acanthifolicin 710 as cytotoxic agent 710 episulfide group of 710 from Pandaros acanthifolium 110 similarity to okadaic acid 710 Acanthodendrilla sp. 702 acanthosterol sulfate A of 702 acanthosterol sulfate B of 702 acanthosterol sulfate C of 702 acanthosterol sulfate D of 702 acanthosterol sulfate F of 702 acanthosterol sulfate G of 702 acanthosterol sulfate H of 702 acanthosterol sulfate I of 702 acanthosterol sulfate J of 702 against Saccharomyces cerevisiae
702 antifimgal activity of 702 A carapis woodi 387,390 in honey bees' tracheal tubes 387 infestations in Minnesota 390 Acaricidal activities 403,406,412,422 against Dermatophagoides pteronyssinus 422 against Psoroptes cuniculi 412 against Rhipicephalus appendiculatus 406 for killing adult ticks 403 of caffeine 422 of Cuminum cyminum 427 of essential oils 412,427 of Eucalyptus camaldulensis All of eugenol 403 of extract prepared by microwave assisted process (MAP) 427 of Lavandula angustifolia 412 of linalool 412 of Margaritaria discoidea 406 of Origanum syriacum var. bevanii All of phenylpropanoid derivatives 403 of Pimenta dioica 403 of Pimpinella anisum All of Tanacetum vulgare 's extracts 427 of P-thujone 427 Acaricidal properties 400,406 of benzaldehyde 406 of carvacrol 406 of cedrene 406 of a-cyclocitral 406 of P-cyclocitral 406 of Euphorbia obovalifolia 's latex 400 of Ficus brachypoda's htex 400 of geraniol 406 of (£)-geranylacetone 406 of a-ionone 406 of linalool 406 of w-cymene 406 of methyl salicylate 406 ofnerol 406
754
of nerolidol 406 of nonanal 406 of P-ocimene 406 of phenylacetaldehyde 406 of phenylacetonitrile 406 of a-terpineol 406 Acaricide 381,429,435 cross-resistance of 429 deguelinas 435 effectiveness of 404 fenazaquinas 429 from Annona squamosa 404 from Azadirachta indica 404 literature about 381 Lonchocarpus urucu as 435 of natural origin 381 pyridabenas 429 rotenoloneas 435 rotenoneas 435 tebufenpyrad as 429 tephrosinas 435 A cams siro 382 as mite species 382 Acrostalamus fungi 455 acrostalidic acid from 455 acrostalic acid from 455 isoacrostalidic acid from 455 3-Acyl tetramic acid 111,112,114 from Alternaha alternata 114 from Alternaha longipes 114 from Alternaha tenuis 114 biosynthetic pathways of 111 from Pyricularia oryzae 114 tautomeric forms of 112 Adenichrome 647 Fe(III)-containing pigment as 647 from Octopus vulgaris 647 Adociasp. 674 adociaquinone A from 678 adociasulfate from 674 from Great Barrier Reef 674 structure of 674 Adociasulfates 1-6 674 as kinesin motor proteins inhibitors 674 from Haliclona (aka Adocia) sp. 674
Adociidae family 664 10-isothiocyanatobiflora-4,15diene of 664 spectral analysis of 664 P-Adrenoceptors 183 with [^H]dihydroalprenolol 183 AflastatinA 127,128 as aflatoxin inhibitor 128 from Streptomyces griseochromogenes 127 structure of 128 AflastatinB 127 as aflatoxin inhibitor 128 from Streptomyces griseochromogenes 127 Anatoxins 128 from Aspergillus flavus 128 from Aspergillus nomius 128 from Aspergillus parasiticus 128 from Aspergillus tamarii 128 African tick species 396 Amblyomma hebraeum as 396 Boophilus decoloratus as 396 Hyalomma sp. as 396 Rhipicephalus appendiculatus as 396 Rhipicephalus evertsi evertsi as 396 Agelas dispar 692 from Bahamas 692 pyridinebetaine B of 692 Agelas nakamurai 670 agelasidine A from 670 agelasidine B from 670 antispasmodic activity of 670 Na^/K^-transporting adenosine triphosphate (ATP)ase inhibitor from 670 spectral data of 670 structure of 670 synthesis of 670 Agricultural pests 423 of forage crops 423 of fruits 423 of ornamentals 423 of timber 423 of vegetables 423
755
Ajoene 432 acaricidal activity of 432 against Tetranyehus urticae 432 anticoagulant properties of 432 Akaterpin 673 as phosphatidylinositolphospholipase C inhibitor 673 from Callyspongia sp. 673 stereochemistry of 673 AlbanolA 17 as aromatase inhibitor 17 Albanol B 234 from Morus uralensis 234 Alcyonidium gelatinosum sp. 619 (2-hydroxyethyl) dimethylsulfoxonium ion from 619 Dogger Bank itch by 619 Aldose reductase inhibitor 691 from Dictyodendhlla sp. 691 a-Alkyl-p-hydroxyproline moiety 367 construction of 367 Allelopathic activity 483 of natural podolactones 484 of synthetic podolactones 484 Allium sativum (LilidicediQ) 415 Alphitolic acid 40 from Licania heteromorpha var. heteromorpha 40 Althiomycin 143 from Cystobacter fuscus 144 frovci Myxococcus xanthus 144 from Streptomyces althioticus 143 from Streptomyces matensis 143 Amblyomma 394,397 by Beauveria bassiana 397 by hyperparasitic fimgi 397 by Metarhizium anisopliae 397 control of 397 in goats 394 in sheeps 394 Amblyomma variegatum 395 repellent properties of 395 [^H]Amine uptake 183 of Ginkgo biloba L. 183 Ancorinoside A 120 fvom Ancorinasp. 120
Ancorinoside A Mg salt 120 ficom Ancorinasp. 120 Ancorinoside B 120 ficom Ancorinasp. 120 Ancorinoside C 120 from Ancorina sp. 120 Ancorinoside D 120 from Ancorinasp. 120 Annona glabra seeds 430 acetogenins from 430 against Dermatophagoides pteronyssinus 430 against Typhlodromus urticae 430 asimicinfrom 430 desacetyluvaricin from 430 squamocinfrom 430 Annona squamosa 415 extract of 415 Anthelmintics 331,332 broad spectrum activity of 332 diminished activity of 331 new class of 332 Anthocyanidins 275 cyanidinas 275 delphinidin as 275 malvidinas 275 pelargonidin as 275 structure of as 275 Anthocyanins 275,276,277,292 cyanidin-3-glucoside as 275 delphinidin-3-glucoside as 275 for coronary heart disease 292 from black grapes 276 in blackberry 277 in blueberry 277 in cabbage, red 277 in cherry 277 inchokeberry 277 in cranberry 277 in currant (black) 277 in food plants 276 in grape (red) 277 in onion 277 in organe, blood O'uice) 277 in raspberry, red 277 in strawberry 277 in wines, porto 277 in wines, red 277
756
malvidin-3-glucoside as 275 pelargonidin-3-glucoside as 275 Anthopleura elegantissima 647 mycosporine-taurine from 647 Anti-apoptotic effects 175 Antiahs toxicaria 203 antiarone A from 203 antiarone B from 203 antiarone E from 204 antiarone J from 203 antiarone K from 203 ficusins A from 204 uses for arrow poison 203 Anti-atherosclerotic activity 257,293 of polyphenols 257,293 Anti-bacterial activity 140 of ikarugamycin 140 Anti-carcinogenic activity 257,293 of polyphenols 257,293 Anti-con vulsant activity 176 ofbilobalide 176 Anti-depressant effects 177 of Ginkgo biloba L. 177 Anti-feedant activity 480 against mammals 480 of l-deoxy-2P,3p-epoxynagilactoneA 480 of nagilactone A 480 of nagilactone C 480 Anti-ftingal activity 66,473,475 of2-hydroxynagilactoneF 475 of intrapetacin A 66 of intrapetacin B 66 of LL-Z1271a 473 of nagilactone C 475 of nagilactone E 475 of oidiodendrolideB 475 ofoidiolactoneD 475 Anti-fungal holothurin 597 from Psolus patagonicus 597 Anti'Helicobacter pylori activities 234,241,243 of 6,8-diprenylorobol 243 of dihydrolicoisoflavone A 243 of formononetin 241 of gancaonini 243 ofgancaonol B 243
of gancaonol C 243 ofglabrene 241 ofglabridin 241 ofglyasperinD 243 ofglycyrin 243 ofglycyrol 241 ofglycyrrheticacid 241 ofglycyrrhizicacid 241 of isoglycyrol 241 of isolicoflavonol 243 oflicochalcone A 241 oflicochalconeB 241 oflicoisoflavoneB 241 of licorice flavonoids 234 of licorice-saponin 241 oflicoricidin 241 oflicoricone 243 ofliquiritigenin 241 ofliquiritin 241 of l-methoxyphaseoUidin 243 of3-(9-methylglycyrol 243 ofvestitol 243 Anti-Human inmiunodeficiency virus (HIV) activity 225,226 of antiarone I 226 of broussoflavonol B 226 of broussoflavonol C 226 ofgancaoninR 226 ofglyasperin A 226 ofglycyrol 226 ofkazinolB 226 of kumatakenin 226 ofkuwanonH 225 oflicochalconeB 226 ofmoracinC 226 of morusin 225 of mulberry tree 225 ofnorartocarpetin 226 ofprenylflavones 225 of wighteone 226 Anti-HIV flavonoids 226 2-arylbenzofiiran as 226 from Glycyrrhiza species 226 from moraceous plants 226 Anti-inflammatory activity 200,257,293 of genus Morw^ 200 of polyphenols 257,293
757
Anti-metastatic activity 559 of natural products 559 Anti-microbial activity 62,224,257,293 ofalphitolicacid 62 of AMOX 224 ofbetulinicacid 62 of formononetin 224 ofgancaonini 224 ofgancaonolB 224 of glabrene 224 of glabridin 224 ofglyasperinD 224 of glycyrin 224 of glycyrol 224 of isoglycyrol 224 of isolicoflavonol 224 of Licania heteromorpha var. heteromorpha 62 of licochalcone A 224 of licochalconeB 224 of licoisoflavoneB 224 of licorice flavonoids 224 of licoricidine 224 of licoricone 224 of liquiritigenin 224 of liquiritin 224 of 3-0-methylglycyrol 224 of 3|3-0-d5-/?-coumaroyl maslinic acid 62 of 3P-0-cw-/?-coumaroyl alphitolic acid 62 of 3 ^'O'tranS'P'C0\xmdC[0y\ alphitolic acid 62 of 3 p-(9-^r<3f/w-/?-coumaroyl maslinic acid 62 of polyphenols 257,293 of vesititol 224 Anti-neoplasic activity 470 against P 388 cells 470 Anti-oxidant activity 63,179,257,293 of Ginkgo bilobaL. 179 of 8-hydroxy-naringenin 64 of kaempferol 3-(9-(2"-P-Dxylopyranosyl)-a-Zrhamnopyranoside 64,65 of kaempferol 3-0-a-Irhamnopyranoside 64
of Licania licaniaeflora 63 of polyphenols 257,293 .of quercetin 3-(9-a-Zarabinopyranoside 64 of quercetin 3-0-a-Zrhamnopyranoside 64,65 of quercetin 3-O-P-Z)galactopyranoside 64 of quercetin-3 -O-a-Lrhamnopyranoside 64 of taxifolin 3-0-a-Zrhanmopyranoside 64 Anti-pyretic activity 200 of genus M(9rw5 200 Anti-radical activity 257,293 of polyphenols 257,293 Anti-stress effects 177 of Ginkgo biloba L. 177 Anti-tumor activity 517,519,559,560, 566,211 of carp oil 566 ofchitosan 560 of Coley's toxin 519 offish oils 566 of lipids 517 ofmorusin 211 of natural products 559 of tuna oil 566 Anti-tumor substances 569 from Agaricus blazei 569 isolation of 569 mechanism of 569 Anti-tussive activity 200 of genus M^rw^ 200 Anti-viral activity 257,293 of polyphenols 257,293 Anxiolytic activity 177 of Ginkgo bilobaL. 177 Apiculture 390 acaricides in 390 Apiguard 391 for thymol-based acaricide 391 ApilifeVAR 391 as Frakno thymol frame 391 a-and P-Apiodionen 117 as topoisomerase inhibitor 117
758
Apis mellifera 387 killed by bee larvae 387 Aplidium pliciferum 635 1,2,3-trithiane derivative from 635 Aplysia kurodai 648 metabolites of 648 neoaplaminone sulfate of 648 AplysillinA 697 as thrombin receptor antagonist 697 from Aplysinafistularis 697 Apoptosis 175 ofbilobalide 175 ofginkgolideB 175 ofginkgolide J 175 using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide 175 Arachidonate 5-lipoxygenase activity 216 of artoninE 217 of morusin 217 Arachidonate oxygenase activity 217 Arachnida 382 class of 382 Arjunic acid 28-P-D-glucosyl ester 40 from Licania licaniaeflora 40 Artemisia absinthium 425 acaricidal activity of 425 against aphids 425 as insecticide 425 Arthropod-borne diseases 394 of humans 394 of livestock 394 zoonotic as 394 Artobiloxanthone 201 from moraceous plants 201 Artocarpesin 201 from moraceous plants 201 y4rrocaA77W5 flavones 216 artoninA 216 artoninB 216 artoninE 216 cycloartobiloxanthone 216 cycloheterophyllin 216 heterophyllin 216 morusin 216 5-lipoxygenase activity of 216
Artocarpus f[dMono\As 203,216,220 against arachidonate 5-lipoxygenase 216 against TNF-a release 220 artocarpesin 203 artonini 203 morachalcone A 203 Artocarpus alt His 202 Artocarpus communis 202 Artocarpus heterophyllus 202 Artocarpus rigida 202 Artocarpus venenosa 202 artobiloxanthone from 202 artoninE from 202 cycloartobiloxanthone from 203 isoprenylated flavonoids from 202 use against inflammation 202 use in malarial fever 202 use as folk medicine 202 ArtoninE 201,215 as 5-lipoxygenase inhibitor 215 from moraceous plants 201 Artonini 201 from moraceous plants 201 Ascidia mentula 642 heneicosane-1,21 -diyl disulfate from 642 3,7,11,15-tetramethylhexadecane1,19-diyl disulfate from 642 Ascosalipyrrolidinone A 148 against antibacterial activity of 148 against chloroquine-resistant Plasmodium falciparum 148 2igmnst Trypanosoma brucei 148 against Trypanosoma cruzi 148 Aspalathus linearis 272 AurantosideA 136 from Theonella swinhoei 136 AurantosideB 136 from Theonella swinhoei 136 Austrovenus stutchburyi 656 brevetoxin Bi from 656 Autotoxic effects 537 of NO on tumor cells 537 Avermectin derivatives 402 from Boophilus sp. 402 Axinella cannabina 658 axisothiocyanates from 658
759
axisothiocyanate 2 from 658 axisothiocyanate 3 from 658 axisothiocyanate 4 from 658 sesquiterpenoid from 658 Axinyssa fenestratus 662 10-isothiocyanato-4,6amorphadiene from 662 4-isothiocyanato-9-amorphene from 662 10-isothiocyanato-5-amorphen-4ol from 662 Azadirachta indica 401 effects on 5oo/7Mw5 sp. 401 Azadirachta indica dust 389 effects on honey production 389 Azadirachta indica oil 388,395 against bee mites 387 against blood sucking ticks 395 from azadirachtin 388 BALB/cmice 534 Meth A sarcoma in 534 Bacillus subtilis ll'i effects of phenols on 223 Bacterial origin 434 acaricidal agents of 434 Barley 260 syringic acid in 260 vanillic acid in 260 Bastaxanthin B 714 as novel carotenoid sulfate 714 from lanthellaflabelliformis 714 from marine sponge lanthella basta 714 Bastaxanthin C 714 as novel carotenoid sulfate 714 from lanthellaflabelliformis 714 from marine sponge lanthella basta 714 Bastaxanthin D 714 as novel carotenoid sulfate 714 from lanthellaflabelliformis 714 from marine sponge lanthella basta lU Bastaxanthin E 714 as novel carotenoid sulfate 714 from lanthellaflabelliformis 714
from marine sponge lanthella basta 714 Bastaxanthin F 714 as novel carotenoid sulfate 714 from lanthellaflabelliformis 714 from marine sponge lanthella basta 1\A Batzella spongQ 685 against Candida albicans 686 antifimgal activity of 686 batzellines A from 685 methyl sulfide groups of 685,686 Bee parasites 387 formic acid in 387 Tropilaelaps clareae as 387 Benzyl benzoate 416 digdmstPsoroptes 416 against Sarcoptes mites 416 against Sarcoptes scabiei 416 effectiveness of 416 in mange control 416 Betulinic acid 17,40 cytotoxic activity of 17 from Licania heteromorpha var. heteromorpha 40 from Licania licaniaeflora 40 bom Licania pittieri 40 Bicyclic phloeodictine B 688 antibacterial activity of 688 cytotoxic activity of 688 cyclic aminoketal from 688 from Phloeodictyon 688 Bicyclo[2.2.2] ring system 363,370 construction of 363,370 Biflustra perfragilis sp. 620 2-methyl-6,7-di(methylthio)-2^isoquinoline-3,5,8-trione from 619 2-methyl-6-methylthio-2i/isoquinoline-3,5,8-trione from 619 Bioactive compounds 3,4,9,16,22 bom Broussonetia gcmis 3 from Broussonetia kazinoki 4,9 from Broussonetia papyrifera 16,22 structure of 9
760
Bioactive monoterpenoids 426 carvacrolas 426 carvomenthenol as 426 carvoneas 426 citronellol as 426 eugenol as 426 geraniol as 426 perillyl alcohol as 426 4-terpineol as 426 thymol as 426 Bioavailability 289 of catechins 289 Biological activity 109,212,228,340 ofalbaninD 229 ofalbaninF 230 ofalbanolA 230 ofalbanolB 229 of alvaxanthone 229 ofangustoneB 228 ofantiaroneB 229 ofantiaroneF 229 ofantiaroneG 229 ofantiaroneH 229 ofantiaronel 229 ofantiaroneJ 229 of artobiloxanthone 229 ofartoninE 229 of bavachalcone 228 ofbroussochalconeB 228 ofbroussoflavonolB 229 of broussoflavonol C 229 ofbroussoflavonolE 230 ofcudraphenoneB 230 ofcudraphenoneD 230 of cycloartobiloxanthone 230 ofdehydroglyasperinC 228 of 3 '-(y,Y-dimethylallyl)-kievitone 228 of8-(Y,y-dimethylallyl)-wighteone 228 of echinatin 228 of edudiol 228 oferythrininB 229 of formaononetin 228 of gancaoinin S 228 ofgancaoninC 228 ofgancaoninE 228
ofgancaoninG 228 ofgancaoninH 228 ofgancaonini 228 ofgancaoninO 228 ofgancaoninP 228 ofgancaoninQ 228 ofgancaoninR 228 ofgancaoninU 228 ofgancaoninV 228 ofgancaoninY 228 of glabranin 228 of glabranine 228 of glabrene 228 of glabridin 228 ofglabrocoumarone A 229 ofglabrol 228 of glisoflavanone 228 ofglyasperin A 228 ofglyasperinB 228 of glyasperin C 228 of glyasperin D 228 ofglyasperin J 228 ofglyasperin K 228 of glycycoumarin 228 ofglycyrdione A 228 of glycyrin 228 ofglycyrol 228 ofglyinflaninA 228 of heterophyllin 230 ofhispaglabridin A 228 of3-hydroxyglabrol 228 of 14a-hydroxy-MFA 340 of3-hydroxyparatocharpinC^ 228 of isoalvaxanthone 230 of isoderrone 228 of isoglycyrol 228 of isoliquiritigenin 228 ofkanzonolB 229 ofkanzonolG 229 ofkanzonolH 229 ofkanzonolP 229 of kanzonol R 229 ofkanzonolS 229 of kanzonol U 229 of kanzonol V 229 of kanzonol W 229 of kanzonol X 229 of kanzonol Y 229
761
ofkazinolB 230 of kazinol E 230 ofkazinol F 230 of kazinol N 230 ofKB-1 229 ofKB-3 229 ofkumatakenin 229 ofkuwanon 212 ofkuwanonC 230 ofkuwanon G 230 ofkuwanon H 212 ofkuwanon M 212 ofkuwanon R 230 oflicochalcone A 229 of licochalconeB 229 of licoflavonol 229 of licoisoflavanone 229 oflicoisoflavone A 229 oflicoisoflavoneB 229 of licoricidin 229 of licoricone 229 of licorisoflavan A 229 ofmacarangaflavanoneB 229 of medicarpin 229 of 1-methoxyficifolinol 229 of l-methoxyphaseoUidin 229 ofmoraceinB 230 ofmoracinC 230 ofmorusin 212,230 of morusinol 230 of mulberrin 230 of mulberrochromene 230 ofmulberrofuranB 230 of mulberrofliran G 212,230 of naringenin 229 of norartocarpetin 230 of3-(9-methylgancaoninP 229 of 4'-0-methylglabridin 229 of oxydihydromorusin 230 of paratocarpin L 229 of phaseoluteone 229 of pinocembrin 229 of 6-prenyleriodictyol 229 of 8-prenyleriodictyol 229 of 6-prenylnaringenin 229 ofsanggenolC 230 ofsanggenolM 230 ofsanggenonA 212,230
ofsanggenonB 230 ofsanggenonC 212,230 ofsanggenonD 212 ofsanggenonM 230 ofsemilicoisoflavoneB 229 of shinpterocarpin 229 ofsigmoidin A 229 ofsigmoidinB 229 ofsoroceinF 230 oftenuifolinB 229 of tetramic acid-containing compounds 109 of topozolin 229 of wighteone 229 Biological properties 518,519 of lipid A 519 Biological studies 35 on Z/cama genus 35 Biosynthesis 15 ofbroussonetine J 15 of broussonetine U 15 5/5^eco-dehydrocyclopiazonic acid 119 BivittosideC 589,590 from Bohadschia bivittata 589 structure of 590 Blasticidin 127 as antibiotic 127 from Streptomyces griseochromogenes 127 Bombesin receptor antagonists 221 from Morw^ species 221 ofkuwanon G 221 ofkuwanon H 221 Boophilus decoloratus 400 for Capsicum sp. 400 for Euphorbia brachypoda 400 for Euphorbia obovalifolia 400 of Solarium incanum 400 Boophilus microplus 399,398 eugenol of 399 isoeugenol of 399 lethal effect on 398 synthetic acaricides for 399 Boophilus ticks 398 in tropical regions 398 sub-tropical regions 398 BrevetoxinB4 656 as toxin 656
762
from Perna canaliculus 656 in shellfish poisoning 656 Bripiodionen 117,118 as human cytomegalovirus protease inhibitors 118 cytotoxicity of 118 from Streptomyces sp. 117 BrosimoneA 201 from moraceous plants 201 BrosimoneD 201 from moraceous plants 201 Broussoaurone A 16,17 antioxidant activity of 16,17 as cyclooxygenase inhibitor 16,17 as neutrophils respiratory burst inhibitor 16 as nitric oxide production inhibitor 16 as platelet aggregation inhibitor 16,17 Broussoflavan A 16 antioxidant activity of 16 as platelet aggregation inhibitor 16 Broussoflavonol E 17 as platelet aggregation inhibitor 17 Broussoflavonol F 17 antioxidant activity of 17 antiproliferative activity of 17 as aromatase inhibitor 17 as cyclooxygenase inhibitor 17 as platelet aggregation inhibitor 17 Broussoflavonol G 17 antiproliferative activity of 17 antioxidant activity of 17 Broussonetia kazinoki 4,5 antifimgal activity of 4 antiinflammatory activity of 4 antioxidant activity of 4 antispasmodic activity of 4 broussonetine C from 5 broussonetine D from 5 broussonetine E from 5 broussonetine F from 5 broussonetine G from 5 broussonetine H from 5 broussonetine K from 5 broussonetine L from 5 broussonetine M from 5
broussonetine N from 5 broussonetine O from 5 broussonetine P from 5 broussonetine Q from 5 broussonetinine A from 5 broussonetinine B from 5 broussonol A from 6 broussonol B from 6 broussonol C from 6 broussonol D from 6 7,4'-dihydroxyflavan from 6 for increased vision 4 kazinol A from 6 kazinolDfrom 5 kazinol E from 6 kazinol K from 5 kazinol Q from 6 kazinol R from 6 sexual potency of 4 Broussonetia papyrifera 4,16 anti-cancer activity of 4 antifungal activity of 4 antioxidant activity of 4 diaphoretic activity of 4 in dyspepsia 4 in pregnancy 4 laxative activity of 4 Broussonetia zeylanica 4,28 aromatase inhibitory substance from 4 broussonetine from 28 cytotoxic flavonoids from 4 3,4'-dihydroxy-2,3 '-bipyridine from 28 8-hydroxyquinoline-4carbaldehyde from 28 8-hydroxyquinoline-4carbaldehyde oxime from 28 glycosidase inhibitory alkaloids from 4 BroussoninA 16 antiftmgal activity of 16 as aromatase inhibitor 16 BroussoninB 16 antifungal activity of 16 Bryozoans 618 as moss animal 618 lophophoreof 619
763
sulflir-containing 618 U-shaped gut of 619 zooids of 619 C-4-O-acetyl derivative 117 Cadlina luteomarginata 659 sesquiterpene isothiocyanate from 659 Caffeic acid 262 in apples 262 in blueberries 262 in cider 262 in coffee beverages 262 Caffeoylquinic acids 262 in apples 262 in blueberries 262 in cider 262 in coffee beverages 262 Califomian sponge 671 absolute configuration of 671 antiinflammatory activity of 671 antimicrobial activity of 671 halisulfates 1-5 from 671 in Halichondriidae family 671 spectral data of 671 Callyspongin A 712 as starfish fertilisation inhibitor 712 of Callyspongia truncata 112 Callyspongin B 712 as starfish fertilisation inhibitor 712 of Callyspongia truncata 112 Calpurnea aurea 400 killing effects on ticks 400 Calyceramide A 713 as neuraminidase inhibitors 713 fvom Discodermia QdXyx 713 Calyceramide B 713 as neuraminidase inhibitors 713 from Discodermia CdXyx 713 Calyceramide C 713 as neuraminidase inhibitors 713 fxom Discodermia Cd\y\ 713 Capsicum species 436,400 capsaicin from 436 killing effects of ticks 400
Capsimycin 138 antifungal activity of 138 from Streptomyces strain 13 8 Carboxyhomoyessotoxin 654 from DSP-infested Mytilus galloprovincialis 654 from mussels 654 structure of 654 Caribbean collection 632 of Didemnum rodriguesi 632 minalemines D from 632 minalemines F from 632 Carp (Cyprinus carpi) 564 antitumor activity of 564 as diuretic 564 for eye fatigue 564 Caspase activity 320 of TNF-a 320 CassigarolA 575 effects on tumor growth 575 Catechins 41,273 (+)-catechin 273 (-)-epicatechin 273 epigallocatechin 273 epicatechin-gallate 273 epigallocatechin-gallate 273 from Licania densiflora 41 from Licania pittieri 41 Cattle babesiosis 398 by Babesia bigemina 398 by Babesia bovis 398 by Ba^^i'/a species 398 Cavemothiocyanate 662 of Acanthella cf cavernosa 662 spectral data of 662 Cell wall conjugates 262 in cereal brans 262 in spinach 262 in sugar beet fibre 262 Cerastoma brevicaudatum 651 (methylthio)furodysinin from 651 dithiofiirodysinin disulfide from 651 Ceratodictyon spongiosum 683 cw,OT-ceratospongamideof 683 Sigmadocia symbiotica of 683 trans, ^/-a/i5-ceratospongamide of 683
764
Chalcomoracin 210 sinapic (3,5-dimethoxy-4-hydroxy) frommoraceousplants 210 as 261 Chemical studies 35 Cinnamon bark 259 on Z/cawa genus 35 gallic acid from 259 Chemical reactivity 488 salicylic acid from 259 ofpodolactones 488 syringic acid from 259 Chemotaxonomic study 36 Cisplatin(CDDP) 559 of Parinari gQnns 36 for cancer chemotherapy 559 Chinese crude drug 202 Classification 281 sanggenon A from 202 of ellagitannin oligomers 281 sanggenon C from 202 ofellagitannins 281 sang-Bai-Pi as 202 ofgallotannins 281 Chinese mulberry tree 202 of galloylated proanthocyanidins isoprenylated flavonoids from 202 281 Morus cathayana as 202 of proanthocyanidins 281 Chlamys hastata 111 ofprodelphinidins 281 from Strongylocentrotus pupuratus of tannins 281 111 Clathsterol 702 Chlorosulfolipid 655 against HIV-1 reverse transcriptase from hepatopancreas 655 (RT) 702 stereochemistry of 655 from Red Sea sponge Clathria sp. Chitosan 563 702 Clavelina cylindhca 644 effects on doxorubicin-induced gastrointestinal toxicity 563 cylindricine F from 644 preventive effects of 563 genistic acid 259 Chitosan inhibitory effects 561 Clove buds 259 on gastrointestinal toxicity 561 protocatechuic acid in 259 on immunocompetent organ syringic acid in 259 toxicity 561 Cnidaria 646 on myelotoxicity of 5-FU 561 corals among 646 Cholesterol 373 gorgonians among 646 oxygenation of 373 hydrozoans among 646 Chorioptes mitQS 410 jellyfish among 646 attack on fetlocks 410 living species of 646 Chromodoris elisabethina 650 sea anemones among 646 icthyotoxic latrunculin A from 650 soft corals among 646 Chronic administration 182 Comantherin sulfate 719 of Ginkgo bilobah. 182 from Comantheria perplexa 719 Chrysanthemum cinerahfolium 384 Comatula pectinata 719 anthraquinones from 719 pesticide properties of 383 Combined effects 562 secondary metabolites from 384 of 5-FU and chitosan 562 Cinnamic acids 261 Complex I inhibitor 436,437 caffeic (3,4-dihydroxycinnamic) annonaceous acetogenins as 437 acid as 261 capsaicin as 436,437 ferulic (3-methoxy-4-hydroxy) as 261 deguelinas 436 monotetrahydrofiiranic derivatives /?-coumaric (4-hydroxy) acid 437 as 261
765
piericidin A as 436 rotenoloneas 436 rotenone as 436 tephrosinas 436 cw-Communic acid 456 /m/w-Communic acid 456 ciS' and /r^A^^-Communic acids 456 Corallistine 688 from Corallistes fulvodesmus 688 X-ray crystallography of 688 Coronaridin 234 Coscinoderma mathewsi 672 as protein phosphatase inhibitor 672 1-methylherbipoline salts from 672 serine protease inhibitors from 672 sesterterpeneshalisulfate-1 from 672 suvaninefrom 672 total synthesis of 672 Cowdria ruminantium 394 as rickettsial pathogens 394 Coxiella burnetii 394 in cattle 394 insheeps 394 Crella sp. 706,707 crellastanin A from 706 crellastatins B-M from 707 Crella spinulata 708 benzylthiocrellidone from 708 dimedone unit of 708 Cribricellina cribraria sp. 619 P-carboline alkaloid from 619 carbolinefrom 619 1 -ethyl-4-methyl-sulfone-Pantimicrobial activity of 619 Crocetin 314 structure of 314 Crocin 315 artificial pathway of 314 Crocin effects 316,319 on LTP-blocking effects of ethanol 316 on TNF-a-induced cell 319 Crocus sativus 315 ethanol extract of 315
effect on central nervous system 315 Crustacea 382 class of 382 Cryptocin 123 against phytopathogenic fimgi 123 Cryptosporiopsis cf. quercina 122 from Tryptergyium wilfordii 122 from Sclerotinia sclerotiorum 123 Cucumaria echinata 720 neuritogenic activity of 720 Cucumariafrondosa 720 2,6-dimethylnonane-1 -sodium sulfate from 720 2,4,6-trimethyl-nonane-1 -sodium sulfate from 720 Cucumariajaponica 595 cucumarioside-Ao-3 from 595 cucumarioside-Ai-2 from 595 cucumarioside-A2-l from 595 cucumarioside-A2-2 from 595 cucumarioside-As from 595 cucumarioside-A4-2 from 595 cucumarioside-A6-2 from 595 cucumarioside-A7-l from 595 cucumarioside-A7-2 from 595 Cucumaria lefevrei 596 from lefevreioside Ai from 596 from lefevreioside A2 from 596 from lefevreioside B from 596 from lefevreioside C from 596 Cucumariosides A7-3 593 from Stichopus chloronotus 593 from Thelenota ananas 593 Cucumariosides A2-4 593 from Stichopus chloronotus 593 from Thelenota ananas 593 Cyanogen iodide (ICN) 336 forcyanation 336 for cyanation of aromatic compounds 336 ofalkenes 336 Cycloartobiloxanthone 201 from moraceous plants 201 Cyclopiazonic acid 119 as calcium uptake inhibitor 119 from Penicillium cyclopium 119
766
Cycloshermilamine D 639 from Cystodytes violatinctus 639 Cylindramide 139 cytotoxic activity of 139 from Halichondria cydindrata 139 Cymbastela hooperi 665 diterpene isothiocyanates from 665 Cymbopogon citratus 398 essential oils from 398 Cymbopogon nardus 398 essential oils from 398 Cystodytes sp. from Fiji 638 dehydrokuanoniamine B from 638 shermilamine C from 638 Cytochrome C 321,325 from mitochondria 321 levels in PC-12 cells 325 Cytoplasmic proteins 321 inapoptosis 321 Cytotoxic activity 65,217,220,227, 468,470 of Antiaris toxicaria (Moraceae) 220 ofantiaronel 227 ofantiaroneJ 220 ofantiaroneK 220 ofantiaroneL 220 of artocarpusflavonoids 220 ofartoninA 220 ofartoninB 220 ofartoninE 217,227,220 ofartoninH 220 ofbroussoflavonolB 227 ofbroussoflavonolC 227 ofcucurbitacinB 65 of cycloheterophyllin 220 of 1 -deoxy-2a-hydroxy-nagilactone A 468 of 3-deoxy-2a-hydroxynagilactone E 470 of 2,3 -dihydro-16-hydroxypodolide and 16-hydroxypodolide 468 ofgancaoninR 227 ofglyasperin A 227 of glycyrol 227 of heterophyllin 220
of2a-hydroxynagilactoneF 470 of inumakilactone A 468 of inumakilactoneB 468 ofkazinolB 227 of I/ca«/a genus 65 ofLicania heteromorpha 65 ofLicania michauxii 65 oflicochalconeB 227 of licoricidin 227 of 15-methoxycarbonyl nagilactoneD 468 of morusin 227 ofnagilactone A 468 of nagilactone B 468 of nagilactone C 468 ofnagilactone D 468 of nagilactone E 468 ofnagilactone F 468 ofnagilactone G 468 ofnorartocarpetin 227 ofpodolactoneE 470 Dakaira subovoidea 620 6//-anthra[ 1,9-6c]thiophene derivatives 620 2,3-Dehydro-16-hidroxi-nagilactone F 464 from Podocarpus nagi 464 2,3-Dehydro-1-deoxy-nagilactone A 460 from Podocarpus nagi 460 Dehydromoracin C 210 from moraceous plants 210 2,3-Dehydro-nagilactone A 460 from Podocarpus nagi 460 Dehydroprenyl flavonoid 203 from Artocarpus s^iQZXQS 203 from Asian Morus species 203 from Brosimopsis oblongifolia 203 Demethylmoracin I 18 as aromatase inhibitor 17 Dendrodoa grossularia 633 dendrodoine from 633 Deoxy-2a-hydroxy-nagilactone A 458 from Podocarpus nagi 458 l-Deoxy-2p,3P-epoxynagilactone A 459 from Podocarpus nagi 459
767
3-Deoxy-2a-hydroxy-nagilactone E 463 from Ileostylus micranthus 463 from Podocarpus nagi 463 1-Deoxy-nagilactone A 459 from Podocarpus nagi 459 3-Deoxy-nagilactone C 459 from Ileostylus micranthus 459 12a-Deoxytetracycline 373 hydroxylation of 373 Dercitinsp. 686 antitumor activity of 686 antiviral activity of 686 immunomodulatory properties of 686 violet acridine alkaloid from 686 Dermatophilus congolensis 394 skin infection of cattle by 394 Desholothurin A 592 from Holothuriaforskali 592 2-Desoxo-15a-methyl-14a-hydroxy-MFA 347 discovery of 347 nematocidal activity of 347 2-Desoxoparaherquamide A 331 nematocidal activity of 331 2-Desoxo-PHA 347 discovery of 347 Detox 543 for immunotherapy 543 P-D-glc-nagilactoside B 460 from Podocarpus nagi 460 P-D-glc-nagilactoside D 460 fxom Podocarpus nagi 460 P-D-glc-nagilactoside E 460 from Podocarpus nagi 460 28-P-D-glucosyl ester 40 from Licania licaniaeflora 40 from Licania pyrifolia 40 Dibenzyltrisulfide 403 as oviposition inhibitor 403 from Petiveria alliacea L. 403 Didemnum chartaceum 631 lamellarin G 8-sulfate from 631 lamellarin sulfates from 631 lamellarin B from 631 lamellarin C from 631 lamellarin L from 631
Didemnum molle 630 comoramide A from 630 comoramide B from 630 cyclodidemnamide from 630 mayotamide A from 630 mayotamide B from 630 mollamide from 630 Didemnum rodriguesi 632 alkaloid caledonin from 632 Didemnum sp. 631,634 anti-human immunodeficiency virus (anti-HIV) activity of 631 didemnaketal C from 631 didemnoline A from 634 didemnoline D from 634 polysaccharide kakelokelose from 631 2,3-Dihydro-16-hydroxypodolide 462 from Podocarpus nagi 462 Dihydrochalcone 234,271 aspalathin from 272 from Dracaena loureiri 234 in apple juice 271 in cider 271 in pomace 271 nothofagin from 272 Dihydrodeoxy-nubilactone A 466 from Podocarpus saligna 466 Dihydromyr 3-rha 41 from Licania licaniaeflora 41 (3',4'-Dihydroxy)benzoylester 40 from Licania pyrifolia 40 (2S)-2',4'.Dihydroxy-2"-(l-hydroxy-lmethylethyl)-dihydrofiiro[2,3A]flavanone 17 as aromatase inhibitor 17 2a,27-Dihydroxybetulinic aicd 40 from Licania pyrifolia 40 3 p,4p-Dihydroxypregn-5-en-20-3sulfate 704 5-pregnene skeleton of 704 from Stylopus australis 704 2a,3a-Dihydroxyurs-12-ene-28-oic 40 from Licania pyrifolia 40 Diketopiperazinecvc/(9-(L-proline-Lthioproline) 692
768
bacterial origin of 692 from ledania ignis 692 Diketopiperazines 362 containing dioxepinoindole ring 362 (3Z)-4,8-Dimethyinon-3-en-1-yl sodium sulfate 721 as sulfated alkene 721 from Microcosmus vulgaris 721 from Ophiocoma echinata 721 Dioxepinooxindole ring system 360 construction of 360 Diplosoma sp. 636 diplamine from 636 Discodermia 138,679 antifungal activity of 139 cytotoxic activity of 139,679 deepwater species of 679 polydiscamide A from 679 Diuretic activity 200 of genus Morw^ 200 DNA topoisomerase I inhibitors 621 ceramide 1-sulfates as 621 p-D-nagilactoside C 460 from Podocarpus nagi 460 Docosahexaenoic acid 565 in fish oils 565 Dolastatin3 648 as antineoplastic agent 648 from Dolabella auriculaha 648 sequence of 648 synthesis of 648 Dolastatin 10 145,649 antiproliferative activity of 145 as antineoplastic agent 649 from Dolabella auricularia 649 Dolastatin 15 145 antiproliferative activity of 145 Doris verrucosa (nudi branch) 651 9-(5-deoxy-5-methylthio-P-DxylofiiranosyOadenine from 651 digestive glands of 651 Doxorubicin 559,560,562 as cancer chemotherapy drug 559 gastrointestinal toxicity by 562 immunotoxicity by 562 myelotoxicity by 562
Doxorubicin plus chitosan 560 tumor growth inhibition by 560 Ds-Penaustroside A 599 from sea cucumber 599 Ds-Penaustroside B 599 from sea cucumber 599 Dysideaavara 668 melemeleone A from 668 spectroscopic analyses of 668 Dysidea herbacea 665,666 dysideathiazole from 665 13-demethylisodysideninfrom 666 13-demethyldysideninfrom 666 13-demethylisodysideninfrom 666 13-demethyldysideninfrom 666 isodysidenir from 665 9-monodechloro-13-demethylisodysidenin from 666 1 l-monodechloro-13-demethylisodysidenin from 666 polychlorinated peptides from 665 Dysidea sp. 667,668 15-acetylthioxyfiirodysinin lactone from 667 P-adrenoreceptor agonist from 668 barbaleucamide A from 668 benzothiazole S1319 from 668 dysideaproline A from 668 dysideaproline B from 668 dysideaproline C from 668 dysideaproline D from 668 dysideaproline E from 668 dysideaproline F from 668 from Okinawa 668 proline-derived analogues of dysideninfrom 668 Dysideathiazole A 667 as polychlorinated amino acid derivative 667 X-ray analysis of 667 Dysideathiazole B 667 as polychlorinated amino acid derivative 667 X-ray analysis of 667 Dysideathiazole C 667 as polychlorinated amino acid derivative 667 X-ray analysis of 667
769
Dysideathiazole D 667 as polychlorinated amino acid derivative 667 X-ray analysis of 667 Dysideathiazole E 667 as polychlorinated amino acid derivative 667 X-ray analysis of 667 Dysidin 142 antifeedant activity of 142 from Dysidea herbacea 142 immunosuppressant activity of 142 moUuscidal activity of 142 Echinoclasterol sulfate 705 as antifungal 705 as cytotoxic steroid 705 from Echinoclathria subhispida 705 phenethylammonium salt 705 Echinoderms 716 classes of 716 histidine derivatives from 716 living species of 716 metabolites of 716 Echinodictyum sp. 697 echinosulfonic acid A from 697 echinosulfonic acid B from 697 echinosulfonic acid C from 697 echinosulfone from 697 Economic viability 345 of 14p-methyl-14-a-hydroxyMFA 345 of 15a-methyl-14a-hydroxyMFA 345 Ecteinascidia turbinata 640,641 antitumor activity of 640 ecteinascidins from 640,641 EGb 185 activity against MPTP 184 effects on neuroendocrine system 185 effects on phospholipid metabolism 185 protective activity of 184
Eiocosapentaenoic acid 565 in fish oils 565 Eliamid 146 cytostatic activity of 146 fiingicidal activities of 146 nematocidal activity of 146 Ellagic acid 280 structure of 280 Engorged toxicity 400 from Hibiscus rosa-sinensis 400 from Nicotiana tabacum 400 from Ocimum micranthum 400 from Quassia simarouba 400 from Ricinus communis 400 from Salvia serotina 400 from Spigelia anthelmia 400 from Symphytum officinale 400 from Stachytarpheta jamaicensis 400 Endotoxins 518 chemistry of 518 Enzymic activity 257 of plant polyphenols 257 (-)-Epicatechin 41,289 from chocolate 289 from Licania pittieri 41 3-£/^/-nagilactone C 459 from Podocarpus nagi 459 3-£/7/-paraherquamide A 354 anthelmintic activity of 354 semi-synthesis of 354 Epingaione 402 growth regulatory activities of 402 Epipolasis sp. 699 halistanol sulfate A from 699 halistanol sulfate B from 699 halistanol sulfate C from 699 halistanol sulfate D from 699 halistanol sulfate E from 699 Epipolasis kushimotoensis 662 P-phenylethylamine of 662 epipolasins A of 662 epipolasins B of 662 epi'StWoWmC 460 from Podocarpus nagi 460 Epitheaflavic acid 274 formation of 274
770
2p,3P-Epoxypodolide 462 from Podocarpus nagi 462 Equisetin 121 antibiotic activity of 121 cytotoxicity of 121 from Fusarium equiseti 121 HIV inhibitory activity of 121 Ergosterol 570,572 as antitumor substance 570 effect on neovacularization 572 from Agaricus blazei 570 Erythroskyrine 134 from Penicillium islandicum 134 Essential oil 415,390,408,421,426 against sarcoptic mange 415 against Varroajacobsoni 390 benzyl benzoate in 415 Cedrus deodara (Pinaceae) 415 from Anonas^p. 392 from Artemisia absinthium 426 from Artemisia tridentata 408 from Calocedrus decurrens 408 from Chamaecyparis lawsoniana (Cupressaceae) 408 from Chamaecyparis nootkatensis 408 from Chenopodium spp. 392 from Foeniculum vulgare 408 from Juniperus occidenatalis 408 from Juniperus occidenatalis 408 from Salvia officinalis 392 from Sequoia sempervirens 408 from Tanacetum vulgare 426 from Thuja plicata 408 from Thymus vulgaris 392 lethality tests of 392 microwave assisted process (MAP) 427 of citronella 421 of eucalyptus 421 of Juniperus viriginiana 408 of Salvia officinalis L. 390 of spearmint 421 of tea tree 421 of Thymus vulgaris L. 390 ofwintergreenoils 421 used as bactericides 390 used as fungicides 390
used as insecticides 390 Estrogen-like activity 227 of phenols from moraceous plants 227 of Glycyrrhiza s^Qcits 227 (25)-Euchrenone 17 as aromatase inhibitor 17 Eudistoma cf. rigida 635 iejimalide C from 635 iejimalide D from 635 Eudistoma gilboverde 634 methyleudistomidin C 634 Eudistoma glaucus 634 eudistomidin C from 634 Eudistoma olivaceum 633 P-carboline alkaloids from 633 A^(10)-methyleudistomin E
from 633 eudistomin K from Ritterella sigillinoides 633 Eudistoma sip. 637 desmethylvaracin from 637 Eupentactafraudatrix 598 from calcigeroside B 598 from calcigeroside Ci 598 from calcigeroside D, 598 from cucumarioside G2 598 Euryspongia 667 15-acetylthioxyfiirodysin from 667 Euscaphic acid 40 from Licania pyrifolia 40 Euthyroides episcopalis 621 euthyroideones from 621 Expectorant activity 200 of genus Morw^ 200 Fabaceae plants 435 isoflavonoids from 435 Fatty acid components 565 of carp oil 565 of tuna oil 565 Ferulicacid 262 in citrus juices 262 in cereal brans 262 in coffee 262 in sugar beet fibre 262
771
Fijian sample 337 of Lissoclinum vareau 631 varacin from 637 Fijian tunicate Polycitorella mariae 634 citorellamine from 634 Flavan-3-ols 273 (+)-catechin 273 epicatechin 273 Flavanols 272 structure of 272 Flavanones 270 eriodictyol 270 hesperetin 270 hesperidin 270 in chickpeas 271 in citrus fruits 271 in cumin 271 in hawthorn berry 271 in licorice 271 in peppermint 271 in rowanberry 271 isosalipurpurin (chalcone) as 271 naringenin 270 naringin 270 phloretin (dihydrochalcone) as 271 Flavones 266,267,285 absorption of 285 apigenin 267 as plant tissue color 267 chrysin 267 diosmetin 267 isoorientin 267 isovitexin 267 lutcolin 267 orientin 267 pinocembrin 267 vitexin 267 Flavonols 264,265,284 from onions 284 glycosides of 284 inkaempferol 265 in plant foods 265 inquercetin 265 structure of 264 Flavonoids 208 from Morus alba root bark 208
5-Fluorouracil (5-FU) 562,559 as cancer drug 559 gastrointestinal toxicity by 562 immunotoxicity by 562 levels in mice plasma 567 myelotoxicity by 562 5-FU plus carp oil 566,568 effects on tumor growth 566 in sarcoma 180-bearing mice 566 5-FU plus tuna oil 566,568 effects on tumor growth 566 in sarcoma 180-bearing mice 566 Forbesin 718 as sulfated glycolipid 718 eicosane-1,16-disulfate from 718 from Asterias forbesi 718 from Asterias vulgaris 718 from Henhcia laeviuscula 118 isorhodopti lometrin-2' -sulfate from 718 Formic acid 384 asmiticide 384 FrondosideB 593 from Stichopus chloronotus 593 from Thelenota ananas 593 FrondosideC 599 from sea cucumber 599 Fungal metabolites 66,473,475,476 from Aspergillus flavus IFM 41934 476 from Aspergillus fumigatus IFM 41243 476 from Aspergillus niger H7160B 476 from Candida albicans 1463D 476 from Candida albicans ATCC 90028 476 from Candida albicans ATCC 90029 476 from Candida dubliniensis CBS 7987 476 from Pichia anomala IFM 47182 476 from Trichophyton mentagrophytes KCH1155 476 from Candida guilliermondii IFM 46823 476
772
from Candida kefyr IFM 46921 476 from Candida parapsilosis IFM 46863 476 from Candida tropicalis IFM 46816 476 from Criptococcus neoformans ATCC 90112 476 from Exophiala dermatitidis 476 Gallic acid 280 structure of 280 GancaoninR 234 from Glycyrrhiza uralensis 234 Gastrointestinal nematodes 349 Haemonchus contortus as 349 Trichostrongylus colubriformis as 349 Gastrointestinal toxicity 559,560 ofbilobalide 171 of5-fluorouracil(5-FU) 560 of chitosan 559 offish oils 559 Gel formulation 385 in Varroajacobsoni 385 8-Geranylapigenin 234 synthesis of 234 Ginkgo bilobah, 165,170 3 '-O-methylmyricetin-3-Orutinoside from 167 5' -methoxybilobetin from 166 amentoflavone from 166 apigeninfrom 167 apigenin-7-O-glucoside from 167 bilobetinfrom 166 biological activities of 165 chemistry of 165 delphidenon from 167 ginkgetinfrom 166 ginkgolide A from 170 ginkgolide B from 170 ginkgolide C from 170 ginkgolide J from 170 ginkgolide M from 170 isoginkgetin from 166 isorhamnetin from 167 isorhamnetin-3-O-glucoside from 167
isorhamnetin-3-O-rutinoside from 167 kaempferol from 167 kaempferol-3-0-(2"-0glucosyl)rhamnoside from 167 kaempferol-3-0-(6"'-0-/7coumaroyl-2"-0-glucosyl) rhamnoside from 167 kaempferol-3.0-[6"'-0-{/7-(7""-0glucosyl)coumaroyl} -2"-0glucosyl]rhamnoside from 167 kaempferol-3-0-[a-rhamnosyl(1 ->2)-a-rhamnosyK 1 ->6)]-pglucosidefrom 167 kaempferol-3-O-glucoside from 167 kaempferol-3-O-retmoside from 167 kaempferol-7-O-glucoside from 167 luteolinfrom 167 luteolin-3'-(9-glucoside from 167 4-0-methylpyridoxine from 172 myricetinfrom 167 myricetin-3-O-rutinoside from 167 quercetinfrom 167 quercetin-3-0-(2"-0-glucosyl) rhamnoside from 167 quercetin-3-0-(6"'-0-/7-coumaroyl2"-0-glucosyl)rhamnoside from 167 quercetin-3-0-[6'"-0-{p-(7""-0glucosyl)coumaroyl} -2"-0glucosyl]rhamnoside from 167 quercetin-3 -0-[6"' -0-/7-coumaroy 12"-0-glucosyl)rhamnosyl-7-0glucosidefrom 167 quercetin-3-0-[a-rhamnosyl(1 ->2)-a-rhamnosyl-( 1 ->6)-Pglucosidefrom 167 quercetin-3-0-glucoside from 167 quercetin-3-O-rhamnoside from 167 quercetin-3-0-rutinoside (rutin) from 167 sciadopitysin from 166
773
Glabrene 234 from Glycyrrhiza glabra 234 Glossodoris quadricolor 650 latruncuiin B from 650 P-Glucuronidase 283 Glucuronides 288 in urine 288 Glycosidase activity 281 of polyphenols 281 Glycoside naringin 288 in urine 288 Glycyrol 234 from Glycyrrhiza uralensis 234 Glycyrrhiza glabra 239,204,206 8-(y,Y-dimethyallyl)-wighteone from 206 3'-(y,y-dimethylallyl)-kievitone from 206 edudiol (3,9-dihydroxy-lmethoxy-2-prenylpterocarpan) from 206 euchrenone from 206 flavanonefrom 239 gancaonin G from 206 gancaonin H from 206 glabrene from 206 glabridin from 206 glabrone from 206 glisoflavanone from 206 glyasperin A from 206 glyasperin C from 206 glyasperin D from 206 glycyrrhizic acid from 239 glyinflanin G from 206 glyinflanin K from 206 hispaglabridin A from 206 3-hydroxyglabrol from 206 3-hydroxyparatocarpin C from 206 isoderone semilicoisoflavone B from 206 kanzonol U from 206 kanzonolVfrom 206 kanzonol W from 206 kanzonol X from 206 kanzonol Y from 206 kanzonol Z from 206 licorice-saponin G2 from 239
liquiritigenin from 239 medicarpin from 206 1 -methoxyphaseollidin from 206 4'-0-methylglabridin from 206 shinpterocarpin from 206 uses as licorice 204 Glycyrrhiza spQCiQS 223 bioactive phenolic compounds
from 223 Glycyrrhiza uralensis 242 4'-0-methylglabridin from 242 1-methoxyphaseollidin from 242 3-(9-methylglycyrol from 242 6,8-diprenylorobol from 242 dihydroisoflavone A from 242 gancaonin I from 242 gancaonol C from 242 gancaonol from 242 glycyrin from 242 hispaglabridin A from 242 isoglycyrol from 242 isolicoflavonol from 242 licoricone from 242 shinflavanone from 242 vestitol from 242 Glycyrrhiza glabra var. 205 glabrene from 205 glabridin from 205 glabrol from 205 3-hydroxyglabrol from 205 pyranoisoflavan from 205 Gymnocrinus richeri 719 gymnochrome C from 719 Gynandropsis gynandra 395 repellent properties of 395 Haemonchus contortus 342 Halenaquinol sulfate 677 absolute stereochemistry of 677 as pentacyclic hydroquinone 677 as yellow pigment 677 from Xestospongia sapra 677 Halichondria species 659,660 diterpenoid from 659 from Marshall islands 660 guai-6-ene skeleton of 660 sesqui- and diterpenoids from 659 sesquiterpenoid from 659
774
Halichondria sponge 663 isothiocyanates from 663 Halidona nigra 684 from Papua New Guinea 684 haligramides A of 684 Halidona sp. 706 haliclostanone sulfate from 706 Halicylindramide A 680 as antifimgal activity 680 as cytotoxic activity 680 from Halichondria cylindrata 680 Halicylindramide B 680 as antifimgal activity 680 as cytotoxic activity 680 from Halichondria cylindrata 680 Halicylindramide C 680 as antifimgal activity 680 as cytotoxic activity 680 from Halichondria cylindrata 680 Halipanicine 661 from Halichondria panicea 661 stereochemistry of 661 total synthesis of 661 Halistanol disulfate B 699 from Pachastrella sp. 699 in the endothelin converting enzyme (ECE) assay 699 stereochemistry of 699 Halistanol sulfate 698 antimicrobial activity of 698 as tris-sodium sulfate salt 698 from Halichondria cf. moorei 698 from Topsentia sp. 698 hemolytic activity of 698 ichthyotoxic activity of 698 pp60v-src protein tyrosine kinase inhibition activity 698 HallactoneA 458 from Podocarpus hallii 458 HallactoneB 462 from Podocarpus hallii 462 from Podocarpus polystachyus 462 from Podocarpus sellowii 462 Halocynthia papulosa 642 6-methylheptyl sulfate from 642 (£)-oct-5-enyl sulfate from 642
Harzianic acid 135 antibiotic activity of 136 from Trichoderma harzianum 136 Hematologic toxicity 559 with immunosuppression 559 with leukopenia 559 Hepatoprotective activity 257,293 of plant polyphenols 257,293 Heptaprenylhydroquinone derivative 676 from Irciniafasciculata 676 Herbal formulation AV/EPP/14 401 Acorus calamus m 401 against larvae 402 against nymphs of Boophilus microplus 402 Azadirachta indica in 401 Cedrus deodara in 402 Eucalyptus globulus in 402 Pongamia pinnata in 401 Hexaprenyl-hydroquinone sulfate 676 as H^/K'^-ATPase inhibitor 676 from Dysidea 676 Himax 416 acaricidal action of 416 against demodectic mites 416 against psoroptic mites 416 against sarcoptic mites 416 Cedrus deodara of 416 Polyalthia excessa of 416 Polyalthia longifolia of 416 Hinokiresinol 234 from Chamaecyparis obtusa 234 Histamine release inhibitor 257,293 polyphenols as 257,293 HMBC and NOB correlations 26 of 5,7,2' ,4 '-tetrahydroxy-3geranylflavone 26 ofisogemichalconeC 25 Holostanol 588 structure of 588 Holothuria pervicax 720 ganglioside HPG-8 from 720 Holothurinoside A 592 from Holothuria forskali 592 Holothurinoside C 592 from Holothuria forskali 592 Holothurinoside D 592 from Holothuria forskali 592
775
HoIothurinA 592 Holothuria leucospilota 592 Homoisoflavone 234 from Dracaena loureiri 234 Honey 385 formic acid in 385 Homer-Wadsworth-Emmons condensation 72 ofstannate 72 House dust mites 417 Actinodaphne lancifolia against 419 benzyl benzoate against 418 Cinnamomum camphora against 419 Cinnamomum japonicum against 419 Dermatophagoides farinae against 417 Dermatophagoides pteronyssinus against 417 Euroglyphus maynei against 417 Lindera umbellata against 419 Melaleuca acacoides dLgSLinst 419 Melaleuca symphycarpa against 419 Neolitsea sericea against 419 Persia thunbergii digmnst 419 Taiwania cryptomerioides against 418 tannic acid against 418 treatment of 418 8-Hydroxy eriodictyol 41 from Licania pyrifolia 41 8-Hydroxy luteolin 41 from Licania pyrifolia 41 8- Hydroxy naringenin 41 from Licania licaniaeflora 41 from Licania pyrifolia 41 2a-Hydroxy ursolic acid 40 from Licania carii 40 from Licania pyrifolia 40 Hydroxybenzoic acid derivatives 259 benzoic acid as 259 gallic acid as 259 4-hydroxybenzoic acid as 259 isovanillic acid as 259 protocatechuic acid as 259
salicylic acid as 259 syringic acid as 259 vanillic acid as 259 2a-Hydroxybetulinic acid 40 from Licania pyrifolia 40 6(3-Hydroxybetulinic acid 40 from Licania pyrifolia 40 lla-Hydroxybetulinic acid 40 from Licania pyrifolia 40 Hydroxycinnamic acid derivatives 260 (2-Hydroxyethyl)dimethylsulfoxonium chloride 716 from Alcyonidium gelatinosum 716 ofDogger Bank Itch 716 from Theonella aff mirabilis 716 3p-Hydroxyholost-7-ene aglycones 594 Cucumariajaponica from 594 14a-Hydroxymarcfortine A 338 synthesis of 338 15a-Hydroxymarcfortine A 338 synthesis of 338 16a-Hydroxymarcfortine A 338 synthesis of 338 3 '-[y-Hydroxymethyl-(£)-ymethylallyl]-2,4,2',4'tetrahydroxychalcone 11 '-0coumarate 16 as aromatase inhibitor 16 14p-Hydroxy-MFA 340 synthesis of 340 14a-Hydroxy-MFA 341 synthesis of 341 3p-Hydroxy-nagilactone A 459 from Podocarpus nagi 459 15-Hydroxy-nagilactoneD 459 from Podocarpus nagi 459 16-Hydroxy-nagilactoneE 463 from Podocarpus nagi 463 1 P-Hydroxy-nagilactone F 463 from Podocarpus nagi 463 3p-Hydroxy-nagilactoneF 463 from Podocarpus nagi 463 2a-Hydroxy-nagilactone F 464,474 against Saccharomyces cerevisiae 474 from Ileostylus micranthus 464
776
16-Hydroxypodolide (salignone H) 462 from Podocarpus saligna 462 Hyperparasites of tick 404 Beauveria bassiana as 404 Bacillus thuringiensis var. kurstaki as 404 Cedecea lapagei as 404 Metarhizium anisopliae as 404 Verticillium lecanii as 404 Hypotensive action 200,209,207 of isoprenylatedflavonoids 207 ofkuwanonG 209 ofkuwanonH 200,209 of Morus alba 200 of A/orw^ species 207 of mulberrofuran C 209 ofmulberrofuranF 209 of mulberroftiran G 209 lO-Hyroxycoronaridine 234 from Tabernaemontana penduliflora 234 Hyrtiomanzamine 691 from Hyrtios erecta 691 immunosuppressive activity of 691 Hysistozoa fasmeriana 635 fasmerianamine A from 635 fasmerianamine B from 635 /ra/75'"5 -hydroxy-4-(4' -hydroxy-3' methoxyphenyl)-4-(2"-imidazolyl)-l,2,3-trithianefrom 635 lanthella basta 695 as Ca^^-channel agonists 695 lantheranA 695 as NaVK^-ATPase inhibitor 695 from/a«//ig//a spp. 695 Ibisterol sulfate 700 against HIV-1 700 from deepwater Topsentia sp. 700 Ichthyotoxic diacylglycerol umbraculumin C 650 from Umbraculum mediterraneum 650 stereochemistry of 650 Ikarugamycin 138 antiprotozoal activity of 138 from Streptomyces
phaechromogenes var ikaruganensin 138 Immunocompetent organic toxicity 560 of5-fluorouracil(5-FU) 560 /« v//ro activity 470,467 ofmilanjilactone A 470 ofmilanjilactoneB 470 ofnagilactoneG 470 ofnagilactoneF 470 of podolactones 467 Industrial syntheses 71 of vitamin A 71 Inhibitory activity 481 of inumakilactone A 481 of inumakilactoneB 481 of inumakilactone C 481 ofnagilactone A 481 of nagilactone B 481 ofnagilactoneG 481 ofpodolactone A 481 of podolactone B 481 of podolactone C 481 of podolactone D 481 ofpodolactone E 481 Insecticidal activity 477 of 14-epi-ponolactone A 477 ofhallactone A 477 of hallactone B 477 ofnagilactone A 477 ofnagilactoneG 477 of nagilactone D 477 ofnagilactone E 477 ofpodolactone A 477 ofpodolactone C 477 of podolide 477 ofsellowinA 477 Integramycin 147 from Actinoplanessxi. 147 as HIV-integrase inhibitor 147 Inumakilactone A 461 from Podocarpus philippinensis 461 from Podocarpus macrophyllus 461 Inumakilactone A glucoside 462 from Podocarpus philippinensis 462
777
from Podocarpus macrophylus 462 Inumakilactone B 461 from Podocarpus macrophyllus 461 from Podocarpus neriifoiius 461 from Podocarpus polystachyus 461 Inumakilactone C 466 from Podocarpus macrophyllus 466 Inumakilactone D 466 from Podocarpus macrophyllus 466 Inumakilactone E 458 from Podocarpus macrophyllus 458 from Podocarpus poly stachyus 458 Irciniafasiculata 673 fasiculatin for 673 Ircinia spinulosa 676 brine shrimp toxicity of 676 2-prenylhydroquinones of 676 Ircinia variabilis 673 22-0-sulfates of palinurin from 673 sulfate esters from 673 Irciniasulfonic acid 714 from/rc/wa sp. 714 reverses multidrug resistance 714 Isobavachalcone 234 from Morus cathayana 234 Isobavachin 234 from Glycyrrhiza pallidiflora 234 Isoflavones 268,269,286,287 acetyldaidzein 269 acetylgenistin 269 aglycones 269 bioavailibity of 286 daidzein 269 genistein 269 glycosides 269 in green split peas 269 inmiso 269 in plant foods 269 in soy foods 287 in soy cheese 269
in soy flour 269 in soy sauce 269 in soya bean 269 in soya milk 269 in textured soya protein 269 intofu 269 in tofti yoghurt 269 metabolism of 287 oestrogenic activity of 268 Isogemichalcone C 16 as aromatase inhibitor 16 Isolicoflavonol 17 as aromatase inhibitor 17 Isoprenylated flavonoids 199,203,244 chemistry of 244 biological activity of 199 effect on testosterone 5a-reductase 244 from Cudrania tricuspidata 203 from Cudrania cochinchinenesis 203 from Glycyrrhiza sp. 199 from medicinal plants 199 kuwanon C as 245 kuwanonEas 245 kuwanon G as 245 kuwanon Has 245 kuwanon Las 245 morusin as 245 mulberrofuran A as 245 mulberrofiiran G as 245 oxydihydromorusin as 245 Isothiocyanates 658 as marine metabolites 658 asterpenes 658 from Axinellida order 658 from Halichondria genera. 658 from Halichondrida order 658 from Lithistida 658 as precursor of formamido group 658 5-Isothiocyanatopupukeanane 661 from Axinyssa species 661 Ivermectin 396 as macrolide antibiotic 396 from Streptomyces avermitilis 396
778
Janolusimide 146 bom Janolus cristatus 146 neurotoxic lipophilic activity of 146 Jaspisin 697 embryos of 697 sea urchin hatching inhibition by 697 Kae 3-(2"-xyl)rha 41 from Licania pyrifolia 41 from Licania licaniaeflora 41 Kae 3-(6"-/?-coum)glc 41 from Licania densiflora 41 Kae3-ara 41 from Licania licaniaeflora 41 from Licania pyrifolia 41 Kae3-rut 41 from Licania apetala var. apetala 41 Kaempferol 41 from Licania pyrifolia 41 KazinolA 17 antioxidant activity of 17 as tyrosinase inhibitor 17 as platelet aggregation inhibitor 17 KazinolB 17 as cyclooxygenase 17 as platelet aggregation inhibitor 17 KazinolC 210 from moraceous plants 210 KazinolE 210 from moraceous plants 210 KazinolF 16,210 antioxidant activity of 16 as tyrosinase by inhibitor 16 from moraceous plants 210 KazinolJ 210 from moraceous plants 210 KazinolM 210 from moraceous plants 210 KazinolN 210 from moraceous plants 210 KeenamideA 652 as cyclic hexapeptide 652 as cytotoxic agent 652 from Pleurobranchus forskalii 642
Ketalized Diels-Alder type adducts 203 sorocenol B as 203 soroceal as 203 Koreoside 599 from Cucumaria koraiensis 599 KuwanonC 210 from moraceous plants 210 KuwanonE 210 from moraceous plants 210 KuwanonG 201 from moraceous plants 201 KuwanonH 201.222,223 as bombesin receptor antagonists 223 as GRP-induced DNA synthesis inhibitor 222 from moraceous plants 201 KuwanonL 210 from moraceous plants 210 KuwanonM 210 from moraceous plants 210 Latrunculia magnifica 689 6,7-epoxy-latrunculin 689 latrunculin M from 689 689 Lissoclinum vareau 638 varamine A from 638 Lactacystin 112 biosynthetic assembly of 112 Lactic acid 386 in honey 386 Lancet-shaped follicular mites 410 as cause of demodectic mange 410 of Demodex gQUXxs 410 Latrunculia 685 Discorhabdin A from 686 Latrunculin A 688 as ichthyotoxins 688 from Latrunculia magnifica 688 2-thiazolidinone moiety of 688 Lavandula angustifolia Miliar 411 essential oil of 411 constituents of 411 LC/ESI-MS analyses 290 of sulfated tea catechins 290 of urinary glucuronid 290 Lepidium sativum 400 in tick toxicity 400
779
Licania apetala var. apetala (E. May) Fritsch 58 kaempferol 3-(9-rutinoside from 58 myricetin 4'-(9-a-Lrhamnopyranoside from 58 phytochemical studies of 38 quercetin 3-0-rutinoside from 58 quercetin 3-0-a-Larabinopyranoside from 58 quercetin 3-0-a-Lrhamnopyranoside from 58 quercetin 3-0-P-Dgalactopyranoside from 58 taxifolin 3-(9-a-Lrhaninopyranoside from 58 Licania carii Cardozo 38,42 betuiinic acid from 42 2a-hydroxyursolic acid from 42 maslinic acid from 42 myricetin 3'-methyl-3-0-rutinoside from 42 myricetin 3-0-(2"-0-P-Dxylopyranosyl)-a-L-rhamnopyranoside from 42 myricetin 3-0-rutinoside from 42 myricetin 3-0-P-Dgalactopyranoside from 42 myricetin 3-0-p-Dglucopyranoside from 42 phytochemical studies of 38 quercetin 3-0-(2"-0-P-Dxylopyranosyl)-a-Zrhamnopyranoside from 42 quercetin 3-O-rutinoside from 42 quercetin 3-(9-P-Dgalactopyranoside from 42 quercetin 3-0-p-Dglucopyranoside from 42 P-sitosterol 3-(9-P-Dglucupyranoside from 42 ursolic acid from 42 Licania densiflora YAtxrAioonXQ 38,49 3 ',4'-dimethylmyricetin-3-0-p-Dglucopyranoside from 49 myricetin 3' ,5' -dimethylether-3 -Oa-L-rhamnoside from 49
myricetin 3 ',5 '-dimethylether-3-0P-D-glucopyranoside from 49 myricetin 3'-methylether-3-0-P-Dgalactopyranoside from 49 myricetin 3-(9-(2"-0-a-Lrhamnopyranosyl)-a-Lrhamnopyranoside from 49 myricetin 4'-methylether-3-0-a-Lrhamnopyranoside from 49 naringenin 8-hydroxy-4'-methyl ether from 49 phytochemical studies of 38 Licania heteromorpha var. heteromorpha Bentham 38,53 alphitolic acid from 53 betuiinic acid from 53 myricetin 3,4'-di-(9-a-Lrhamnopyranoside from 53 myricetin 3-0-a-Lrhamnopyranoside from 53 myricetin 3-0-P-Dgalactopyranoside from 53 myricetin 4'-methylether-3-0-a-Lrhanmopyranoside from 53 myricetin 4'-methylether-3-0-P-Dgalactopyranoside from 53 myricetin 4'-methylether-3-0-P-Dglucopyranoside from 53 myricetin 7-methylether 3,4'-di-0a-L-rhamnopyranoside from 53 3P-0-cw-/?-coumaroyl alphitolic acid from 53 3P-0-c/5'-/7-coumaroyl maslinic acid from 53 3 P-(9-cw-/7-coumaroy l-2ahydroxy-urs-12-en-28-oic acid from 53 3P-0-/ra/75-/7-coumaroyl alphitolic acid from 53 3P-(9-rra/M'-/7-coumaroyl maslinic acid from 53 3 P-0-rra«5-/7-coumaroyl-2ahydroxy-urs-12-en-28-oic acid from 53 phytochemical studies of 38 Licania intrapetiolaris Spruce (ex. Hook) 38
780
cucurbitacin B from 59 intrapetacin A from 59 intrapetacin B from 59 phytochemical studies of 38 Licania licaniaeflora (Sagot) Blake 38,56 arjunic acid 28-P-D-glucosyl ester from 57 betuiinic acid from 57 maslinic acid from 57 olean-12-ene-2a,3P-diolfrom 57 oleanolic acid 3-O-a-Larabinopyranoside from 57 oleanolic acid from 57 pomolic acid from 57 phytochemical studies on 38 tormentic acid 28-P-D-glucosyl ester from 57 ursolic acid from 57 Licania pittieri FrmiCQ 38,42 catechinfrom 42 epicatechin from 42 phytochemical studies of 38 quercetinfrom 42 quercetin 3-0-a-Larabinopyranoside from 42 quercetin 3-0-a-Lrhamnopyranoside from 42 quercetin 3-O-P-Dgalactopyranoside from 42 quercetin 3-0-P-Dglucopyranoside from 42 ursolic acid from 42 Licania pyrifolia GnsobdLch 38,42 a-amyrin from 42 betulin from 42 betuiinic acid from 44 2,3-dihydroxylup-12-en-28-oic acid 3-(3',4'-dihydroxybenzoyl ester) from 44,45 2a,3a-dihydroxyurs-12-ene-28-oic acid from 45 euscaphic acid 28-p-Dglucopyranosyl ester from 45 euscaphic acid from 45 1 la-hydroxybetulinic acid from 44 6P-hydroxybetulinic acid from 44
2a-hydroxyursolic acid from 45 lupeol from 42 maslinic acid from 45 oleanolic acid from 44 phytochemical studies of 38 p-sitosterol 3-0-P-Dglucopyranoside from 45 P-sitosterol from 42 tormentic acid 28-P-Dglucopyranosyl ester from 45 tormentic acid from 45 2,3,27-trihydroxylup-12-en-28-oic acid 3-(3'-4'-dihydroxybenzoyl ester) from 45 ursolic acid 3-0-a-Larabinopyranoside from 45 ursolic acid from 44 uvaolfrom 44 Licorice 204,205 anti-hepatitic principles from 205 antitussives from 205 anti-ulcer compounds from 205 dihydrophenanthrene from 205 dihydrostilbenes from 205 flavor from 205 Glycyrrhiza species from 205 isoliquiritigenin from 205 isoliquiritin from 205 liquiritigenin glycosides from 205 liquiritin apioside from 205 liquiritin from 205 sweetening agents from 205 Linalool 413 analysis of 413 efficacy of 413 Liouvilloside A 608 massfragmentationof 608 Lipid A 518,519,523,525,526,529,534, 537,538 acute effects of 523 antitumor activities of 518 association with immunomodulators 534 effect on acquired immunity 529 effect on cytokine production 526 effect on innate immunity 525
781
from Escherichia coli 519 from Salmonella typhimurium 519 in cancer vaccination 537 to treat tumor-bearing animals 538 treatment with 534 Lipid A DT 5461 534 for C38 colon carcinoma 535 for Lewis lung carcinoma 535 for MH134 hepatoma 535 for MM46 mammary carcinoma 535 for murine Meth A fibrosarcoma 535 treatment with 534 Lipid A OM-174 536 antitumor activity of 536 treatment with 536 Lipid A ONO-4007 535 treatment with 535 Lipid A tolerance 521 by anti-LPS antibodies 522 a-Lipomycin 135 from Staphylococcus aureofaciens 135 as antibacterial agent 135 p-Lipomycin 135 from Staphylococcus aureofaciens 135 as antibacterial agent 135 Lipopolysaccharides (LPS) 518,520,521, 523,528,533,539 acute effects of 523 antigenic properties of 518 biological properties of 518 by PROb colon cancer cells 533 chemical properties of 518 detoxificaiton of 520 effects on angiogenesis 528 effects on blood flow 528 from Pantoea agglomerans 539 from Salmonella abortus 539 in human macrophages 521 in peritoneal carcinomatosis 533 phase I trials with 539 receptor for 520 Rhodobacter sphaeroides lipid A as 521
Liquiritigenin 234 from Glycyrrhiza species 234 Lissoclinum bistratum 628 bistratamide A from 628 bistratamide B from 628 bistratamide C from 628 bistratamide D from 628 Lissoclinumjaponicum 637 dimethyl-5-(methylthio) varaein from 637 Lissoclinum patella 622,623,625,626, 627,629 ascidiacyclamide from 622 heptapeptide ulicyclamide from 622 patellamide A from 623 patellamide B from 623 patellamide C from 623 patellamide E from 627 patellamide F from 627 patellamide G from 627 patellazole A from 625 patellazole B from 625 patellazole C from 625 patellins 1-6 from 629 prepatellamide A from 626 tawicyclamide A from 629 tawicyclamide B from 629 trunkamide A from 629 ulithiacyclamide B from 623 ulithiacyclamide from 622 Lissoclinum perforatum 636 1,2,3-trithiane derivative from 636 Lissoclinum sp. 637 trithiane from 637 Lissoclinum sp. from Great Barrier Reef 636 lissoclinotoxin A from 636 lissoclinotoxin C from 636 lissoclinotoxin D from 636 LL-Z1271a 465 from Acrostalagmus 465 from Oidiodendron griseum 465 LL-Z1271Y 465 from Acrostalagmus 465 from Oidiodendron griseum 465
782
Luteolin 286 absorption of 286 Luteolin-7-(9-p-glucoside 286 absorption of 286 Lycopersicon hirsutum f. glabratum (Solanaceae) 433 2-undecanone from 433 methyl ketones: 2-tridecanone from 433 Lydicamycin 126 as antibacterial agent 126 from Streptomyces lydicus 126 Lyngbya majuscula 143 pukeleimide C from 143 Lyrophagus longior 419 against 1,8-cineole 420 against camphor 420 against Eucalyptus globulus (Myrtaceae) 419 against fenchone 420 against Lavandula stoechas 419 against Lavandula angustifolia 419 against Imalool 420 against linalyl acetate 420 against Mentha x piperita (Lamiaceae) 419 against menthol 420 against menthone 420 Malic conjugates 262 in grapes 262 in lettuce 262 in spinach 262 in wines 262 Malonomicin 118 antiprotozoal activity of 118 from Streptomyces rimosus var. paromomycinus 118 Maltophilin 140 as antiftingal agent 140 from Stenotrophomonas maltophilia 140 MalyngamideA 142 structure of 142 Mange mites 409 transmission of 409 treatment of 409
Marcfortine 331 synthesis of 331 Marcfortine A (MFA) 331,332,336,372 antiparasitic activity of 332 asnematocide 331 conversion to paraherquamides 372 from Penicillium roqueforti 331,332 microbial hydroxylation of 346 reaction of 336 spiroalkyl analogs of 355 structure of 332 Marcfortine A's Reaction 336 with cyanogen iodide (CNI) 336 Margaritaria discoidea 395 acaricidal properties of 395 against Amblyomma variegatum 395 Marmesin 18 antifungal activity of 18 Maslinicacid 40 from Licania carii 40 from Licania licaniaeflora 40 from Licania pyrifolia 40 Matrigel 573 photograph of 573 MauritamideA 689 as taurine-containing metabolite 689 from Agelas mauritiana 689 Melaleuca species (Myrtaceae) activity 418 against Dermatophagoides pteronyssinus 418 against Melaleuca argentea 418 against Melaleuca dealbata 418 against Melaleuca saligna 418 Melinis minutiflora (Poaceae) 398 a- and p-pinene from 398 MelophlinB 117 Membranotropic action 588 of triterpenoid glycosides 588 Menthol 391 effects on honey bees 391 Metabolism 283 of dietary flavonoids 283
783
Meth A fibrosarcoma 534 antitumoral effects of 534 15-Methoxycarbonyl-nagilactone D 458 from Podocarpus nagi 458 14P-Methyl-14a-hydroxy-MFA 340 synthesis of 340 15a-Methyl-14a-hydroxy-MFA 342 synthesis of 342 15P-Methyl-14a-hydroxy-MFA 342 synthesis of 342 1 -Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 184 dopaminergic neurotoxicity of 184 Microspinsodamide 680 as cyclic peptide 680 from Sidonops microspinosa 681 p-hydroxy-/7-bromophenylalanine residue of 681 in HIV-1 infection 681 structure of 681 Microxine 691 as Cdc2 kinase inhibitor 691 from Microxina sp. 691 Milanjilactone A 462 from Podocarpus milanjianus 462 Milanjilactone B 464 from Podocarpus milanjianus 464 Mirabimide E 144,145 effects on solid tumours 145 from Scytonema mirabile 144 Mites 423 as Oligonychus sp. (Acari: tetranychidae) 423 as Phyllocoptruta oleivora 423 as Tegolophus australis (Acari: Eriophyidae) 423 as Tetranychus sp. 423 Miticidal properties 390,414 as Azadirachta indica 414 ofestragole 414 ofeugenol 414 of linalyl acetate 414 ofmenthol 390 ofterpenes 414 Mitogenicity 530 of lipid A 530 to B lymphocytes 530
Mitomycin 559 as anticancer drug 559 Molluscicidal activity 59 of Licania carii 60 of Licania densiflora 60 of Licania heteromorpha var. heteromorpha 60 of Licania licaniaejlora 60 of Licania pittieri 60 of Licania pyrifolia 60 Molluscs 647 outer body of 647 Monoamine oxidase activity 181 of Ginkgo bilobaL. 181 Monophosphoryl lipid A 537 from Salmonella minnesota 537 Morachalcone A 201 from moraceous plants 201 MoracinN 18 as aromatase inhibitor 18 Morusin 201,202 from moraceous plants 201 from Morus alba L. root bark of 202 Morusin hydroperoxide 210 from moraceous plants 210 Morwi" flavonoids 215 effects on arachidonate metabolism 215 Mulberroftiran C 210 from moraceous plants 210 Mulberroftiran F 210 from moraceous plants 210 Mulberroftiran G 210 from moraceous plants 210 Mycalesp. 712 as cytotoxic glutathione adducts 712 thiomycalolide A of 712 Mycothiazole 710 as novel lipid 710 anthelmintic properties of 710 from Spongia mycofijiensis 110 Myelotoxicity 559 ofchitosan 559 offish oils 559 Myr3-(2"-rha)rha 41 from Licania densiflora 41
784
Myr 3-(2"-xyl)rha 41 from Licania carii 41 from Licania pyhfolia 41 Myr 3,4'-dirha 41 from Licania heteromorpha var. heteromorpha 41 Myr3'4-diOMe-3-glc 41 from Licania densiflora 41 Myr3'4-diOMe-3-rha 41 fvom Licania densiflora 41 Myr3'OMe-3-gal 41 from Licania densiflora 41 Myr3'OMe-3-glc 41 fvom Licania densiflora 41 Myr3'OMe-3-rut 41 from Licania carii 41 Myr3-ara 41 from Licania licaniaeflora 41 Myr 3-gal 41 from Licania carii 41 from Licania densiflora 41 from Licania heteromorpha var. heteromorpha 41 from Licania licaniaeflora 41 Myr3-glc 41 from Licania carii 41 from Licania densiflora 41 Myr3-rha 41 from Licania densiflora 41 from Licania heteromorpha var. heteromorpha 41 from Licania pyrifolia 41 Myr3-rut 41 from Licania carii 41 Myr3-xyl 41 from Licania densiflora 41 Myr 4'OMe-3-gal 41 from Licania heteromorpha var. heteromorpha 41 Myr4'OMe-3-glc 41 from Licania heteromorpha var. heteromorpha 41 Myr4'OMe-3-rha 41 fvom Licania densiflora 41 from Licania heteromorpha var. heteromorpha 41
Myr4'-rha 41 from Licania apetala var. apetala 41 Myr 7-OMe-3,4'-dirha 41 from Licania heteromorpha var. heteromorpha 41 Myricetin (Myr) 41 fvom Licania densiflora 41 from Licania pyrifolia 41 Mytilus galloprovincialis 654 from Adriatic coast of Italy 654 from Mytilus edulis 654 in fish oils 565 n-3 polyunsturated fatty acid from 565 NagilactoneA 458 from Podocarpus macrophyllus 458 fvom Podocarpus nagi 458 from Podocarpus philippinensis 458 from Podocarpus polystachyus 458 NagilactoneB 458 from Podocarpus nagi 458 NagilactoneC 458 from lleostylus micranthus 458 from Podocarpus halli 458 from Podocarpus nagi 458 from Podocarpus nivalis 458 from Podocarpus macrophyllus 458 fvom Podocarpus purdeanus 458 NagilactoneD 458 from Podocarpus nagi 458 NagilactoneE 461 fvom Podocarpus nagi 461 NagilactoneF 464 from Podocarpus macrophilus 464 from Podocarpus milanjianus 464 from Podocarpus nagi 464 from Podocarpus sellowii 464 Nagilactone G 462 from Podocarpus milanjianus 462 from Podocarpus sellowii 462
785
Nagilactonel 464 from Podocarpus nagi 464 NagilactoneJ 466 from Podocarpus nagi 466 Nagilactoside A 459 from Podocarpus nagi 459 Nagilactoside F 460 from Podocarpus nagi 460 Nagilactoside G 460 from Podocarpus nagi 460 Namenamicin 645 from Polysyncratn lithostrotum 645 Naphthoquinones 408,429 action of 429 from Calceolaria andina 408 mitochondrial respiration inhibition by 429 structure of 408 Narains 697 from Jaspis sipQCiQS 697 in ascidian larvae 697 metamorphosis induction by 697 (25)-Naringenin 17 aromatase inhibition by 17 Naringenin 288 Naringenin chalcone 271 in juice 271 in ketchup 271 in tomato skin 271 Natural podolactones 457 7,9(ll)-dienolideas 457 7a,8a-epoxy-9(ll)-enolideas 457 a-pirone [8(14),9(11)-dienolide] as 457 Natural products 573,617 antimetastatic activity of 573 antitumor activity of 573 from marine invertebrates 617 in tumor-bearing mice 573 sulfiir-containing 617 Neamphius huxleyi 688 neamphine from 688 Neoflavone dimer 1 234 from Pistacia chinensis 234 Neoflavone dimer 2 234 from Pistacia chinensis 234
Neolitsea sericea 422 24Z-ethylidenelanost-8-en-3one from 422 24-methylenelanost-8-en-3one from 422 lanostanes from 422 Nephteis fasicularis 644 source of fasicularin 644 Neuronal cell 315 crocin's effect on 315 Neuroprotective properties 173,177 of Ginkgo biloba L. 173,177 Neurotrophic activity 174,177 in neural injury 174 of Ginkgo biloba L. 177 New lupane derivatives 46 from Licania pyrifolia 46 Nicotiana tabacum, Solanaceae 415 A^-Methyl-D-aspartate (NMDA) receptors 184 EGb as antagonist of 184 radioligand binding of 184 NMDA-induced currents 319 in hippocampal neurons 319 Non-holostane triterpenoids 600 bivittosideB 600 kurilosideC 600 psolusosideB 600 Nor- or bisnorditerpQnoids 454 nagilactone C 454 nagilactone E 454 oidilactone C 454 NubilactoneA 463 from Podocarpus nubigena 463 (3/?)-Nyasol 234 from Anemarrhena asphodeloides 234 (35)-Nyasol 234 from Anemarrhena asphodeloides 234 Nyasol (cw-hinokiresinol) 234 from Anemarrhena asphodeloides 234 Oceanapiasp. 713 dithiocyanates from 713 nematocidal activity of 713 thiocyanatin A from 713
786
thiocyanatin B from 713 thiocyanatin C from 713 total synthesis of metabolites of 713 3P-0-c/.y-/7-coumaroyl alphitolic acid 40 from Licania heteromorpha var. heteromorpha 40 3p-0-c/6:-/7-coumaroyl maslinic acid 40 from Licania heteromorpha var. heteromorpha 40 3 P-0-c/5'-/7-coumaroyl-2a-hydroxy ursolic acid 40 from Licania heteromorpha var. heteromorpha 40 Oestrogenic activity 257,293,302 of plant compounds 257,302 of polyphenols 293 Oidiodendrolide B 465 from Oidiodendron truncatum 465 Oidiodendrum griseum All terpenoid dilactones from 472 Oidiolactone C 465 from Oidiodendron griseum 465 from Oidiodendron truncatum 465 Oidiolactone D 465 from Oidiodendron griseum 465 from Oidiodendron truncatum 465 Oidiolactones 475 antiftingal activities of 475 from Oidiodendrum truncata 475 01ean-12-ene-2a,3p-diol 40 from Licania licaniaeflora 40 Oleanolic acid 40 from Licania pittieri 40 from Licania licaniaeflora 40 Oleficin 135 antibiotic activity of 135 from Streptomyces parvulus 135 Oligoglycosides 589 from starfishes 589 Oligosaccharide moiety 611 of patagonicoide A 611 NOESY correlations of 611 8-OMe apigenin 41 from Licania densiflora 41
OncoVax-P 545 against prostate cancer 545 monophosphoryl lipid A in 545 Ophioxanthin 721 5,6,5' -6' -tetrahydro-P, P-carotene3,4,3',4'-tetraol 4,4'-disulfate as 721 2is carotenoid sulfate 721 from Ophioderma longicaudum 721 Order aspidochirota 594 stichloroside A from 594 stichloroside Ai from 594 stichloroside A2 from 594 stichloroside B from 594 stichloroside Bi from 594 stichloroside C\ from 594 stichloroside C2 from 594 Stichopus chloronotus 594 Oriamide 682 as cytotoxic peptide 682 Oteromycin 147 from unidentified fiingus 147 3p-0-rra«5-p-coumaroyl alphitolic acid 40 from Licania heteromorpha var. heteromorpha 40 3P-0-/ra«5-/7-coumaroyl maslinic acid 40 from Licania heteromorpha var. heteromorpha 40 3 P-0-/ra/75-/7-coumaroyl-2a-hydroxy ursolic acid 40 from Licania heteromorpha var. heteromorpha 40 Ovothiols 716 from marine invertebrate eggs 716 ofmercaptohistidines 716 Oxalic acid 386 in honey 386 toxicity of 386 4-Oxo-flavonoids 263 structure of 263 16-OxoparaherquamideB 372 conversion of 372 3-(9-a-L-arabinoside 40 from Licania licaniaeflora 40 from Licania pyrifolia 40
787
Paraherquamides 331,332 fromMFA 332 synthesis of 332 Paraherquamide A 331,367 antiparasitic activity of 332 asymmetric synthesis of 367 conversion of PHB to 335 from Penicillium paraherquei 331 hydroxylationofMFA 335 structures of 332 total synthesis of 367 (+)-Paraherquamide B 359 conversion of MFA to 333 stereocontrolled synthesis of 358 Patagonicoide A 603 NOESY correlations of 603 Pateamine 709 as cytotoxin 709 dilactone functionality of 709 from New Zealand species of Mycale 709 total synthesis of 709 Patellamides 624 as macrophage scavenger receptor inhibitor 624 from Lissoclinum patella 624 X-ray crystallography of 624 p-Coumaric acid 262 in cereals brans 262 in spinach 262 in sugar beet fibre 262 p-Coumaroylquinic acids 262 in sweet cherries 262 Penares sp. 713 as a-glucosidase inhibitor 713 penarolide sulfate A from 713 Pentaglycosides 601 from sea cucumbers 601 Pentamera calcigera 598 from calcigeroside B 598 from calcigeroside Ci 598 from calcigeroside Di 598 from cucumarioside G2 598 Petrosia weinbergi 705,706 antiviral activity of 705 antiviral metabolites from 706 orthoesterol disulfate A
from 705 orthoesterol disulfate B from 705 orthoesterol disulfate C from 705 weinbersterol disulfate A from 706 Phakellistatin 5 683 as cytotoxic heptapeptide 683 from Phakellia costata 683 methyl sulfide group of 683 solid-phase synthesis of 683 Phase I trial 540 with monophosphoryl lipid A 540 Phenolic compounds 200 from Morus alba 200 from Morus bombycis 200 from Morus Ihou 200 kuwanon G as 200 Pheromones 428 against Tetranychus mites 428 famesol as 428 naphthoquinones as 428 nerolidolas 428 structure of 428 Phomasetin 122 as HIV inhibitor 122 from P/zowa sp. 122 PhylUdia pustulosa 651 4a-isothiocyanogorgon-l 1-ene from 651 Phyllospongia foliascens 709 in serological reactions 709 sulfated galactolipid M-6 from 709 Phylum echinodermata 587 asteroideain 587 crinoideain 587 echinoideain 587 holothuroidea in 587 ophiuroidea in 587 secondary metabolites from 587 Physarorubinic acid 133 from Physarum polycephalum 13 3 Phytolacca dodecandra 400 tick toxicity of 400 Piericidinas 435 as inhibitor of 435
788
Pimenta dioica 398 against Boophilus microplus 398 essential oils from 398 Pimpinella specks 431 (1 £)-propeny 1-4-hydroxybenzene from 431 against Typhlodromus telahus 431 epoxy-anoltiglate from 431 epoxy-pseudoisoeugenolisobutyrate from 431 epoxy-pseudoisoeugenoltiglate from 431 isoeugenolisobutyrate from 431 phenylpropanoids from 431 pseudoisoeugenolisobutyrate from 431 Piperazinedione 359 Piquerol A 404,405 acaricidal activity of 404 from Piqueria trinervia 404 toxicity to gravid female ticks 404 Piquerol B 404 acaricidal activity of 404 from Piqueria trinervia 404 toxicity to gravid female ticks 404 Plant polyphenol 257,281,300 anthocyanins as 257 bioactivity of 257 epidemiological studies on 300 flavanolsas 257 flavanones as 257 flavonesas 257 in humans 281 isoflavones as 257 occurrence of 257 proanthocyanidins as 257 structure of 257 Plant regulatory activity 4809 ofpodolactoneE 480 Platelet-activating factor (PAF) 188 as potent phospholipid inflammatory mediator 188 PNU-141962 353 isotopic labeling of 353 Podocarpus totara 454 podolactones from 454 PodolactoneA 461 from Podocarpus neriifolius 461
PodolactoneB 461 from Podocarpus neriifolius 461 PodolactoneC 461 from Podocarpus neriifolius 461 from Podocarpus milanjianus 461 PodolactoneD 461 from Podocarpus neriifolius 461 Podolactone E from Ileostylus micranthus 463 from Podocarpus neriifolius 463 Podolactones 453,454,455,495 antifeedant activity of 454 anti-inflammatory activity of 453, 454 anti-tumor activity of 453,454 biogenetic pathway for 455 from fimgi 465 from Ileostylus micranthus 454 from Podocarpus spQQxts 453 fiingicidal activity of 453,454 growth regulatory activity of 454,453 insecticidal activity of 453,454 synthesis of 495 Podolide 462 from Podocarpus gracilor 462 Pohnpei 661 13-isothiocyanatocubebane from 661 1 -isothiocyanatoaromadendrane from 661 2-thiocyanatoneopupukeanane from 661 4-thiocyanatoneopupukeanane from 661 thiocyanates from 661 Polonovski-Potier reaction 337 Polycarpamine A 640 as disulfide alkaloid 640 as sulfiir-containing antifiingal agent 640 from Polycarpa aurata 640 from Polycarpa auzata 640 from Polycarpa clavata 640 polycarpine as 640 synthesis of 640 Polycarpamine B 640 as disulfide alkaloid 640
789
as sulfur-containing antifungal agent 640 from Polycarpa aurata 640 from Polycarpa auzata 640 from Polycarpa clavata 640 polycarpine as 640 synthesis of 640 Polycarpamine C 640 as sulfur-containing antifungal agent 640 from Polycarpa aurata 640 from Polycarpa auzata 640 from Polycarpa clavata 640 synthesis of 640 Polycarpamine D 640 as sulfur-containing antifungal agent 640 from Polycarpa aurata 640 from Polycarpa auzata 640 from Polycarpa clavata 640 synthesis of 640 Polycarpamine E 640 as sulfiir-containing antifungal agent 640 from Polycarpa aurata 640 from Polycarpa auzata 640 from Polycarpa clavata 640 synthesis of 640 Polycephalin B 134 structure of 134 Polycephalin C 134 structure of 134 Polyclinum planum 643 polyclinal from 643 Polyenoyltetramic acids 132 Polymastiamide A 704 as antimicrobial steroid 704 from Polymastia boletiformis 704 Polyphenol glycoside 281 hydrolysis of 281 PonalactoneA 463 from Podocarpus nakaii 463 Ponalactone A glucoside 463 from Podocarpus nakaii 463 Potato tubers 259 in gallic acid 259 in protocatechuic acid 259 in salicyclic acid 259
in syringic acid 259 in vanillic acid 259 PR 1388 465 from Oidiodendron griseum 465 from Oidiodendron truncatum 465 Pramanicin 149,150 as antifiingal agent 149 biosynthesis of 150 ftom g^^nus Stagonospora 149 Predator mites 434 as pathogen 434 Neozygites adjarica as 434 Neoseiulus fallacis as 434 Phytoseiulus persimilis as 434 Proanthocyanidins 278,292 in apples 278 degradation of 292 in grapes 278 in pears 278 in strawberrys 278 structure of 278 4-Propenoyl-2-tyrosylthiazole 682 from Theonella sp. 682 Propionibacterium acnes 534 Proton-translocating NADH:Q oxidoreductase 435 high-affinity inhibitors of 435 Psammaplin A (bisprasin) 693 effects on Bacillus subtilis 693 from Dysidea species 693 from Psammaplysilla 693 Psammaplysilla purpurea 693 antimicrobial activity of 693 as tyrosine kinase inhibitor 693 presammaplin A from 693 psammaplin B from 693 psammaplin C from 693 psammaplin D from 693 Pseudaxinyssa 665 a,a)-bis-isothiocyanates of 665 Fijian species of 65 a-isothiocyano-(9-formyl analogues of 665 Pseudaxinyssa pitys 659 famesyl isothioycyanate from 659 PsolusosideB 598 from Psolus fabricii 598
790
Psoroptes mites 410 in bull fattening management 410 spread of 410 Ptilocaulis spiculifer 690 alkyl sulfate from 690 diS Senegalese s^iongQ 690 dakaramine from 690 Ptilometrasp. 719 anthraquinones from 719 Pulcherrimine 717 from Hemicentrotus pulcherrimus 1\1 Pyrethrins 405 against Hyalomma anatolicum 405 against Rhipicephalus haemaphysaloides 405 Pyridoacridine alkaloids 639 cytotoxic effects on KB cells 639 from Chelynotus semperi 639 kuanoniamine A as 639 kuanoniamine B as 639 kuanoniamine C as 639 kuanoniamine D as 639 NMR spectral analysis of 639 Pyrrole-imidazole alkaloid taurodispacamide A 690 antihistaminic activity of 690 from Agelas oroides 690
Que3-rha 41 from Licania apetala var. apetala 41 from Licania densiflora 41 from Licania licaniaeflora 41 from Licania pittieri 41 from Licania pyhfolia 41 Que 3-rut 41 from Licania apetala var. apetala 41 from Licania carii 41 from Licania densiflora 41 Quercetin 41,283 from Licania densiflora 41 from Licania pittieri 41 from Licania pyrifolia 41 in plasma 283 Quercetin glucuronides 284 as plasma metabolites 284 Quercetin-3-glucoside 285 bioavailability of 285 Quercetin-3-rutinoside 285 in foods 285 Quercetin-4'-glucoside 285 in foods 285 Quercetin-glycosides 285 bioavailability of 285 from onions 285
Que 3-(2"-xyl)rha 41 from Licania carii 41 from Licania pyrifi)lia 41 Que3'OMe-3-glc 41 from Licania apetala var. apetala 41 ^om Licania densiflora 41 from Licania licaniaeflora 41 Que3-ara 41 from Licania pittieri 41 from Licania pyrifolia 41 Que3-gal 41 from Licania apetala var. apetala 41 from Licania carii 41 from Licania densiflora 41 from Licania licaniaeflora 41 from Licania pittieri 41
Rec-assay 225 of 6-prenyleriodictyol 225 of 8-prenyleriodictyol 225 ofgancaoninC 225 of isoliquiritigenin 225 of licoisoflavanone 225 of licoisoflavoneB 225 of semilicoisoflavone B 225 Red wine 274 (+)-catechin in 274 (-)-epicatechin in 274 Resveratrol 582 effects in LLC-bearing mice 582 effects on tumor volume 582 effects on tumor weight 582 Retinoid chemistry 69 recent progress in 69 Retinol (vitamin A) 69 in mammalian food 69
791
Reutericyclin 127 as antibiotic 127 Rosmarinic acid 262 in culinary herbs 262 in mixed herbs 262 in stuffing 262 Rotenoneas 435 as inhibitor of 435 RT-PCR analyses 323 of tumor necrosis factor TNF-a 323 Sa mae sam 574 antitumor activity of 574 as mild cathartic 574 Cassia garrettiana Craib 574 from cassigarol A 574 from piceatannol 574 from piceatannol acetate 574 Saffron {Crocus sativus L.) 313,314,315 anti-tumor activity of 314,315 as coloring agent 313 for flavoring 313 uses in medicine 313 SalignoneA 466 from Podocarpus saligna 466 Salignone B 466 from Podocarpus saligna 466 Salignone! 462 from Podocarpus saligna 462 Salignone J 466 from Podocarpus saligna 466 Salignone K 466 from Podocarpus saligna 466 Salignone L 466 from Podocarpus saligna 466 Salignone M 463 from Podocarpus saligna 463 Salvia officinalis 391 essential oils from 391 Sang-Bai-Pi 209 as herbal medicine 209 sanggenon C from 209 sanggenon D from 209 Sanggenon A 201 from moraceous plants 201 Sanggenon B 210 from moraceous plants 210
Sanggenon C 201 from moraceous plants 201 Sanggenon D 210 from moraceous plants 210 Sanggenon M 234 from Morus cathayana (root bark) 234 Sanggenon O 201 from moraceous plants 201 Saponin 204 Glycyrrhiza aspera from 204 Glycyrrhiza eurycarpa from 204 Glycyrrhiza glabra from 204 Glycyrrhiza inflata from 204 Glycyrrhiza korshinskyi from 204 Glycyrrhiza uralensis from 204 Sarcoptes mitts 410 cause of human scabies 410 symptoms of 410 Sarcoptes scabiei 409 cause of mange mites 409 cause of scabies 409 Sea cucumbers 597 calcigeroside Cj from 597 calcigeroside D2 from 597 cucumarioside Ao-1 from 597 cucumechinoside A from 597 cucumechinoside B from 597 cucumechinoside C from 597 cucumechinoside D from 597 cucumechinoside E from 597 cucumechinoside F from 597 triterpene glucosides from 601 Secondary metabolites 3,432,617 acaricidal activity of 432 against Tetranychus urticae 432 biological activity of 617 epitaondiol as 432 epitaondiol diacetate as 432 epitaondiol monoacetate as 432 from Broussonetia kazinoki 3 from Broussonetia papyrifera 3 from Broussonetia zeylanica 3 from Stypopodium flabelliforme 432 halogenation of 617 ofbryozoans 617 of marine invertebrates 617
792
ofnudibranchs (sea slugs) 617 of ophistobranchs (sea hares) 617 ofsponges 617 oftunicates 617 stypetriol triacetate as 432 SellowinA 461 from Podocarpus sellowii 461 SellowinB 461 from Podocarpus sellowii 461 Sellowin C 458 from Podocarpus sellowii 458 Sesquiterpene cadina-4,10( 15)-dien-3-one 402 from Hyptis verticillata 402 Sesquiterpenes 419,659 from Neolitsea sericea 419 from Taiwania cryptomeriodes 419 ShaagrockolB 675 anifimgal activity of 675 from red sea sponge Toxiclona toxius 675 spectral data of 675 Sidnyum turbinatum 643 1-heptadecanyl sulfate from 643 1-hexyl sulfate from 643 1-octadecanyl sulfate from 643 sodium (25)-2,6,10,14-tetramethylpentadeca-1,18-diyl sulfate from 643 SigmoidinB 234 from Glycyrrhiza uralensis 234 Sinapic acid 262 in broccoli 262 in citrus juices 262 in kale 262 in leafy brassicas 262 Siphonodictyon coralliphagum 669 bis(sulfato)cyclosiphonodictyol A 669 siphonodictyal D from 669 siphonodictyols G from 669 Sodium (or potassium) 2,6-dimethylheptyl configuration of 642 from Halocynthia roretzi 642 from Polycitor adriaticus 642 sulfate 642
Sokotrasterol sulfate 700 from Halichondriidae family 700 Sophoraflavanone 234 from Anaxagorea luzonensis 234 Soroceal 201 from moraceous plants 201 Soybean isoflavones 286 in humans 286 Spasmolytic activity 257,293 of plant polyphenols 257,293 SpinosynA 405 SpinosynB 405 from Gynandropsis gynandra (L.) 405 structure of 405 Spinosyns 404 from Saccharopolyspora spinosa 404 Spiro oxmAoXt 371 formation of 371 Spiroalkyl analogs 357 biological activity of 357 ofMFA 357 Sponges (Porifera) 657 filter-feeding organisms of 657 living species of 657 Stellettamine 687 A^-deacetylkuanoniamine C from 687 from Stelletta sp. 687 total synthesis of 687 Stereoselective synthesis 74 of i^ retinal 74 Steroidal monoglycosides 587 from Ophioderma longicaudum 588 6a-Sterol sulfate 702 cytotoxicity against 702 from Dysideafragilis 702 from Venetian lagoon 702 Sterol sulfates haplosamate A 703 as HIV-1 integrase inhibitors 703 Stilbene derivatives 579 2,3,5,4'-tetrahydroxystilbene-2-0D-glucoside as 579 from Polygonum species 579 in LLC-bearing mice 579 piceidas 579
793
resveratrol as 579 tumor growth inhibition by 579 Stomphia coccinea 1\1 benzyltetrahydroisoquinoline imbricatine from 717 by starfish Dermasterias imbricata causes 717 Storage mites 423 Acarus siro as 423 Lepidoglyphus destructor as 432 Streptolygidin 130 antibiotic activity of 130 as bacterial RNA polymerase inhibitor 130 as terminal DNA transferase inhibitor 130 from Streptomyces lydicus 130 structure elucidation of 130 Structural elucidation 601 of sea cucumber triterpene glycosides 601 Structure/activity relationship 404 of eugenol 404 Stylophora pistillata 646 palythrine-threonine-sulfate from 646 Stylosanthes sp. 400 against Boophilus microplus 400 against Haemaphysalis intermedia 400 against Rhipicephalus sanguineus 400 Stylosanthes hamata 399 against Boophilus microplus 399 Stylosanthes humilis 399 against Boophilus microplus 399 Stylosanthes scabra 400 against Boophilus microplus 400 against Haemaphysalis intermedia 400 against Rhipicephalus sanguineus 400 34-Sulfatobastadin 13 695 as endothelin A receptor inhibitor 695 from Janthella sp. 695 Sulfide-containingpyrroloiminoquinones 685
as antileukaemic agents 685 from Prianos melanos 685 prianosin A as 685 structure of 685 Sulfircin 672 as antifimgal sesquiterpene sulfate 672 as A^, A^-dimethylguanidinium salt 672 hipposulfates A from 672 Sulfolane 715 from Batzella sp. 715 from Lissoclinum tunicate 115 Sulfone 715 Anchinoe tenacior 715 as constituent of Miditerranean sponge 715 Sulfiir-containing cyclic peptides 678 biological activities of 678 cytotoxicity of 678 Discodermin A as 678 Discodermin B as 678 Discodermin C as 678 Discodermin D as 678 from sponges 678 Suvanine 670 as acetyl cholinesterase inhibitor 670 from Coscinoderma species 670 from Ircinia species 670 Suzuki cross-coupling reaction 73 Swem oxidation 340 Synapic potential mediation 318 by non-NMDA receptors 318 Synthesis 503,508 of (±)-3p-hidroxinagilactone F 503 of3p-hydroxy-13,14,15,16tetranorlabda-7,9(l l)-dien(19,6p),(12,17)-diolide 508 ofLL-Z1271a 495 of nagilactone F 499 ofOidiolactoneC 505 Tannic acids 280 structure of 280
794
Tartaric conjugates 262 in coffee 262 Tauropinnaic acid 657 as phospholipase A2 inhibitor 657 from Pinna muricata 657 stereochemistry of 657 Taurospongin A 690 as DNA polymerase inhibitor 690 as HIV reverse transcriptase inhibitor 690 as sulfated acetylenic fatty acid derivative 690 Hippospongia sp. 690 Taxifolin 3-rha 41 from Licania apetala var. apetala 41 from Licania licaniaeflora 41 Taxisol 559 as anticancer drug 559 Tea tree {Melaleuca alternifolia, Myrtaceae) 415 activity against Psoroptes cuniculi 415 oil of 415 Tenuazonic acid 114 analogues of 115 antibacterial activity of 115 antiviral activity of 115 as growth inhibitor 115 biosynthesis of 114 for peptide inhibition 115 from Alternaria alternata 114 from Alternaria longipes 114 from Alternaria tenuis 114 from Phoma sorghina 114 from Pyricularia oryzae 114 Tertiary amines 373 oxidation of 373 Tetrahydroglabrene 234 synthesis of 234 2,4,2',4'-Tetrahydroxy-3'-prenylchalcone 16 aromatase inhibition by 16 5,7,2' ,4' -Tetrahydroxy-3 -gerany Iflavone 17 aromatase inhibition by 17 (2S)-5,7,2',4'-Tetrahydroxyflavanone 17 aromatase inhibition by 17
Tetramicacid 109,110 biological activity of 110 biosynthesis of 110 ^^C-NMRof 113 derivatives of 109 1,5-dihydro-4-hydroxy-2H-pyrrol2-oneas 109 from Chaetomium globosum 125 from lactic acid bacteria 126 from Melophlus sarassinorum 116 IR values of 113 2,4-pyrrolidinedione as 109 spectral properties of 111 structure of 110,111 synthesis of 110 UV spectra of 113 Tetramic acid magnesium salt 115 from Pseudomonas magnesiorubra 115 Tetranichus milQS 433 neem extracts against 433 Tetranorditerpenoid dilactones 454 from Acrostalagmus genus 454 from Aspergillus wentii 454 from Oidiodendron griseum 454 from Oidiodendron truncatum 454 Tetranychus urticae AIA^AIS adults of 424 effects of Amaryllidaceae family on 425 effects of Combretum glutinosum on 424 effects of Combretum micranthum on 424 effects of Glossostemon bruguieri on 425 effects of Juglans regia 424 effects of Melia azedarach extracts on 424 effects of Prosopis chilensis on 424 effects of Sambucus nigra on 424 effects of Taraxacum officinale (Asteraceae) on 424 QffQCts of trigyna on 424 leaf-dipping method against 424 reared on Brassica rapa 425 reared on Canna indica 425
795
reared on Conyza dioscoridis 425 reared on Trigonellafoenum' graecum 425 Theaflavin 274 formation of 274 Theaflavingallates 274 formation of 274 Thearubigins 274 formation of 274 Theonella sponges 681 keramamides F from 681 theonellapeptolide congener from 681 thiazole-containing cyclic peptides from 681 Theonezolides 710 as cytotoxic agents 710 from Theonella sp. 710 Therapeutic vaccines 541 against cancer 541 Thioacetates 666 absolute configuration of 666 conversion to fiirodysin 666 from Z>v^Wea species 666 (I5*,45*,65*,7/?*)-4-Thiocyanato-9cadinene 660 from Trachyopsis aplysinoides 660 X-ray analysis of 660 5-Thio-D-mannose 715 as naturally occurring 5-thiosugar 715 from Clathria pyramida 115 Thiofiirodysinin 667 asfiiranosesquiterpene667 from Dysidea avara 667 Thymol 391 employed as Frakno thymol frame 391 Thymovar 391 employed as Frakno thymol frame 391 Thymus vulgaris 391,415 against Knemidocoptes pilae 415 essential oils from 391,415 Tick-borne diseases 394 in livestock 394
Ticks toxicity 403 byeugenol 403 byisoeugenol 403 by methyleugenol 403 bysafrole 403 Tirandamycin A 131 biological activity of 131 from Streptomyces tirandis 131 Topsentiasp. 701 in guanosine diphosphate/G-protein RAS exchange assay 701 sulfates of 701 topsentiasterol sulfate A from 701 topsentiasterol sulfate B from 701 topsentiasterol sulfate C from 701 topsentiasterol sulfate D from 701 topsentiasterol sulfate E from 701 Tormentic acid 40 from Licania licaniaeflora 40 from Licania pyrifolia 40 Total synthesis 502 of nagilactone F 502 ToxadocialA 711 as sulfated long chain alcohols 711 from Toxadocia cylindrica 711 thrombin inhibition by 711 ToxadocialB 711 as sulfated long chain alcohols 711 from Toxadocia cylindrica 711 thrombin inhibition by 711 ToxadocialC 711 from Toxadocia cylindrica 111 thrombin inhibition by 711 Toxic essential oils 393 from Apis mellifera 393 from Varroajacobsoni 393 Toxiclona toxius 675 toxicol A from 675 toxiusol from 675 Trachyopsis halichondrioides 700 26-norsokotrasterol sulfate from 700 Tracheal mites 389 effects ofvegetable oils on 389 Triandamycin B 131 from Streptomyces flaveolus 131 Trichostrongylus colubriformis 342
796
Tridacna maxima 652 arsenic-containing sugar sulfate from 652 Tridentata marginata 646 tridentatol A from 646 tridentatol B from 646 tridentatol C from 646 Trididemnum sp. 638 from Guam 638 shermilamine A from 638 P-Triketone 117 from Apiosordaria effusa 117 Triterpene glycoside 596,587,589 antifiingal activity of 589 cytotoxic activity of 589 cytostatic activity of 589 from Cucumaria echinata 596 from Pentamera calcigera 596 from sea cucumbers 587 hemolytic activity of 589 immunomodulatory activity of 589 Tropical ixodid ticks 404 Hyalomma genera as 404 Rhipicephalus genera as 404 Tryptophan derivative 369 construction of 369 Tumor 532 effectof lipid A on 532 in host response to LPS 531 Tumor growth in LLC-bearing mice 581 effects of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside on 581 effects of piceid on 581 Tumor necrosis factor-a (TNF-a) 219 as tumor promoter 219 by okadaic acid 219 Tunicates (Ascidians) 621 active metabolites of 622 ascidiacyclamide from 622 bistratamides from 622 from Phylum chordata 621 lissoclinamides from 622 patellamides from 622 Tyrindoxyl sulfate 652 as Tyrian purple dye 652 from Murex truncatus 652
Tyrophagus putrescentiae 421 against 1,8-cineole 421 against fenchone 421 against isomers of caryophyllene 421 against linalool 421 against linalyl 421 against menthone 421 against myrtanol 421 against Pinus halepensis 420 digdxnsX Pinus nigra 420 against Pinus pinaster 420 against Pinus pinea 420 against pinene 421 against pulegone 421 against a-terpinene 421 against y-terpinene 421 against terpineol 421 against valencene 421 UoamineA 644 from Aplidium uouo 644 UoamineB 644 of Aplidium uouo 644 3-thiomethylacrylate ester group of 644 Urbalactone 459 from Podocarpus urbanii 459 Ursolic acid 18,40 from Licania carii 40 from Licania licaniaeflora 40 from Licania pittieri 40 from Licania pyrifolia 40 inhibition of HI V-1 protease dimerization by 18 Uvaria pauciovulata ATI effects on Dermatophagoides pteronyssinus All squamocin from 422 structure of 422 Vancoresmycin 150 activity against gram-positive bacteria 150 from Amycolatopsin sp. 150
797
Varroajacobsoni 387 honey bees tolerant to 387 toxicity of 387 VarroaxmiQS 383,384,392 as honey bee parasites 384 biological activity of 392 Vasoprotective effects 302 of green tea 302 Vermisporin 125 antimicrobial activity of 125 from Ophiobolus vermisporis 125 Vernonia amygdalina 400 tick toxicity of 400 Veterinary medicine 383 for ectoparasites control 383 Vetiver grass 399 for controlling ticks 399 Phetchabunas 399 'Si Sa Kef as 399 'Uthai Thani' as 399 VirenamideA 644 as cytotoxic linear peptides 644 from Diplosoma virens 644 Virenamide B 644 as cytotoxic linear peptides 644 from Diplosoma virens 644 Virenamide C 644 as cytotoxic linear peptides 644 from Diplosoma virens 644,645 Vitamin A synthesis 71 by Baadische Anilin by 71 by Hoffrnann-LaRoche 71 by Rhone-Poulenc 71 SodaFabrik 71 Waiakeamide 684 as cyclic hexapeptide 684 from Ircinia dendroides 684 Watersipora subtorquata 620 5,7-dihydroxy-6-oxo-6//anthra[ 1,9-6c]thiophene-1 carboxylic acid from 620 Wentilactone A 465 from Aspergillus wentii 465 Wentilactone B 465 from Aspergillus wentii 465
Wheat flour 259 vanillic acid in 259 syringic acid in 259 Xanthobaccin A 140 asfiingitoxicmetabolite Xanthobaccin B 140 asftmgitoxicmetabolite Xanthobaccin C 140 asfiingitoxicmetabolite Xestoquinolide B 677 from Xestospongia cf. carbonaria 611 protein kinase activity of spectral data of 677
140 140 140
677
Yessotoxin 653 analogues of 653 from Patinopecten yessoensis 653 in diarrhetic shellfish poisoning (DSP) 653 Zoanthus sp. 646 sphingolipid hariamide from 646 zoanthid A from 646 Zyzzya cf marsailis 686 discorhabdin A from 686 makaluvamine F from 686 total synthesis of 686
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