Studies in Natural Products Chemistry Volume 22 Bioactive Natural Products (Part C )
Studies in Natural Products Chemistry edited by Atta-ur-Rahman
Vol. 1 Stereoselective Synthesis (Part A) Vol. 2 Structure Elucidation (Part A) Vol. 3 Stereoselective Synthesis (Part B) Vol. 4 Stereoselective Synthesis (Part C) Vol. 5 Structure Elucidation (Part B) Vol. 6 Stereoselective Synthesis (Part D) Vol. 7 Structure and Chemistry (Part A) Vol. 8 Stereoselective Synthesis (Part E) Vol. 9 Structure and Chemistry (Part B) Vol. 10 Stereoselective Synthesis (Part F) Vol. 11 Stereoselective Synthesis (Part G) Vol. 12 Stereoselective Synthesis (Part H) Vol. 13 Bioactive Natural Products (Part A) Vo1.14 Stereoselective Synthesis (Part I) Vol. 15 Structure and Chemistry (Part C) Vo1.16 Stereoselective Synthesis (Part J) Vol. 17 Structure and Chemistry (Part D) Vol. 18 Stereoselective Synthesis (Part K) Vol. 19 Structure and Chemistry (Part E) Vol.20 Structure and Chemistry (Part F) Vo1.21 Bioactive Natural Products (Part B) Vo1.22 Bioactive Natural Products (Part C)
Studies in Natural Products Chemistry Volume 22 Bioactive Natural Products (Part C)
Edited by
Atta-ur-Rahman
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
2000 ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon
- Singapore
- Tokyo
ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands 92000 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 Science Global Rights Department, PO Box 800, Oxford OX5 1DX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-maih
[email protected]. You may also contact Global Rights directly through Elsevier's home page (http://www.elsevier.nl), by selecting 'Obtaining Permissions'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (978) 7508400, fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WlP 0LP, UK; phone: (+44) 171 631 5555; fax: (+44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Global Rights Department, at the mail, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2000 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for.
ISBN:
0 444 50588 1
Transferred to digital printing 2005 Printed and bound by Antony Rowe Ltd, Eastbourne
Foreword Plant and animal organisms offer a wide diversity of compounds which can serve as exciting new pharmacophores and which can reveal new mechanisms of action for controlling disease processes. Vol. 22 of Studies in Natural Products Chemistry comprises articles written on bioactive natural products by leading authorities in their respective fields. Robinson has reviewed the metabolism and pharmacology of alkaloids found in animals. Chemical ecology can be an attractive tool for identifying antifungal natural products and this area has been reviewed by Graham et al. The chapter by R ios and co-workers presents studies carried out on triterpenes which have shown anti-inflammatory activity. Malaria continues to be a major health problem in developing countries and there are a large number of deaths each year caused by it. The review by Kawanishi et al. presents the current status of work done in this field as well as on natural anti-diabetic compounds. Synthetic approaches involving intramolecular diyl trapping reaction are described by Little et al. for the synthesis of linearly fused tricyclopentanoids. The use of classical and biocombinatorial approaches to bioactive fungal natural products is discussed by Jiang et al. F010p has presented the chemistry of 2aminocyclopentanecarboxylic acid. Antioxidants continue to attract attention in medicine and many interesting flavonoids have been found in nature which possess antioxidant and pro-oxidant properties. These studies have been reviewed by Vanden Berghe and co-workers, while antioxidant activity found in South American plants is reviewed by Desmarchelier. Other interesting reviews include those on insect juvenile hormones in plants by Tobe, antiulcer and gastroprotective activity of flavones by Martin et al, biological activity of simple flavones by Tahara et al. Anti-convulsant plants by Raza and biological activity of anthracenones of the Karwinskia genus by PiSeyro-Lopez. A number of different species of plants of Polygonumspecies possess interesting biological activities. The bioactive compounds in such plants are reviewed by Adamczeski. Hypericum perforatum (St. Johns wort) is one of the extensively studied plants because of its wide range of biological activities, specially its use for the treatment of mild to moderate depression. The review by Erdelmeier describes the research carried out on this plant. Finally tropane alkaloids are reviewed by Christen in respect of their chemistry and biological activity. I would like to express my thanks to Dr. Shakil Ahmad and Dr. Durre Shahwar for their 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. It is hoped that this volume, which represents the third volume of this series devoted to bioactive natural products, will be of great interest to organic chemists, medicinal chemists and pharmacologists.
Atta.ur.Rahman February, 2000
Ph.D. (Cantab), Sc.D. (Cantab)
This Page Intentionally Left Blank
Preface In his fascinating and beautifully written autobiography (For the Love of Enzymes, Harvard University Press, Cambridge, MA, 1989), Arthur Kornberg repeatedly emphasizes the central role of chemistry in understanding life processes. Nowhere is this connection clearer and more direct than in the field of "Natural Products" chemistry. From the early stereochemical studies on tartaric acid carried out by Louis Pasteur one-and-a-half centuries ago, through the subsequent pioneering work of chemists such as Emil Fischer, Otto Wallach, Robert Robinson, Vlado Prelog, R.B. Woodward, D.H.R. Barton .... (the list could go on and on), we have seen again and again how the careful study of naturallyoccurring compounds has, on the one hand, enriched our understanding of the science of organic chemistry itself, and on the other hand, provided deep insights into biological phenomena. Despite wide swings in the popularity or even "trendiness" of natural products research, the field continues to advance around the world. It is not a coincidence that natural products have played, and will continue to play, a seminal role in the discovery and development of pharmaceutically and agrochemically important agents. Three billion years of biological evolution have resulted in the development of metabolic pathways leading to the synthesis of hormones, pheromones, antibacterial, anti-fungal, anti-protozoan, and antiinsectan agents, as well as many other bioactive compounds that are of adaptive value in the lives of the organisms that produce them. The natural products chemists of the world (in some respects indistinguishable from "chemical ecologists") isolate these molecular entities, focussing chiefly on compounds with particularly interesting biological activities either from the human point of view or from that of the producing organism. They establish their structures and define their biosynthetic pathways. They study their mechanisms of action and their metabolic pathways. They devise synthetic methods which make novel target structures accessible for further research and for application. While much of this research is driven by the entirely worthy desire to obtain "useful knowledge," it is clear that scientists entering the field of natural products chemistry are often deeply motivated by their love of nature in general, of chemistry in particular, and by their fascination with understanding as much of life as possible at the molecular level. The "Studies in Natural Products Chemistry' series, now in its twenty-second volume, documents an incredible diversity of research. If we bear in mind the fact that for some of the most important groups of organisms (i.e. soil dwelling microbes; insects and other arthropods), most species have not yet been described, let alone subjected to chemical investigation, we can look forward eagerly to many future volumes in this series. With respect to the present volume, the reader can expect a veritable chemical feast.
Jerrold Meinwald
Cornell University Ithaca, NY 14853 USA
This Page Intentionally Left Blank
CONTENTS Foreword
V
Preface
vii
Contributors
xi
The Metabolism and Biochemical Actions of Alkaloids in Animals T. ROBINSON Using Chemical Ecology to Locate New Antifungal Natural Products STEPHANIE J. ECKERMAN AND KATE J. GRAHAM
55
Natural Triterpenoids as Anti-Inflammatory Agents J.L. RJOS, M.C. RECIO, S. MAlqEZ AND R.M. GINER
93
Current Status of the Chemistry and Synthesis of Natural Antimalarial Compounds and natural Substances used to Alleviate Symptoms of Diabetes (Aldose Reductase and A-Glucosidase Inhibitors) K. KAWANISHI AND N.R. FARNSWORTH 145 A Diradical Route to Bioactive Natural Products and their Analogs R. DANIEL LITTLE AND MICHAEL M. OTT
195
Bioactive Fungal Natural Products Through Classic and Biocombinatorial Approaches ZHI-DONG JIANG AND ZHIQIANG AN
245
The Chemistry of 2-Aminocyclopentanecarboxylic Acid FERENC FOLOP
273
Structure-Activity Relationship of Flavonoids as Antioxidant and Pro-Oxidant Compounds P. COS, M. CALOMME, L. PIETERS, A.J. VLIETINCK AND D. VANDEN BERGHE
307
Recent Advances in the Search for Antioxidant activity in South American Plants C. DESMARCHELIER, G. CICCIA AND J. COUSSIO
343
Insect Juvenile Hormones in Plants JACQUELINE C. BEDE AND STEPHEN S. TOBE
369
Antiulcer and Gastroprotective Activity of Flavonic Compounds" Mechanisms Involved M.J. MARTIN, C. ALARCON DE LA LASTRA, V. MOTILVA AND C. LA CASA
419
Simple Flavones Possessing Complex Biological Activity S. TAHARA AND J.L. INGHAM
457
Medicinal Plants with Anticonvulsant Activities MOHSIN RAZA, M. IQBAL CHOUDHARY AND ATTA-UR-RAHMAN
507
Chemistry, Structure and Biological Activity of Anthracenones of the Karwinskia Genus A. PllqEYRO-LOPEZ AND N. WAKSMAN
555
Bioactive Natural Products Derived from Polygonum Species of Plants: Their Structures and Mechanisms of Action NWAKA OGWURU AND MADELINE ADAMCZESKI
607
Hypericum Perforatum - St. John's Won:Chemical, Pharmacological and Clinical Aspects C.A.J. ERDELMEIER, E. KOCH AND R. HOERR
643
Tropane Alkaloids: Old Drugs used in Modem Medicine P. CHRISTEN
717
Subject Index
751
CONTRIBUTORS Madeline Adamczeski
Department of Chemistry, American Washington, D.C. 20016-8014, USA
University,
Zhiqiang An
Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge, MA 02139-1562, USA
Atta-ur-Rahman
International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
Jacqueline C. Bede
Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, M5S 3G5, Canada
D. Vanden Berghe
Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium
M. Calomme
Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium
M. Iqbal Choudhary
International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
P. Christen
University of Geneva, Laboratory of Pharmaceutical Analytical Chemistry, 20, Boulevard d'Yvoy, CH-1211 Geneva 4, Switzerland
G. Ciccia
C6tedra de Microbiologia Industrial y Biotecnologia, Facultad de Farmacia y Bioqulmica, Universidad de Buenos Aires, Junin 956 1113 Buenos Aires, Argentina
P. Cos
Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium
J. Coussio
C~tedra de Farmacognosia, IQUIMEFA-CONICET, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Jun|n 956 1113 Buenos Aires, Argentina
C. Alarc6n de la Lastra
Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcfa Gonz~lez s/n, 41012Seville, Spain
xii
C. Desmarchelier
C6tedra de Microbiologta Industrial y Biotecnologta, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 1113 Buenos Aires, Argentina
Stephanie J. Eckerman
Chemistry Department College of St. Benedict/St. John's University 37 S. College Avenue, St. Joseph, MN 56374, USA
C.A.J. Erdelmeier
Dr. Willmar Schwabe GmbH & Co., Research and Development, Karlsruhe, Germany
N.R. Famsworth
Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Illinois, USA
Ferenc Ft~lOp
Institute of Pharmaceutical Chemistry, Albert SzentGy0rgyi Medical University, H-6701, Szeged, POB 121, Hungary
R.M. Giner
Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Avda. Vicent Andr/~s Estell~s s/n., 46100 Burjassot (Valencia), Spain
Kate J. Graham
Chemistry Department College of St. Benedict/St. John's University 37 S. College Avenue, St. Joseph, MN 56374, USA
R. Hoerr
Dr. Willmar Schwabe GmbH & Co., Research and Development, Karlsruhe, Germany
J.L. Ingham
Department of Food Science and Technology, University of Reading, Whiteknights, P.O. Box 226, Reading RG6 2AP, England, U.K.
Zhi-Dong Jiang
Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge, MA 02139-1562, USA
K. Kawanishi
Kobe Pharmaceutical University, Kobe, Japan
E. Koch
Dr. Willmar Schwabe GmbH & Co., Research and Development, Karlsruhe, Germany
C. La Casa
Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonzfilez s/n, 41012Seville, Spain
~176176
XIU
R. Daniel Little
Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
S. M~iflez
Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Avda. Vicent Andr6s Estell/~s s/n., 46100 Burjassot (Val/mcia), Spain
M.J. Martin
Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonz~ilez s/n, 41012Seville, Spain
V. Motilva
Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonz~ilez s/n, 41012Seville, Spain
Nwaka Ogwuru
Department of Chemistry, American Washington, D.C. 20016-8014, USA
Michael M. Ott
Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
L. Pieters
Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium
A. Pifleyro-Lopez
Departmento de Farmacologia, Y Toxicologia, Apdo. Postal 146, Col. del Valle, 66220, Garza Garcia, N.L. Mexico
Mohsin Raza
International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
M.C. Recio
Departament de Farmacologia, Facultat de Farmhcia, Universitat de Valb,ncia, Avda. Vicent AndrOs Estell6s s/n., 46100 Burjassot (Valencia), Spain
J.L. Rios
Departament de Farmacologia, Facultat de Farmb.cia, Universitat de Valencia, Avda. Vicent AndrOs Estell~s s/n., 46100 Burjassot (Valencia), Spain
T. Robinson
Lederle Graduate Research Center, Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst Box 34505, Amherst, MA 010034505, U.S.A.
University,
xiv
S. Tahara
Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589, Japan
Stephen S. Tobe
Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, M5S 3G5, Canada
A.J. Vlietinck
Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium
N. Waksman
Departmento de Farmacologia, Y Toxicologia, Apdo. Postal 146, Col. del Valle, 66220, Garza Garcta, N.L. M~xico
Bioactive Natural Products
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Pror Chemistry Vol. 22 9 2000 ElsevierScience B.V. All rights reserved
THE METABOLISM AND BIOCHEMICAL ALKALOIDS IN ANIMALS
ACTIONS OF
T. ROBINSON
Lederle Graduate Research Center Department of Biochemistry and Molecular Biology University of Massachusetts, Amherst Box 34505, Amherst, MA 01003-4505 U.S.A A B S T R A C T : The metabolism and pharmacology of naturally-occurring alkaloids are reviewed, with emphasis on work of the last ten years during which there have been many important advances. It is recognized that the final effects of alkaloids on animals may result from activity of their metabolic products rather than of the original substance. Therefore one section discusses how alkaloids are metabolized, first in terms of the general processes and then with many examples of the metabolism of particular types of alkaloid structures. The second section on pharmacology deals with detailed biochemistry of alkaloid action at the level of molecule-to-molecule, rather than describing behavioral or gross physiological effects. Modern molecular biological methods have revealed the intimate structures of neural receptors and other cellular molecules with which the alkaloids interact. In this section of the review the material is organized according to the processes being affected rather than according to types of alkaloid. Thus there are subsections for the various neural receptors, structural components of cells, enzymes, etc.
GENERAL INTRODUCTION The toxic and stimulatory effects of alkaloid-containing plants o11 the behavior of animals have been observed for many centuries; and since the early 19th Century it has been possible to attribute many of these effects to specific, chemically characterized substances, which became known as "alkaloids" because many of them formed salts with acids. Late in the 19th Century the action of certain alkaloids on specific physiological systems became clear. Since the middle of the 20th Century the availability of isolated and well-characterized biochemical systems has made possible still further refinement in our knowledge of how alkaloids act as they do in terms of membrane structures, enzymes, receptors, transporters, and so on. It is such systems of molecule vs. molecule that are the focus of this review. Behavioral effects, and even effects on gross physiological systems, have a vast literature that will be mostly passed over here. "Animals" is used here in a broad sense, to include insects, molluscs, and other lower life forms, as well as mammals. Microorganisms are, however, excluded. With the growing realization that some effects of alkaloids are, in fact, effects of their metabolic products rather than of the originally
4
T. ROBINSON
administered compound, it became essential to include information about how alkaloids are metabolized. METABOLISM
Introduction, Common Processes Animals have processes for dealing with foreign compounds, some of which are assimilated because of their nutritional importance, while others are of no value or even detrimental. Alkaloids fall into the latter group, but they are treated by processes that are not unique to them but have general roles in metabolism. Such processes as hydrolysis, oxidation, reduction, and conjugation are applied to alkaloids just as they are to such nutritionally important molecules as carbohydrates or proteins. The following sections review these processes, first in general and then as they apply to alkaloids. As a general rule, it appears that the metabolism of alkaloids in animals does not proceed to complete breakdown yielding carbon dioxide but that a few small modifications of the structure are produced.
A. Hydrolysis Several alkaloids contain ester groups, and an early step in their metabolism is the hydrolysis of the ester bond. Additional reactions may then occur to complete the metabolism. As will be shown in specific examples, esterases both in blood serum and the liver are active in different cases.
B. Oxidation Oxidative processes are well-known in animal metabolism; and they include pocesses that abstract hydrogen atoms from the substrate as well as those that add oxygen atoms to the substrate. For alkaloid molecules, though, oxygenation is more usual than dehydrogenation. This may be because the dehydrogenase enzymes are closely matched with the structures of their usual substrates, while oxygenases are less specific. Cytochrome P-450 Enzymes occur in all classes of organisms, and a single species may have several dozen different types, acting on hundreds of different substrates as dioxygenases. Some use flavoprotein as a reductant and some use cytochrome Bs. They have roles both in normal metabolism of steroids, eicosanoids, nitric oxide, etc. and also in oxidizing exogenous compounds, including certain alkaloids. Some exogenous compounds induce the formation of P-450 enzymes through a complex
ALKALOIDS IN ANIMALS
5
series of molecular events [1 ]. As well as introduction of oxygen atoms, the P-450 systems are also responsible for removal of N-methyl groups, converting them to carbon dioxide. Not all oxidations of alkaloids can be ascribed to microsomal P-450 systems. Mammalian liver contains a flavoprotein oxidase, first discovered in rabit liver and called "quinine oxidase". It is also an aldehyde oxidase, and its mechanism of action on heterocyclic nitrogen compounds probably involves addition of hydroxide ion to a suitable ring position, followed by dehydrogenation. Thus, the introduced oxygen atom comes from water rather than from molecular oxygen, in contrast to the P-450 oxygenases.
[2]. C. Conjugation Conjugation describes a process in which some exogenous molecule becomes joined to a common metabolite. Examples are the addition of acyl groups, amino acid residues, carbohydrate groups or sulfate groups. Conjugation may follow preliminary oxidative or hydrolytic processes that release hydroxyl groups suitable for the derivatization, there are also some cases of addition of methyl groups [3]. Besides metabolic process that degrade alkaloids, there are also process that produce alkaloids in animals. Some of these are straightforward condensations of amines with aldehydes or ketones as in the formation of isoquinolines or harman derivatives by condensation of an aldehyde or ketone with, respectively, dopamine or tryptamine [4, 5, 6]. More complex reactions must also occur to account for the formation of such complex structures as morphine, which is now well-established as an endogenous compound in animals, although at very low concentration [7, 8]. Amaryllidaceae Alkaloids
Galanthamine products found in human plasma and urine result from epimerization of the hydroxyl group and then dehydration to a ketone [9]. Caffeine and other Purines
Methylated xanthines like caffeine are degraded in humans and rats by oxidative removal of methyl groups [10]. Thus caffeine goes to 1,7dimethylxanthine and 1-methylxanthine [11]. After the first demethylation there is an alternate pathway producing 5-acetylamino-6-formylamino-3methyluracil. This pathway is more active in people with a more active acetylation system [ 12, 13]. There are individual, quantitative differences in the activity of this pathway in humans. In rats a major metabolite is
6
T. ROBINSON
1,3,8-trimethylallantoin, in which all three methyl groups are retained [ 14]. In rabbits the major urinary products are, in order, 1-methylxanthine, 1methyluric acid, 7-methylxanthine, and 1,7-dimethylxanthine [15]. Pretreatment of rats with caffeine increased the activities of the P-450 enzymes; so that demethylation and C-8 oxidation were doubled as compared with untreated rats [ 16]. Pretreatment with polycyclic aromatic compounds causes a similar increase in this P-450 activity [ 17]. Experiments with liver slices and cultured cell lines have corroborated this pathway of purine degradation. In human liver slices 16 xanthine derivatives were produced from caffeine by action of P-450 system. Demethylation at N-3 was the most prominent process [18, 19]. Comparison of cell lines from humans, hamsters, mice, and rats show some interspecies differences but all of them demethylated and oxidized caffeine [20]. Human liver cells give 1,3,7-trimethylurate as the major metabolite of caffeine, but also made were the intermediate products theobromine, theophylline, and paraxanthine [21]. Human liver microsomes convert theophylline to 1-methylxanthine, 3-methylxanthine, and 1,3-dimethyluric acid [22, 23]. Human kidney microsomes produced each of the three possible demethylated products as well as 1,3,70
CH3
0
(~H3 Caffeine
(~H3 Theophylline
1
O
O
CH3....,J~
iCH3
1,7-Dimethylxanthine
1
H
G 1-Methylxanthine
O
CH3~ ~
~I"[~C)C;H3 5-Acetylamino-6-formylamino-3-methyluracil
H Caffeine Metabolism
ALKALOIDS IN ANIMALS
7
trimethyluric acid from caffeine [24]. It appears that two distinct P-450 systems are involved in these p r o c e s s e s - - - o n e responsible for demethylation and the other for oxidation at C-8 [16, 25, 26, 27]. In premature human infants the reverse of the demethylation process occurs, in that theophylline, which is 1,3-dimethylxanthine, becomes methylated at N-7 to make caffeine [28].
Cocaine and Other Tropane Alkaloids Because of its wide use as a drug of abuse, cocaine's metabolism has been studied more than that of other alkaloids of the tropane group. Isolation of breakdown products from the urine following administration of cocaine has indicated that processes of demethylation, ester hydrolysis, and hydroxylation of the aromatic ring account for the more than a dozen products that have been identified [29, 30, 31, 32]. Studies on the enzymology of these processes have localized these processes both histologically and, in some cases, intracellularly. Esterases that hydrolyze the two ester groups are widely distributed in tissues ~ for instance in serum, liver, and brain. It is thought that hydrolysis in the serum is slow and contributes little to the overall rate of degradation [33, 34, 35]. The enzyme in serum that is responsible for this hydrolysis is 2000 times more active in hydrolyzing the unnatural isomer (+)-cocaine than the natural (-)-cocaine, and this difference may account for the low pharmacological activity of the (+)-isomer [36]. An interesting sidereaction occurs in subjects (rats or humans) who consume ethanol at the same time as cocaine. The esterase present in the microsomal fraction of liver catalyzes a transesterification reaction that replaces the methyl ester /0-13 N~-..~
H %jL.;LX.X.,,~3
N ~ ~3
H (-)-Cocaine ~
H,,,j~2H5
R Cocaethylene drolysis
Ecgonine
8
T. ROBINSON
grouping with an ethyl ester, giving the compound known as cocaethylene [34, 37, 38]. Cocaethylene persists in the serum longer than cocaine itself and is more potent in some actions [39, 40]. Antibodies raised to a cocaine analog in its hydrolysis transition state were tested for hydrolytic activity, and one of them was found to be active [41 ]. Oxidation is the second major process in degradation of cocaine, and it occurs in the liver as a result of activity of microsomal cytochrome P-450 enzymes. Oxidative reactions account for hydroxylation of the aromatic ring of the benzoic acid moiety and for N-demethylation [30, 42, 43]. Specifically, it appears to be the 3A form of P-450 that is responsible, since feeding inhibitors of this isoform to mice inhibited the hepatotoxicity caused by the demethylated product [44]. There is less information about the metabolism of other tropane alkaloids; but for those that are esters (e.g. hyoscyamine, scopolamine) hydrolysis of the ester bond takes place to some extent, yielding bases, which may be further transformed [45, 46]. Rabbit serum has been found to have esterases that are relatively specific for esters of tropic acid, and this may account for the ability of rabbits to feed with impunity on leaves of Atropa belladona [47]. Mice transform released tropic acid to a glucuronide [48]. Oxidation also occurs, both in rats and humans, with the production of N-oxides and oxidative removal of the N-methyl group to make nor-derivatives [45, 46].
Colchicine Colchicine is demethylated i n the liver, forming 2-demethyl and 3demethyl products A microsomal P-450, NADPH-dependent system is responsible for this demethylation [49]
Cyclopeptide Alkaloids As might be expected, peptidases can catalyze hydrolysis of cyclopeptides ~ for example frangufoline [50].
CH3
Frangufoline
ALKALOIDS IN ANIMALS
9
Ellipticine Group
CH3
Ellipticine
In the intact rat or liver microsomes ellipticine is hydroxylated at C-9 or C-7. The resulting hydroxyl derivative is then converted to a glucuronide or dehydrogenated to a keto derivative [51, 52]. 9Methoxyellipticine is demethylated and conjugated with glucuronic acid at the C-9 position or it is conjugated with glutathione at the C-10 position [52, 53]. 9-Hydroxy-2-methylellipticine is similarly conjugated, not only with glutathione but also with N-acetylcysteine [54]. Oxidation of elliptinium salts produces a quinone that reacts with amino compounds to form oxazoles [55].
Indole Alkaloids Serotonin is metabolized to bufotenin, and much of this is excreted in the urine as a glucuronide conjugate [56]. Vindoline and related alkaloids of C a t h a r a n t h u s roseus are extensively metabolized by mammals through the actions of esterases, peroxidases, and P-450 oxidases [57]. Oxidation of vindoline and vinblastine occurs in human serum, catalyzed by ceruloplasmin [58]. The major metabolite of yohimbine is l lhydroxyyohimbine, but the 10-hydroxy derivative is also produced [59]. Strychnine injected into rats gives as its major metabolite its 21,22epoxide, but other epoxidized and hydroxy derivatives are also prodced [60]. Harman is hydroxylated at C-6 by mice [61 ].
Isoquinoline and Morphinan Alkaloids Laudanosine is O-demethylated to various products by dogs, rabbits, and humans [62]; but 1,2,3,4-tetrahydroisoquinoline in human brain is Nmethylated [63]. Several isoquinoline alkaloids are O-methylated by the action of catechol O-methyl transferase [64]. It is now well accepted that small amounts of morphine and related alkaloids are not only present in many foods but are also synthesized normally by animals. Therefore the metabolism of exogenous morphine and related molecules calls for no processes outside of what must be usual metabolism [65, 66]. Normal human plasma has been found to contain
10
T. ROBINSON
80pg/ml of endogenous morphine [67]. The concentration is higher in anorexic and bulimic subjects [68]. The pathway for biosynthesis of morphine in animals is similar to that in plants with the final steps being" (R)-reticuline - : > ( + ) - s a l u t a r i d i n e codeinone > codeine > morphine
>-----> thebaine
>
The oxidative reactions of this sequence are catalyzed by the microsomal P-450 system [69, 70, 71]. A P-450 system from rat liver can also oxidize morphine [72]. One product of this oxidation is morphinone, a highly toxic electrophile that couples with thiol groups. The latter reaction may deplete glutathione and in other ways may account for the hepatotoxicity of morphine [73, 74]. The demethylation of codeine to morphine probably accounts for the analgesic action of codeine, and people with a defect in this demethylating system probably get no analgesia from codeine [75, 76]. Rats, too, show strain differences in the ability to demethylate codeine to morphine [77]. Quinidine, quinine, or sparteine inhibit the conversion of codeine to morphine, presumably by inhibiting the P-450 enzyme [71, 78]. While O-demethylation converts codeine to morphine, N-demethylation also occurs and produces norcodeine [79]. Conjugation with glucuronic acid is a major process for detoxification of codeine and morphine. For morphine both the 3- and 6-glucuronides are produced, but the ratio between them varies widely with the species. Generally the 3-glucuronide predominates; but in humans and guinea pigs there is also significant formation of 6-glucuronide, while rats make very little of that isomer [80]; but the unnatural isomer (+)-morphine is glucuronidated by rats more at the 6-position than the 3-position [81 ]. In the presence of low concentrations of ethanol more 6-glucuronide is made, but higher concentrations of ethanol inhibit the glucuronidation reaction [82]. Two enzymes that catalyze the glucuronidation have been characterized in liver microsomes [83, 84]. Along with the glucuronides as excretion products in the urine, the adduct of morphinone with glutathione can be prominent in some species [85]. Major excretion products from codeine are norcodeine, morphine, and codeine-6-glucuronide [86]. Nicotine
and
Related
Alkaloids
The metabolism of nicotine is a much-studied area in which metabolic products were first identified in the urine and later the mechanisms for their formation were characterized in detail. The major products obviously result from oxidations, some that introduce oxygen atoms, and some that remove methyl groups. The half-life of nicotine in blood can vary greatly. In mice a half life of only 6 minutes has been found [87], but in humans it is about 30 minutes [88]. Moreover there are strain to strain differences within a species [87] and even between males and females or between
11
ALKALOIDS IN ANIMALS
smokers and nonsmokers [89]. While the major metabolic products are the same in all animals, there are quantitative differences from species to species.
HO.!~
H3CO"~
//~-----~N_CH3
3CO
(-)-Reticuline
HOOx O,
N'-CH3
_
~
H3CO O (+)-Salutaridine
o ~ . /~
.......
.~ O, ~
"-CH3
H3CO Thebaine
. HO N--OH
H
Codeinone
13-GIucosyl. N-'CH3 H Morphine-6-glucuronide Morphine-RelatedPathway
N
Codeine
HO.~ H
Morphine
12
T. ROBINSON
Generally found in significant amounts are nornicotine, cotinine, cotinine N-oxide, trans-3'-hydroxycotinine (and its glucuronide), quaternary N-methylated derivatives, nicotine l'-N-oxide, nicotine Nglucuronide, and some unchanged nicotine [90, 91, 92, 93, 94] . Interestingly, there are somewhat diferent patterns of metabolites depending on the route of nicotine administration [95] or on preadministration of ethanol or phenobarbital [96, 97, 98]. The natural form, S-(-)-nicotine gives rise to a different pattern of products than unatural R-(+)-nieotine [99, 100, 101 ].
CH 3
"~
H
Nornicotine
Nicotine
1
5'Hydroxynicotine
I
CH3
Cotinine
CH3
3-Hydroxycontinine
T
o
CH3
Cotinine-N-oxide
Nicotine Metabolism
At the subcellular level the importance of the P-450 system of microsomes in metabolizing nicotine has been clearly established [98, 102] 9The first step is oxidation by P-450 at C-5' to give the 1'-5'-iminium ion that is then oxygenated using water as the source of oxygen [103, 104]. A flavoprotein is implicated in this later step, or possibly also in an alternative pathway [ 104, 105, 106]. The N-demethylation and N-oxideforming reactions are also catalyzed in microsomes, but the details of these
ALKALOIDS IN ANIMALS
13
processes are not characterized [98]. Formation of N-methyl quaternary ammonium derivative is catalyzed by a cytosolic enzyme from human liver but not from rat liver [ 107]. Quatemization abolishes the ability of nicotine to penetrate the blood-brain barrier [108].
Pyridines Arecoline is rapidly metabolized, the first step being hydrolysis of the ester group through action of carboxylesterase [ 109].
Pyrrolizidines The pyrrolizidine alkaloids are well-known as liver toxins. They not only poison livestock that eat plants containing them but have also been implicated in human cases where large amounts of certain medicinal herbs (e.g. comfrey) have been used. The reason for discussing them in this section is that the actual toxins are not the native alkaloids themselves but metabolic products of them produced in the liver by the action of microsomal enzymes. The esters may be hydrolyzed and N-oxides reduced, but it is dehydrogenation of the saturated pyrrolidine ring to a pyrrole that gives rise to the toxicity [1 10, 111]. For instance, monocrotaline in rat liver gives rise to monocrotalic acid and several pyrroles [112]. Retrorsine N-oxide is converted to dehydroretronecine [113]. In the liver these pyrroles react with thiol groups of essential molecules to form thioethers [114]. They may also be detoxified by conjugation with glutathione [ 115]. For instance dehydroretronecine is converted in rat liver to 7glutathionyldehydroretronecine. [ 116]. Dehydromonocrotaline, produced from monocrotaline is believed to be the active toxic metabolite. It reacts at its C-9" or C-7" position to alkylate the N-3 position of several purine nucleosides or various amino groups [117]; but it can be detoxified by conjugation with glutathione. Apparently an early action of the alkaloid is
t,. ~ I . ~
~CH2CH o
~H2CH v
o
Retrorsine-N-oxide
Dehydroretronecine
14
T. ROBINSON
a diversion of cysteine metabolism away from conversion to taurine and toward synthesis of glutathione [ 118]. Senecionine is converted to the Noxide not by a P-450 enzyme but by a flavin-containing oxidase in pigs and guinea pigs [ 119, 120]. An interesting sidelight on the metabolism of pyrrolizidines is that several butterfly and moth larvae that feed on plants containing these alkaloids metabolize them to products that serve the insects as pheromones or defensive substances. Thus the butterfly ldea leuconoe converts alkaloids from Parsonia laevigata to N-oxides [ 121 ]. The moth Creatonotos transiens makes its pheromone, R(-)-hydroxydanaidal from7(S)-heliotrine found in its host plant, Seneciojacobaea [122].
Quinolines Dictamnine fed to rats is demethylated and the ring system oxidized [123]. Quinidine is oxidized to 3-hydroxyquinidine and other products [ 124].
Quinolizidines The metabolism of sparteine by humans produces as the chief products 2and 5-dehydrosparteine as well as 2- and 7-oxo derivatives [124, 125]. Somewhere less than 10% of the Caucasian population have little ability to metabolize it at all [126, 127, 128]. Pachycarpine, the optical antipode of (-)-sparteine is converted in rats to (+)-(4S)-hydroxysparteine as the major product [ 129].
Tyramine Derivatives Cathinone is metabolized by humans to (R,S)-(-)-norephedrine and (R,R)(-)-norpseudoephedrine [130]. Mescaline is metabolized by mice to 3,4,5trihydroxyphenylacetaldehyde, which is oxidized to the corresponding acid by hepatic microsomes [ 131 ]. BIOCHEMICAL ACTIONS
Introduction As compounds that are hydrophilic and basic, it is no surprise that alkaloids interact readily with essential biochemical constituents such as proteins, polysaccharides, and nucleic acids, forming hydrogen bonds and ionic bonds. Less obvious reactions may also be relatively common. For example, essential thiol groups of proteins can undergo nucleophilic reactions with positively charged alkaloids, and pi complexes are possible
ALKALOIDS IN ANIMALS
15
between aromatic rings of alkaloids and those of biological molecules. Metal ion coordination could play a role with some alkaloids that can complex such essential metals as iron or magnesium. Among the vast range of possibilities, some may have little to do with the actual, physiological effects of alkaloids; but others may provide exactly the mechanisms for the pharmacological effects. Biochemistry starts with knowledge of the nature of the reactants and their concentrations at the site of action. Access to this information is often difficult because administered alkaloids may be metabolically modified before becoming active, as discussed in the first part of this review. Moreover, physical barriers and complex transport systems may intervene between administration and action. The literature has many examples of drugs that at concentrations of 0.001M or higher have effects on purified biochemical systems in vitro, yet in vivo the effective concentration may be orders of magnitude lower. While injection can provide a more direct delivery of alkaloid to its site of action than can oral administration, alkaloids may become bound to serum proteins and thus be made less available to tissues. More and more it has become evident that subtle aspects of molecular shape and electron distribution must be considered in any explanation of drug action, and computer modeling has become an essential tool in visualizing molecular interactions, although it appears to have been applied more to the development of synthetic drugs than to naturally-occurring alkaloids [ 132]. In the following sections I have chosen to organize the information according to the type of biochemical system acted upon rather than according to structural types of the alkaloids. While there are arguments possible for either arrangement, I have been persuaded by observing that quite disparate structures can act on the same system; so that if they were treated separately, there would be much redundancy in describing the biochemical system each time that a different class of alkaloid was considered. Recent information on the toxicology of alkaloids can be found in [133]. CHOLINERGIC TRANSMISSION Transmission of nervous impulses by way of acetylcholine release and action is widespread, occurring not only in higher animals but also important in arthropods. In higher animals acetylcholine is the most important neurohormonal transmitter. It functions in the autonomic system, in motor nerves, and in some parts of the central nervous system. It functions not only in synapses between neurons but also on muscles or glands that are controlled by the neurons. After its action the acetylcholine is removed rapidly through hydrolysis by the enzyme acetylcholine esterase. Drugs, including some alkaloids, can interact with this process at several levels:
16
T. ROBINSON
1. As inhibitors of acetylcholine synthesis 2. As inhibitors or stimulators of acetylcholine release 3. As substances that mimic the action of acetylcholine (agonists) 4. As substances that block the action of acetylcholine (antagonists) 5. As blockers of ion channels that the agonist opened 6. As inhibitors of acetylcholine esterase One case of a natural alkaloid that affects acetylcholine synthesis is sanguinarine, which inhibits choline acetyl tranferase [134]. There are several alkaloids that affect release of acetylcholine, but they may not owe their primary pharmacological effects to this activity [135]. Among the agonists and antagonists of the cholinergic system there are many alkaloids, and several different structural types. To undertand their actions it is first necessary to understand that there are two main types of cholinergic receptors in the nervous system ~ nicotinic and muscarinic. Both respond to acetylcholine; but the nicotinic class has nicotine as a specific agonist, while the muscarinic class has the fungal alkaloid muscarine as its specific agonist. There are also different specific antagonists for the two classes. Nicotinic receptors respond faster than muscarinic ones and occur where fast responses are important to the organism. Molecular biology has now provided detailed information about the structures and reactions at the two types of cholinergic receptors; and the actions of alkaloids can be correlated with the nature of the receptors. Ion channel blockers and acetylcholinesterase inhibitors are considered in later sections of this review.
Nicotinic Receptor A symposium publication covers all aspects of nicotine pharmacology [136]. The nicotinic acetylcholine receptor, among other effects, comrols the passage of sodium and potassium ions across the membranes that contain it. It is composed of 2 or more alpha subunits and 2 or more other subunits arranged around the actual ion channel. The overall molecular mass of each subunit is approximately 50kDa. In the muscle receptor there are two identical alpha subunits and one each of the beta, gamma, and delta subunits. Receptors from other tissues also have similar structures, but the combination of subunits can be different and result in somewhat different responses to agonists and antagonists [137, 138]. The binding sites for acetylcholine are dependent on cysteine residues at or near positions 193 and 195 of the alpha subunit [ 139]. When acetylcholine becomes bound to two alpha subunits, the channel opens. The other subunits influence the affinity for acetylcholine, though not binding it themselves [140]. The prototypical antagonist at the nicotinic receptor is, of course, nicotine itself, which stimulates at low concentration but blocks the receptor at high concentration. Competitive binding experiments have shown that
ALKALOIDS IN ANIMALS
17
acetylcholine and (-)-nicotine bind to the same high-affinity site [141 ]. The unnatural isomer, (+)-nicotine, is much less active [142, 143, 144]. The quinolizidine alkaloid (-)-cytisine and the piperidine alkaloid (-)-lobeline evidently bind to the same site as nicotine. It has been pointed out that all three of these alkaloids share in the relationship of a charged nitrogen atom to an aromatic ring and that while the cationic nitrogen associates with a nucleophilic cysteine side-chain in the high affinity site, the aromatic ring may interact with aromatic side-chains of the receptor [145, 146]. Arecoline, a piperidine derivative from the betel nut, is also an agonist [ 147]. Physostigmine, best-known as an inhibitor of acetylcholinesterase, is also an activator of the nicotinic receptor; but strictly speaking, it is not an agonist because it does not act at the same site as acetylcholine [ 148]. d-Tubocurarine, a powerful antagonist, is bound at two sites ~ a high affinity site using alpha and gamma subunits, and a site with 1/400 as much affinity using alpha and delta subunits [149]. By measuring the affinity of analogues, it was shown that the 12' and 13' hydroxylated positons of the alkaloid are important for binding [150]. Thermodynamic studies have shown that the binding is entropy-controlled [ 151 ]. Nicotine and alpha-bungarotoxin from cobra venom both bind in the region of residues 173-204. Nicotine becomes bound to tyrosine residue 198 of the Torpedo receptor [152]; but competitive antagonists are bound to tyrosine 190 and a neighboring cysteine [153]. The nor-diterpenoid alkaloids of Delphinium spp. are powerful antagonists, and they also compete with alpha-bungarotoxin, which, like them, prefers receptors made from 7 alpha subunits [ 138,154]. Indeed, the most potent, non-protein antagonist is the Delphinium alkaloid methyllycaconitine [155]. Structure-activity studies with the aconite alkaloids have revealed the importance of a C-3 hydroxyl group and an oxygen at C-8 [156]. (+)-Sparteine is an antagonist mostly competitive with acetylcholine but also to some extant a blocker of the open ion channel [157]. Despite the potency of these antagonists, they all show reversible binding [ 154]. H --...
C2H5N,,~, ~
,
jOC_~
l
I HaOCH3Aconitine C While some agonists and antagonists may bind to the acetylcholine site, others owe their activity to association with other s i t e s - - e v e n on different subunits. A pyrrolizidine from dendrobatid frogs blocks the ionic
18
T. ROBINSON
channels but does not compete with nicotine for a binding site in rat cerebral membranes [137]. Epibatidine, a chlorinated alkaloid from these frogs, has a structure resembling nicotine and acts as an agonist rather than antagonist [158]. Cocaine has myriad effects; and one of the less important ones comes from its action on the ion channel of the nicotinic receptor, thus antagonizing the effects of nicotine, though not binding at the same site [159]. Strychnine, known primarily for its antagonism of the glycine receptor, may also act on a regulatory site of the nicotinic receptor [ 160]. Physostigmine, known more for its inhibition of acetylcholinesterase, also acts on the nicotinic receptor at a site distinct from the acetylcholine site [ 148]. It and some other alkaloids seem to act as sensitizing modulators for the natural transmitter [ 161 ]. Many compounds structurally related to nicotine have been tested for activity with the nicotinic cholinergic receptor; but. as with the (+)- isomer of nicotine, they generally less activity as agonists, while showing other activities such as blocking the ion channel. Metabolic derivatives of nicotine may account for some of the actions attributed to nicotine, but not necessarily actions at the nicotinic receptor [162]. Nicotine does, indeed, act on other tissues; and some of these actions will be cited in other sections of this review. Although controlling ion channels is the best-known function of the nicotinic receptor, there are examples of other processes under control of this receptor. There is one, for instance, that mediates the release of dopamine or other catecholamines and is inhibited by ibogaine or strychnine [ 163, 164, 165].
Muscarinic Receptor The muscarinic cholinergic system has quite a different mode of operation in that the receptor is connected to the final action by a chain of events. Thus its response is slower than the nicotinic, where the receptor and ion channel are closely connected. Five distinct muscarinic receptors have been identified in mammals, based on anatomical location, genetic analysis, function, and amino acid sequence. All of them have seven transmembrane domains [166, 167, 168, 169]. The N- terminal domain outside the cell binds acetylcholine or other ligands at a site that includes an aspartate residue, while the C-terminal domain inside the cell is coupled to a socalled "G-protein", which is initially bound to guanosine diphosphate (GDP), but exchanges it for guanosine triphosphate (GTP) when activated by its transmitter. The activated G-protein then activates phospholipase C, which hydrolyzes phosphoinositides to release 1,4,5-inositol triphosphate [170]. The final action depends on which type of cell is involved; so that in some types ion channels are opened just as with the nicotinic receptor, but in other cases other processes are affected, for example the release of dopamine [171]. Since there are these differences
ALKALOIDS IN ANIMALS
19
among receptors, it is not surprising that different alkaloids act upon them in different ways. Moreover, stimulation of the muscarinic receptor can make nicotinic receptors of the same cell more responsive to nicotine, perhaps by increasing the concentration of cytosolic calcium [172]. Pilocarpine is long-known as a muscarinic antagonist [173]. Imperialine, a steroid alkaloid, himbacine, a piperidine, and ebeinone, a quinolizidine, are all antagonists at M2 receptors [174, 175]. Quinidine appears to act not on the receptor itself but on the ion channel that is opened by the receptor [176, 177]. Veratridine, similarly, acts on sodium channels [178]. Cocaine, atropine (DL-hyoscyamine), and other tropane alkaloids are wellrecognized antagonists of the muscarinic receptor. Although (-)-cocaine is the naturally occurring form, (+)-cocaine was found to be the more potent with both M~ and M2 receptors. [ 179, 180, 181 ]. Tropanes may not all act in the same way, but atropine causes dissociation of the complex between receptor and its G-protein in heart membranes, and this may be its mechanism of action [182]. Although opioids have their own, specific receptors, they can also act on muscarinic cholinergic receptors [183]. Yohimbine, best-known as an adrenergic blocker, also acts on muscarinic receptors, stabilizing a non-permeable form of the sodium channel [ 184]. ADRENERGIC RECEPTORS The transmitter molecule in the adrenergic system is norepinephrine, but the overall structures and mechanisms of response are like those of the muscarinic cholinergic system [ 185]. That is, the receptor molecule spans the cell membrane with seven transmembrane domains. The outer segment of the protein is responsible for interacting with the transmitter, and the inner segment is associated with a G-protein, which in the inactive state is bound to GDP. When activated, the receptor converts the G-protein to a form that exchanges the GDP for GTP, and the G-protein then activates a particular effector such as an adenylate cyclase. Activation is terminated by hydrolysis of the bound GTP to GDP. Based on location and specific responses to particular agonists and antagonists the adrenergic receptors have been classified first into alpha and beta types, then further subdivided; so that there are two alpha and three beta types. Further, alpha 1 and alpha 2 both have several subtypes [186, 187]. The alpha l receptors activate phospholipase C, while the alpha 2 receptors inhibit adenylate cyclase. The beta receptors are coupled to G-proteins that stimulate adenylate cyclase [188, 189, 190]. This complexity means that no alkaloid can be described simply as "acting on the adrenergic system". Most information regarding the action of alkaloids relates to the alpha 2 receptor, where the indole alkaloids yohimbine and its isomer, rauwolscine, bind with high affinity and block the receptor [187, 191]. Dihydrocorynantheine, an indole alkaloid of similar structure preferentially binds to the alpha 1 receptor, as do some aporphines [192,
20
T. ROBINSON
193, 194]. For all of these alkaloids that bind to alpha receptors coplanarity of the A, B, C, and D rings is important. Then, specificity for 1 or 2 subtype depends on the conformation of groups on the E-ring [195]. The seventh hydrophobic domain of the receptor has been identified as the major determinant for alkaloid binding [ 189].
I
o-I Yohimbine
Protoberberines are also antagonists at alpha 2 receptors [134, 196]. Tetrandrine and dicentrine interact antagonistically with alpha 1 receptors [192, 197, 198]. One of cocaine's many actions may also be on an alpha receptor, since its vasoconstrictive action is antagonized by yohimbine [199]. PURtNERGIC RECEPTORS The class of purinergic receptors has been recognized more recently than other types of receptors, and it incorporates both the receptors known as "adenosine receptors" (P I) as well as receptors responding to adenine nucleotides such as ADP (P2). Although there is much overlap, by and large the P I group is antagonized by methylxanthines, like caffeine. The P2 group has a low affinity for methylxanthines [200, 201,202, 203]. The P l group of adenosine receptors is further subdivided into A l, A2 and A3 by their different preferences for different xanthines and synthetic drugs [204, 205]. A2 has been subdivided further into A2a and A2b. The adenosine receptors have been cloned and sequenced. All have the characteristic structure of receptors working through G-proteins ~ seven transmembrane segments with the N-terminal extracellular and the Cterminal intracellular. There are cysteine residues and carbohydrate on the extracellular loops. Histidine residues on transmembrane segments 6 and 7 are important for antagonist binding. Molecular modeling has suggested that the N-1 position of adenosine and the N-9 of xanthine antagonists occupy the same site on the receptor [206]. The A1 group finds its greatest expression in the central nervous system, but is also in other organs. The alkaloid best-known as an antagonist of adenosine receptors
ALKALOIDS IN ANIMALS
21
is caffeine; b u t , in general, 8-substituted xanthines such as 1,7dimethylxanthine or theophylline, are active [203, 207, 208, 209]. Adenosine receptors regulate diverse functions, with downregulation of adenylate cyclase, calcium channels, and phospholipase A2, upregulation of potassium channels, and ambiguous efects on phospholipase C [210]. The inhibitory effects of the A l receptor being overcome by caffeine makes caffeine appear to be a stimulant of, for instance, the release of calcium. Tolerance to caffeine may come from upregulation or an increased number of adenosine receptors [210, 211 ]. Abrupt withdrawal of caffeine causes a fall in cyclic AMP and sensitization to adenosine [210]. There are some extremely complex interactions in this area. To follow just two examples: The A~ activation of phospholipase C causes increased hydrolyis of inositol lipids so that inositol-l,4,5-trisphosphate is produced and can induce calcium release from microsomes. Caffeine can directly inhibit opening of the calcium channel in the microsomes beyond any action it may have on the initial A l receptor [212, 213]. Caffeine acting on hippocampal A I receptors appears to stimulate release of acetylcholine, but it is actually blocking adenosine receptors that inhibit the release [214, 215]. The end-result is increased wakefulness produced by caffeine. While many effects of caffeine can be attributed to its antagonism at adenosine receptors, its effect on various ion channels is not always mediated by adenosine receptors, and caffeine will appear again in the section on ion channels. SEROTONERGIC RECEPTORS The identification of an active substance named "serotonin" as 5hydroxytryptamine in the mid-twentieth century has been followed by much progress in understanding the distribution and function of this substance in particular groups of neurons in the central nervous system, where it appears to function predominantly as an inhibitory transmitter. At this writing 14 distinct receptors for serotonin have been identified and some of them cloned and sequenced. They have been subdivided into 7 major types, with further subdivisions. All of them have seven membranespanning domains and are coupled with G-proteins [216, 217, 218, 219, 220]. Detailed information from one receptor that has been sequenced (5HT6) shows that a threonine residue at position 196 in the polypeptide chain is important for ligand binding. This threonine side-chain has been found to form a hydrogen bond with the N-1 of an indole ring in either serotonin itself or other, exogenous molecules. Substitution of alanine at this position greatly reduces affinity for agonists and the stimulation of adenylate cyclase, since the alanine side-chain lacks hydrogen bonding capability [220].
22
T. ROBINSON
The best-known of the agonists for serotonergic receptors are several ergot alkaloids, such as ergovaline, and their synthetic derivative LSD [221,222]. By testing a series of these compounds it has been found that a reduced five-membered indole ring increased specific binding to serotonin receptors [223]. A fiat conformation of the D-ring is essential for biological activity [224]. Alkaloids that act as serotonin antagonists include rauwolscine and its isomer yohimbine, which are also noted as adrenergic antagonists [225, 226, 227]. The non-ergot alkaloids asimilobine and lirinidine are also antagonists [228]. In addition to its receptors, serotonin has transporter molecules; and there are alkaloids that interfere with the transport and uptake process. DNA's for serotonin transporters of rat leukemia cells have been cloned and sequenced. One of them has a 653 residue protein with 12-13 transmembrane domains. Interestingly they show considerable homology with transporters of some other transmitter substances; and are sensitive to cocaine, reserpine and several other alkaloids [229, 230, 231,232]. Thus the inhibitory action of serotonin can be reduced not only by substances that act on its receptor but also by substances that hinder its access to the receptor [233]. DOPAMINE RECEPTORS AND TRANSPORTERS Dopamine, or 3,4-dihydroxytyramine is increasingly recognized as a neurotransmitter having a number of distinguishable receptors. It is also reactive with several other metabolites to produce alkaloidal products that may have significant physiological effects [4, 234, 235, 236]. There are at least five types of dopamine receptors in two families [237]. All of them have seven membrane-spanning alpha helices, and all but D3 are coupled to G-proteins. Dl and D5 activate adenylate cyclase. D2 inhibits adenylate cyclase. Within each type there are probably subtypes [238, 239, 240]. Genetic variants in the level of D2 receptors have important behavioral consequences. A low level leads to craving for substances that release more dopamine in the brain [241 ]. The binding of fifteen bisbenzylisoquinoline alkaloids to Dl and D2 receptors has been measured, and it was found that the most active ones had a 11,12'-ether bridge, as in thaligrisine. The configuration of chiral centers also makes a difference [242]. (-)Tetrahydropalmatine blocks brain dopamine receptors, while the (+)-form depletes dopamine levels [243, 244]. Tests of the activity of aporphines on D l and D2 receptors have shown that (R)- configured compounds had greater affinity than their (S)-antipodes because of the orientation of the lone pair on N-6. The most potent antagonists have hydroxyl at C-11, but a hydroxyl at C-10 makes an agonist rather than an antagonist [245]. Some of the ergot alkaloids interact with dopamine receptors that are not linked to adenylate cyclase [222, 246]. Reduction of the five-membered indole
ALKALOIDS IN ANIMALS
23
ring results in decreased affinity for the receptor, in contrast to the effect with the serotonin receptor [223]. Another important aspect of dopamine neurochemistry is the role of dopamine transporters in moving dopamine across cell membranes. Techniques of molecular biology have permitted the manufacture of chimeric dopamine transporters having interchangeable segments of the molecule. In this way the requirements for specific sites or actions can be determined. From this, it appears that the amino terminus is important for ionic dependence and uptake mechanism, the carboxyl terminus for determining substrate affinity and stereoselectivity, and the middle section for interaction with inhibitors [247, 248]. Dopamine and cocaine probably bind to both shared and separate domains. A transporter from rat brain has 620 amino acid residues and 12 membrane-spanning domains. Both termini are intracellular. Segments 1-3 are important for both substrate and inhibitor interactions; segments 5-8 are involved only with inhibitors. [249, 250]. Another one from bovine brain has 693 residues [251 ]. Cocaine is the most-studied inhibitor of the dopamine transporter [252, 253]. It inhibits reuptake of dopamine at D l and D2 receptors so that action of the transmitter is prolonged and accentuated [229, 237, 254, 255, 256]. Testing of seven possible stereoisomers of cocaine showed that the natural form, (-)-cocaine, binds more than sixty times better than the next best isomer. Binding requires an aryl group connected directly or indirectly to C-3 and an ester group at C-2, both of them in the beta orientation [257]. Cocaine binds slowly to the carrier, while dopamine binds rapidly [258], and their binding is mutually exclusive [259]. Spermine inhibits the binding of cocaine [260]; and sodium ion concentration makes a difference [261 ]. A transporter from striatal synaptosomes has a binding site for ATP, which affects the structure but does not become hydrolyzed [262]. Reserpine acts on the dopamine transporter to cause release of the amine, so that free dopamine accumulates extracellularly. The binding appears to be non-covalent [247, 263]. (-)-Cathinone acts similarly [264]. Nicotine and, even more, some quaternary, N-methylated products of nicotine metabolism, inhibit dopamine uptake. This may help to explain why smoking relieves some symptoms of Parkinsonism [265, 266, 267]. In another way, nicotine acting on certain acetylcholine receptors evokes release of dopamine from rat striatal cells [268]. Veratridine has a similar effect [ 142]. THE OPIATE RECEPTOR The name "Opiate Receptor" has stuck even though it has been thought since the early 1970's that the endogenous transmitters acting on this receptor are oligopeptides, enkephalins and endorphins. It is also now accepted that morphine, the classical opiate, itself occurs naturally in animals and may act as an endogenous transmitter [66, 269, 270]. There
24
T. ROBINSON
are several different opiate receptors, distinguished by anatomical locations and by differential responses to selected agonists and antagonists [271 ]. As a rough simplification: mu receptors mediate the analgesic effect delta receptors mediate emotional effects kappa receptors mediate sedative effects sigma receptors mediate psychomimetic effects All of them have seven transmembrane, hydrophobic segments and are linked to regulatory G-proteins. Mu, delta, and kappa receptors have been purified, cloned, and sequenced [66, 272, 273]. The mu receptor is subclassified into two subtypes. Type 1 is a high affinity receptor for both morphine and enkephalins. Type 2 is the classic morphine receptor [66, 274]. The delta receptor has been reconstituted in lipid vesicles [275]. The enkephalins prefer delta receptors, and dynorphins prefer kappa receptors [276]. The synthetic drugs (R)-methadone and naloxone prefer mu receptors [277, 278]. The mu and delta receptors are associated with addiction and cause dopamine release, whereas kappa receptors cause aversion and decrease the release of dopamine [279, 280]. The morphine receptor in human lung has low affinity and perhaps does not fit into the established classification [281]. Cocaine indirectly raises the level of extracellular dopamine in the sustantia nigra by decreasing the number of kappa receptors [282. For binding to all receptors the free 3-hydroxyl group of morphine is important, so that codeine or morphine-3glucuronide have low affinity. The 3-glucuronide, lacking analgesic properties by itself, can nevertheless increase the analgesia produced by morphine. Thus it could be considered a regulator of morphine action [283]. The 6-glucuronide has affinity equal to that of morphine for mu receptors but higher for delta receptors and lower for kappa receptors [271,284]. Some actions attributed to morphine may, in fact, be due more specifically to the 6-glucuronide [66]. However, the 6-glucuronide penetrates poorly into brain capillary endothelial cells, except in the presence of vincristine, which inhibits P-glycoprotein that may bind the glucuronide [285]. Several alkaloids not structurally similar to morphine apparently act at opioid receptors. Harmaline binds to the delta receptor of rat synaptosomal membranes, whereas salsolinol binds preferentially to mu receptors [286]. Ibogaine and related indoles have low affinity for opioid receptors except moderate affinity for sigma2 [287]. Mitragynine appears from responses in Guinea pig ileum to act on an opioid receptor [288]. Some events following activation of opiate receptors are complicated because different concentrations of the agonist produce diffferent effects even on the same group of cells ~ in the release of catecholamines from chromaffin cells, for instance [269]. A transient increase in intracellular
ALKALOIDS IN ANIMALS
25
free calcium caused by morphine results from specific stimulation of the delta receptors [289]. One way that morphine may decrease pain responses is that it inhibits release of the pain transmitter substance P from cells of the spinal cord [290]. On the other hand, it increases release of neuropeptide FF [291]. Symptoms of morphine withdrawal are accompanied at the cellular level with increased activity of phospholipase C and the various effects mediated by the product of this enzyme, inositol 1,4,5-triphosphate [292]. Morphine causes release of arachidonic acid from mouse macrophages; and since arachidonic acid is a precursor of prostaglandins and thromboxanes, there may be an indirect effect of morphine on processes affected by these hormones [293]. There are numerous results suggesting an interaction between morphine and adenosine receptors. Perhaps adenosine acts as a mediator of morphine effects [294]. AMINO ACID AND PEPTIDE RECEPTORS There are many small oligopeptides that serve as neurotransmitters and hormones. The enkephalins, endorphins, etc. have already ben mentioned in the section on opiate receptors. Two amino acids that serve in this way are glycine, an inhibitory transmitter, and glutamic acid, an excitatory transmitter, gamma-Aminobutyric acid (GABA) is less well established, but probably an inhibitory transmitter. Peptide hormones like vasopressin and somatostatin are made in the pituitary gland or hypothalamus and influence processes elsewhere in the body. Strychnine has been known for a long time as a blocker of a glycine receptor in the brain. There are also some glycine receptors insensitive to strychnine [295, 296, 297, 298]. A subunit of the glycine receptor that binds strychnine has been cloned and sequenced. It has sequence and structure homologies with the nicotinic cholinergic receptor; and the strychnine-binding sequence is around residue 200 of a 48kDa subunit, a region corresponding to the acetylcholine-binding region of the nicotinic receptor [299]. Tyrosine 197 and/or 202 is probably involved [300]. This site is not identical with the glycine site but interacts closely with it [301 ]. The binding of strychnine to this receptor is regulated by inorganic anions [302]. Corynine, another indole alkaloid, is also an inhibitor of the glycine receptor that acts at a site removed from the glycine-binding site [303]. Although not normally classed as alkaloids, the natural products kainic acid, quisqualic acid, and N-methyl-D-aspartic acid (NMDA) are established, specific agonists for glutamic acid receptors. They can be seen as conformationally restricted analogues of glutamic acid. At least six recognition sites have been recognized on this receptor [304]" glutamate site, glycine-modulatory site, magnesium-binding site, zinc-binding site, polyamine-modulatory site, cation channel.
26
T. ROBINSON
Ibogaine appears to be an antagonist with this receptor [305, 306]; and, in an indirect way, repeated administration of morphine causes increased expression of a glutamate receptor subunit [307]. There are also probably some glutamate receptors for which NMDA is not an agonist [304]. GABA receptors are sometimes refered to as benzodiazapine receptors because this synthetic drug is an agonist at the GABA receptor [308]. The best-known alkaloid inhibitor of the GABA receptor is bicuculline, which is a competitive antagonist of GABA [309, 310]. The securinine group of indolizidines is also active but less so than bicuculline [311 ]. Forty-five alkaloids structurally related to bicuculline have been tested for activity at this receptor on rat brain synaptic membranes. Both agonists and antagonists were found [312]. Some effects of morphine have been ascribed to the blocking of a GABA receptor, thus causing excitation; but this is a low affinity binding [313]. Both glycine and glutamic acid stimulate movement of chloride ion in hippocampal neurons; and caffeine inhibits this process, although it has no effect on the glycine-binding site of the NMDA receptor [314].
Bicuculline
There are a few cases of alkaloids that act at the receptors for peptide hormones. Spiroquinazoline inhibits the binding of substance P to its receptor on astrocytoma cells [315]. Chelerythrine and sanguinarine compete with vasopressin for receptors on rat liver cells [316]. Psycholeine is an antogonist at somatostatin receptors on cultured pituitary cells [317, 318]. Caffeine inhibits the binding of thyrotropinreleasing hormone to pituitary cells [319]. NUCLEIC ACIDS AND PROTEIN SYNTHESIS The much-studied systems of nucleic acid replication and transcription, followed by translation of the nucleic acid code into protein structure has many points that can be influenced by exogenous compounds, including some alkaloids. To begin with the structural integrity of stored
ALKALOIDS IN ANIMALS
27
deoxyribonucleic acid, several alkaloids have been shown to bind to DNA in ways more specific than by the self-evident ionic attraction that would be expected between the anionic nucleic acid and the (usually) cationic alkaloid. Intercalation into the DNA double helix occurs with a variety of compounds and is a result of hydrophobic pi interactions between the stacked base residues of the DNA and appropriate functional groups of the foreign substance. Intercalation may follow an initial ionic attraction of the two molecules and can be measured by observing spectral changes [320, 321,322] or changes in chromatographic or electrophoretic mobility [323,324]. Binding can shift equilibria among the various conformations of the double helix and affect replication and transcription [325, 326]. Because of their promise as antitumor drugs ellipticine and derivatives are probably the most investigated alkaloids that interact with DNA. While it is the cationic form of ellipticine that binds, hydrophobicity rather than electrostatic attraction is the main driving force [327]; and intercalation at low concentration is accompanied by other binding modes if the concentration increases [328]. Ellipticine itelf intercalates by a noncooperative neighbor exclusion model without any specificity for base sequence [321]. The most active antitumor compounds in the ellipticine group are 9-hydroxy derivatives, and these are the ones that show the highest affinity binding to DNA. They have special afinity for a doublet sequence of two guanine-cytosine pairs [329]. A 9-methyl group is also important [323]. 9-Aminoellipticine interacts with single-stranded DNA, specifically at apurinic sites [330]. There are several other alkaloids that have been indicated by physical measurements to intercalate into the DNA double helix. Among them are melinone F and normelinone F, quaternary beta-carbolines from Erythrina melinoniana [331]. Aristolactam,N-beta-D-glucoside from Aristolochia indica has a very high affinity for intercalation [332]. Berberine and sanginarine intercalate, berberine preferentially at alternating adeninethymine stretches in the DNA chain [181,320, 333]. Hydrophobicity is believed to be the predominant driving force for the binding [322]. Several xanthine derivatives intercalate and cause swelling of DNA. The most effective are unnatural, synthetic compounds; but caffeine and theophylline have some effect, caffeine more than theophylline [326]. Fagaronine and nitidine appear to intercalate into initiation complexes, thus inhibiting the action of DNA polymerase [334]. The presence of a quaternary nitrogen in fagaronine is important for its activity [335]. Several sponge alkaloids containing the 2-aminoimidazole group intercalate into DNA but have other effects as well, such as inhibiting DNA polymerase [336]. In addition to the intercalation mechanism, other types of binding occur between DNA and alkaloids. Following administration to rats various isotopically labeled pyrrolizidines have been found to contribute covalently bound label to DNA of liver, lung, and kidney perhaps because
28
1". ROBINSON
their pyrrole metabolites alkylate nitrogen atoms of the bases [117, 337]. N-Acylated pyrrolidines from Chamaesaracha conioides bind to DNA with high affinity [338]. Camptothecin forms alkali-labile linkages with closed, supercoiled DNA. The active site on the alkaloid for this binding is the hydroxyl group at C-20 in the E-ring, but C-7 of the B-ring is also involved in an interaction with adenine residues [339]. Dictamnine forms adducts with DNA when irradiated [340]. Harmine may act similarly [341]. Budmunchiamines bind in an unknown way [342]. The steroidal alkaloids solasodine and O-acetylsolasodine apparently alkylate DNA by means of their spiro-aminoacetal group, which can open to make an electrophilic iminium species [343]. Isogravacridonchlorine from Ruta graveolens is a mutagen that has been shown to act as a frame shifter in Salmonella [344]. There is less information in the literature about binding of alkaloids to ribonucleic acid than there is for deoxyribonucleic acid; but such binding does occur. However, since ribonucleic acid is single-stranded, the intercalation mechanism does not apply. Binding of the purine alkaloids caffeine and theophylline follows the reverse order from their binding to DNA .... theopylline binds with 10,000 times the affinity of caffeine [345]. Elliptinium acetate, best-known for its intercalation into DNA, is easily oxidized to an A-ring quinone amine that binds to the 2'-oxygen atom of ribose units in RNA. The first product then cyclizes to a spiro derivative [346]. In addition to alkaloids that interact with already formed nucleic acids and thus inhibit replication and transcription, there are several that inhibit these processes by other mechanisms. Topoisomerases are essential enzymes in the replication process. They make a break in supercoiled DNA, allowing one segment of the DNA to pass through, and then reseal the break, thus permitting the DNA to uncoil and become accessible to polymerases. Two types of topoisomerase are recognized. Type I is monomeric, breaks a single strand, and is poisoned by camptothecin. Type II has multiple subunits, breaks both strands, and is poisoned by ellipticine. Both poisons act by prolonging the life of a transient intermediate in which a broken strand is held by the enzyme [347, 348]. As already noted, the ellipticines intercalate in DNA, but this alone may not be sufficient to inhibit topoisomerase action; an oxidizable phenolic group is necessary to prevent the enzyme from resealing breaks that it has made [349]. With camptothecin the topoisomerase I becomes linked to the 3' end of the broken DNA, so that the rejoining step is blocked [350]. Camptothecin interacts specifically with guanine residues [351 ], and in its presence cleavage occurs most frequently at the dinucleotide pair thymineguanine, whereas some sites that are normally cleaved are not cleaved when the alkaloid is present [352]. Interestingly the topoisomerase I of yeast is not inhibited by camptothecin, as a result of a different amino acid
ALKALOIDS IN ANIMALS
29
sequence at its active site [353]. Fagarone and monomargine are other alkaloids that inhibit DNA topoisomerases [354, 355]. There are several others that inhibit DNA replication. Fagaronine and nitidine have ben mentioned previously as intercalating agents, and this intercalation evidently inhibits the action of DNA polymerase [334]. Several benzophenanthridines and protoberberines inhibit reverse transcriptase, which catalyzes the synthesis of DNA with an RNA template [356, 357]. Mimosine at the cellular level, rather than directly on the process, is an inhibitor of DNA synthesis because it chelates iron [358] and because it blocks the synthesis of thymidylate, an essential precursor of DNA [359]. Vincristine may also act as an inhibitor of thymidylate synthesis, perhaps because it alkylates an essential thiol group of an enzyme [360]. The translation step where proteins are assembled following the code of messenger RNA has a few instances of inhibition by alkaloids [361]. Emetine and tubulosine block peptide bond formation, acting similarly to the antibiotic cycloheximide by blocking translocation of the growing peptide chain from the A site to the P site of the ribosome. They evidently bind to a specific ribosomal site [362]. Homoharringtonine may act similarly [363]. Lycorine may act at the level of termination [364]. Narciclasine and related alkaloids of the Amaryllidaceae prevent binding of the 3' end of aminoacyl-tRNA to the peptidyl transferase site of the ribosome [365, 366]. Mescaline may act similarly [367]. Following the ribosomal process many proteins are modified further by being phosphorylated, and several alkaloids affect this phosphorylation step, among them reserpine and sanguinarine, which inhibit [368, 369]; veratridine, which stimulates [370]; and chelerythrine, which in some cases inhibits and in other cases stimulates [369, 371]. ENZYME INHIBITORS There are innumerable reports of enzymes being inhibited by alkaloids, and this should not be surprising because enzymes and alkaloids both have functional groups that could be expected to interact in various ways. Probably at some concentration almost any alkaloid could be found to inhibit almost any enzyme. The crucial question though is, "Does a particular enzyme-alkaloid interaction help to explain the larger physiological effect of the alkaloid?" In a few cases the answer is that it does, and in some other cases that it might. Perhaps the most famous instance of enzyme inhibition explaining a gross effect of an alkaloid is the inhibition of cholinesterase by (-)physostigmine (eserine). Since acetylcholine is such a widespread and essential neurotransmitter, an increase in its concentration at synapses has serious consequences; and physostigmine by inhibiting the hydrolysis intensifies and prolongs the action of acetylcholine on its receptors [372].
30
T. ROBINSON
Several derivatives of physostigmine have been tested for activity. The (+)-isomer had little activity, but (-)-N-methylphysostigmine was more active than the natural alkaloid [373]. Testing of many other alkaloids has found few with significant activity [374, 375]. Galanthamine, although less potent than physostigmine, is longer lasting [376]. Huperzine A, similarly, is slower to bind to the enzyme and slower to dissociate than physostigmine; but it is potent and has pharmacological use [377, 378, 379]. d-Tubocurarine, known best as an antagonist at the nicotinic cholinergic receptor, is also an inhibitor of acetylcholinesterase, where it appears to act at two different sites [380, 381]. A group of 3alkylpyridinium polymers from the sponge Reniera sarai has recently been found to have potent anticholinesterase activity [382]. Acetylcholinesterase binds its substrate at a site containing tryptophan and aromatic residues [383]. The catalytic site, as in other esterases, contains serine and aspartate residues. In its action the enzyme forms a transient intermediate in which the acetyl group of substrate is esterified with the serine residue, and then rapidly hydrolyzed. Physostigmine, in contrast, forms a carbamylated enzyme that is hydrolyzed very much more slowly. Several alkaloids owe their toxicity to specific inhibition of enzymes that catalyze the hydrolysis of glycosidic bonds in carbohydrate oligomers and polymers. Thus there are glucosidase inhibitors, mannosidase inhibitors, etc. [384, 385, 386]. Almost all of these alkaloids can be describd as indolizidines with a large number of hydroxyl groups in their structure, but there is at least one pyrrolizidine [387], one piperidine [388], and one tropane [389, 390]. Swainsonine and castanospermine are the best known alkaloids in this group. Swainsonine is an inhibitor of alpha mannosidase II of the Golgi apparatus. As a result of its action Nlinked oligosaccharides are made with extra mannose groups [391,392, 393]. Castanospermine inhibits beta-mannosidase I and several glucosidases [391 ].
slieR Swainsonine
The oligosaccharides affected are used in making glycoproteins, which are then impaired in their functions [392, 393]. Many other alkaloids have been reported to be enzyme inhibitors, but little detail is available about their action or pharmacological significance. They are listed in Table 1.
ALKALOIDS IN ANIMALS
31
Enzyme Inhibition by Alkaloids
Table 1.
,
Inhibited Enzyme
Alkaloid
|m i
l
Reference
i
quinidine
P-450 oxidases
quinine
monoamine oxidase
quinine
phosphodiesterase
406
mannosidase
403
phosphodiesterase
407,408
xanthines
glutamic dehydrogenase
409
404 l
405 I
swainsonine
.
caffeine
t
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
beta-carbolines
dioxygenases
410
vinblastine, vincristine
lipoxygenase
411
vinblastine, vincristine
monoamine oxidase
412
papaverine
cAMP phosphodiesterase
413
chelerythrine
protein kinase C
414
beta-carbolines
monoamine oxidase
415
aldose reductase
416
sucrase
417
.
.
.
.
.
.
.
.
.
berberine, palmatine
.
.
castanospermine
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
xanthines
phosphodiesterase
408
berbamine
ATPase
418
allocryptopine
phosphodiesterase
419
nicotine, continine
3-OH-steroid dehydrogenase
420
O-methylase
421
.
cocaine .
.
.
.
.
.
.
.
purpurone
ATP-citrate lyase
422
bis-indoles
tyrosine kinase
423
harmaline
monoamine oxidase
424
bastadins
inosine-5'-phosphate dehydrogenase
425
protoberberines
tyrosine hydroxylase
426
chelidonine
monooxygenases
427
cerveratrums
cAMP phosphodiesterase
428
protoberberines
elastase
429
strychnine, brucine
lactic dehydrogenase
430
,
i
,
i
ii
CYTOSKELETON AND MEMBRANE DISRUPTORS
The cytoskeleton of cells is composed of several components that are responsible for maintaining the physical structure of the cell. It comprises
32
1".ROBINSON
the filamentous structures microtubules composed of the protein tubulin, microfilaments composed of the protein actin, and intermediate filaments that vary in composition. The cell membrane is a complex structure composed of proteins, lipids, and carbohydrates. All of these structures have been found to be adversely affected by particular alkaloids. Many detergent-like molecules are well known to destroy the orderly lipid structure of cell membranes. More subtle than gross effects on the membrane, there may be effects on transmembrane processes such as ion transport. The alkaloids that also show these effects are those with an amphipathic structure whose hydrophobic part associates with the membrane lipids, while the hydrophilic part resides in the aqueous medium. There are not many such alkaloids with these properties, and they are mainly glycosides of nitrogen-containing steroids that act specifically on sterol-containing membranes, where they form 1:1 complexes with the sterols. For this activity the alkaloid must be glycosylated at the 3-beta position, and additional specificity is conveyed by the nature of the glycosyl group and by the nature of the membrane sterol. The most active alkaloid in this respect is alpha-tomatine [421,422, 423]. Quinidine is a strong perturber of model lipid membranes that contain acidic phospholipids [424]. Synergism between alkaloids has been reported, and this may be important because plants that contain this type of compound usually have more than one of them [425]. Although the effects of these alkaloids on transmembrane ion transport may result from nonspecific membrane disruption, other effects may be more specific for certain ions; and they are therefore treated in the section on ion transport effects in this review. Oxidation of the lipid components of membranes is another process that is deleterious to membrane stability; and there are alkaloids that favor this oxidation and those that inhibit it. In the former group are sanguinarine [426, 427] and orellanine [428]. In the latter group are boldine [429], reserpine [368], colchicine and colchieeine [430] The effect of colchicine on inhibiting mitosis by disrupting microtubules has been known for many years, and now the detailed mode of action is becoming clear. The structural protein of tubules, tubulin, has alpha and beta subunits as well as subtypes of the beta foma. Colchicine binds specifically to the beta subunit, and kinetics of the binding change
0"t30 / ~ ~ 0 Colchicine
~3
ALKALOIDS IN ANIMALS
33
somewhat with the beta subtype [431,432]. Following binding there is local unfolding of the beta-subunit helix in the carboxyl terminal region around arginine 390 [433]. By studying binding of derivatives of the alkaloid the requirements for binding have been characterized. Methoxy, or other oxygen-based, functional groups at C-1, C-2, and C9 are required; and the acetamido group must be at C-7 [434, 435, 436]. Binding is temperature-dependent and occurs in at least two steps initially with the trimethoxybenzene ring and later with the tropolone ring [437, 438, 439]. The B-ring is important for immobilizing the A and C rings [440]. Kinetically a fast, reversible binding is observed, followed by a slow conformational change [441 ]. In the binding interaction the ring A of colchicine becomes juxtaposed to the alpha-beta subunit surface [442], but the alkaloid molecule is bound covalently to the beta-subunit, perhaps making a bridge between two different regions of this subunit [443]. In the tubulin molecule residue 316 is directly involved in binding the trimethoxyphenyl ring. Tubulins have a hydrophobic residue at this location, either valine or isoleucine [444]. Residues 214-241 are also involved [445]. Colchiceine, which differs from colchicine by having a hydroxyl group instead of methoxyl on ring C, also binds to tubulin and inhibits assembly of microtubules; but, strangely, the two alkaloids bind at different sites and do not compete with each other [446]. Besides colchicine, there are other alkaloids that disrupt microtubules. The most studied are the dimeric indole alkaloids vinblastine and vincristine. They also bind to the beta subunit but not at the same site as colchicine [447, 448, 449]. Vincristine has the highest affinity in this group [450]. The related, monomeric alkaloids vindoline and catharanthine bind much more weakly [451 ]. The vinblastine high affinity binding site is at residues 175-213, while colchicine is nearby at 214-241, as well as at other loci [445]. In addition, there is a low-affinity binding site for vinblastine [452]. There are about 1.5 binding sites for vinblastine per molecule of tubulin (mol. wt. ca. 110kDa) [453]. Modifications of the vinblastine structure have permitted some inferences about the requirements for binding [454]. Magnesium and guanosine triphosphate at less than millimolar concentration increase the rate and stability of binding [455, 456]. Rather than causing unfolding, vinblastine apparently induces oligomerization of the tubulin [457]. Maytansine binds at a tubulin site where guanine nucleotides exchange and causes unfolding [457, 458]. Nonalkaloidal inhibitors such as paclitaxel and macrolides bind at different sites from those used by the alkaloids, but binding of the alkaloids prevents binding ofpaclitaxel [459, 460]. There is litle information about alkaloids that may act on other elements of the cytoskeleton. Reserpine binds to G-actin but not to Factin [461]. Nicotine at 10 micromolar causes disassembly of actin filaments [462].
34
T. ROBINSON
MISCELLANEOUS CHANNEL TRANSPORT EFFECTS In previous sections the membrane transport of inorganic ions controlled by receptors to endogenous transmitters has been discussed. There are many effects of alkaloids on ion transport where the normal mechanism of control is obscure or where the effect of the alkaloid appears to be directly on an ion channel rather than on a controlling receptor. Some of these effects are gathered together in this section. Ryanodine barely rates as an alkaloid, although it is often called one. It is a complex diterpenoid whose only nitrogen atom is in an esterified proline group, and it shows no basic properties. Since there is no known endogenous transmitter, the system that is acted on by ryanodine is called the "ryanodine receptor"; and as with the opiate receptor, it will probably continue to be called that even if an endogenous transmitter substance is discovered [463]. Ryanodine receptors have been most thoroughly studied in skeletal muscle, although they are also present (with some differences) in other tissues. In skeletal muscle ryanodine acts on a calcium release channel in the sarcoplasmic reticulum to provide intracellular calcium ions required for muscle contraction. There is a high affinity site where binding locks the channel in an open position and a low affinity site where binding closes the channel at high concentrations of the alkaloid [464, 465]. In both cases calcium must be present to open the channel and allow ryanodine to bind [466]. Isolation of the receptor protein from skeletal muscle, cloning the DNA for it, and deducing the amino acid sequence have all been acomplished; so that much is known about the mechanisms involved [467, 468, 469, 470, 471,472]. Adding up the masses of the 5037 residues in the sequence gives a monomeric molecular mass of 565,223Da. The ion channel is made from a symmetrical arrangement of four of these units. The purified receptor has been inserted into artificial membranes and responds there to ryanodine just as it does in vivo [473, 474, 475]. The receptor molecules are arranged so that both the N- and C-terminals are on the cytoplasmic side of the sarcoplasmic reticulum vesicles [476]. Each monomer of the receptor has a binding site for ryanodine, and binding to only one of the sites is inhibitory [477]. Partial digestion has narrowed the binding site down to a 135kDa fragment [478]. Several structural analogues of ryanodine have been tested to clarify the structural requirements for binding. The pyrrole group controls the orientation of binding, but the large, hydrophobic terpenoid moiety is also important [479, 480]. Spermine increases both the rate and affinity of ryanodine binding to the receptor [481 ]. Caffeine antagonizes the action of ryanodine, acting on the same pool but stimulating calcium ion release. It does not act at the same receptor site; and once the site has been blocked by ryanodine, caffeine is unable to unblock it, although excess calcium ion can [477, 482, 483,484, 485]. Caffeine increases the affinity for calcium of the calcium activator site [486]. The above discussion applies to the action of ryanodine on
ALKALOIDS IN ANIMALS
35
skeletal muscle. There are differences observed in heart muscle, smooth muscle, brain, adrenal glands, and liver [487, 488, 489, 490, 491]. The release of calcium by caffeine leads to further effects that are dependent on calcium ion for example the activation of ATPase and oxidative phosphorylation [492, 493]. Caffeine is apparently influential on other ion transport process that are not under the control of adenosine receptors or affected by ryanodine. It increases the concentration of free calcium ion in pancreatic beta cells and also inhibits potassium channels [494]. Sparteine has a similar effect on the same cells [495]. In another example caffeine stimulates release of calcium from intracellular stores in liver cells and is not competitive with ryanodine [496]. Senecionine acts similarly [497]. Conversely, caffeine inhibits a calcium channel in rat cerebellar microsomes [212]. Quinine and quinidine block potassium channels, both calcium independent ones and calcium-activated ones in several types of membranes [498, 499, 500]. Quinine also has less specific effects on other ion channels ~ chloride as well as cations [501]. Blocking of potassium channels has a secondary effect of stimulating synthesis of phosphatidylserine and affecting other processes that are controlled by potassium concentration [289, 502, 503]. The bisbenzylisoquinoline alkaloid dauricine seems to act similarly to quinidine [504]. Some effects of quinine and quinidine, rather than showing actions on specific ion channels may come from more general effects on membrane structure. Quinidine does decrease the fluidity of liver plasma membrane [505]; and it does interact with lipid bilayers [506]. Some other alkaloids may have specific effects on ion channels or ion tranporters. Tetrandrine and hernandezine inhibit calcium entry into human vascular cells [507, 504, 508]. Tetrandrine binds directly to calmodulin [509]. alpha-Solanine inhibits active transport of calcium in rat intestine [510]. Certain diterpenoid alkaloids inhibit calcium-activated potassium channels in aorta muscle [511]. Hirsutine blocks voltagedependent calcium influx in aorta muscle [512]. Yohimbine and berberine inhibit ATP-sensitive potassium channels [513, 514]. Crambescidin is a calcium channel blocker that inhibits the cholinergic contraction of ileum muscles [515]. In contrast to these inhibitors of ion transport, veratridine and homobatrachotoxin activate sodium and calcium channels in several cell types [516, 517, 518, 519, 520]. Veratridine binds both a high affinity site and a low affinity site on fast sodium channels [521]. The toxicity of veratridine may result from calcium ions passing in through an open sodium channel [522]. A secondary effect of increased calcium concentration is increased phosphorylation of tyrosine residues that then modify the activity of several enzymes [370]. Caffeine stimulates chloride ion efflux from epithelial cells, independent of its activity on adenosine receptors [523]. Agonists of the mu opioid receptor activate inward conductance of potassium ions [524]. Cocaine causes loss of magnesium
T. ROBINSON
36
and calcium from smooth muscle cells [525, 526], but it has also been found to block sodium channels [537]. Harmaline inhibits a sodium/iodide symporter in thyroid cells [528]. Aside from inorganic ions, amino acids and other small organic molecules have transport systems to move them across membranes. Human erythrocytes have two different amino acid transporters, and both of them are affected by harmaline, though in different ways [529]. Caffeine and theophylline both inhibit glucose uptake by insulin-treated adipocytes [5301. Other alkaloids that appear to have effects on ion chanels may, more accurately, be general disrupters of membrane structure. For example alpha-solanine and alpha-chaconine affect ion channels, but are wellestablished as having general effects on membranes [531,532]. ABBREVIATIONS ATP ADP GTP GDP cAMP DNA RNA GABA
= = = = = = = =
Adenosine triphosphate Adenosine diphosphate Guanosine triphosphate Guanosine diphosphate Cyclic adenosine monophosphate Deoxyribonucleic acid Ribonucleic acid gamma-Aminobutyric acid
REFERENCES
[1] [2] [31 [41 [5] [61 [7] [8] [91 [lO] [ii]
Guengerich, F. P. Amer. Scientist ,1993, 81, 440-447. Stanulovi'c, M.; Chaykin, S. Arch. Biochem. Biophys.,1971,145, 27-34, 35-42. Collins, A. C.; Cashaw, J. L.; Davis V. E. Biochem. Pharmacol., 1973, 22, 2337-2348. Collins, M. A; Cheng, B. Y. Arch. Biochem. Biophys.,1988, 263, 86-95. Haber, H.; Roske, I.; Rottmann, M.; Georgi, M.; Melzig, M. F. Life Sci.,1997, 60, 79- 89. Breyer-Pfaff, U.; Wiatr, G.; Stevens, I.; Gaertner, H. J.; Mundle, G.; Mann, K. Life Sci.,1996, 58, 1425-1432. Cashaw, J. L. J. Chromatog.,1993, 613, 267-273. Kajita, M; Niwa, T.; Maruyama, W.; Nakahara, D.; Takeda, N.; Yoshizumi, H.; Tatematsu, A.; Watanabe, K.; Naoi, M.; Nagatsu, T. J. Chromatog. B 1994, 654, 263-269. Tencheva, J.; Yamboliev, I; Zhivkova, Z. J. Chromatog., 1987, 421, 396-400. Agfindez, J. A. G.; Luengo, A.; Benitez, J. Drug Metab. Dispos., 1992, 29, 343349. Stavric, B. Food Chem. Toxic., 1988, 26, 645-662.
ALKALOIDSIN ANIMALS [12]
[13] [14] [15]
[16] [17] [18] [19] [20] [21] [22] [23]
[24] [25] [26] [27]
[28] [29] [30] [31] [32] [33] [34]
[35] [36] [37] [38] [39]
37
Mellini, D. W.; Caporaso, N. E.; Issaq, H. J. J. Liq. Chromatog., 1993, 16, 1419- 1426. Miceli, J. N; Chapman, W. J. Liq. Chromatog., 1990, 13, 2239-2251. Arnaud, M. J.; Zvi, Z. B.; Yaari, A.; Gorodischer, R. Res. Commun. Chem. Pathol. Pharm., 1986, 52, 407. Beach, C. A.; Mays, D. C.; Sterman, B. M.; Gerber, N. J. Pharmacol. Exp. Therap., 1985, 233, 18-23. Berthou, F.; Goasduff, T.; Dr6ano, Y.; M~nez, J.-F.Life Sci. 1995, 57, 541-549. Campbell, M. E.; Grant, D. M.; Inaba, T.; Kalow, W. Drug Metab. Dispos., 1987, 15, 237-249. Berthou, F; Ratanasavanh, D.; Riche, C.; Picart, D.; Voirin, T.; Guiliouzo, A. Xenobiotica 1989, 19, 401-417. Berthou, F; Flinois, J. P.; Ratanasavanh, D.; Beaune, P.; Riche, C.; Guillouzo, A. Drug Metab. Dispos., 1991, 19, 561-567. Fuhr, U; Doehmer, J.; Battula, N.; W61fel, C.; Kudla, C.; Keita, Y.; Staib, A. H. Biochem. Pharmacol., 1992, 43, 225-235. Roberts, E. A.; Furuya, K. N.; Tang, B. K.; Kalow, W. Biochem. Biophys. Res. Commun., 1994, 201, 559-566. Rasmussen, B. B.; Nielsen, K. K.; Brasen, K. Anal. Biochem., 1994, 222, 9-13. Zhang, Z.-Y.; Fasco, M. J.; Kaminsky, L. S. J. Chromatog. B 1995, 665, 201208. Grant, D. M.; Campbell, M. E.; Tang, B. K.; Kalow, W. Biochem. Pharmacol., 1987, 36, 1251-1260. Tassaneeyakul, W.; Birkett, D. J.; McManus, M. E.; Tassaneeyakul, W.; Verpnesr M. E.; Anersson, T.; Tukey, R. H.; Miners, J. O. Biochem. Pharmacol., 1994, 47, 1767-1776. Tjia, J. F.; Colbert, J.; Back, D. J. J'. Pharmacol. Expt. Therap., 1996, 276, 912-917. Konishi, H.; Morita, K.; Yamaji, A. Biol. Pharm. Bull. Tokyo, 1996, 19, 593598. Brazier, J. L.; Ribon, B.; Desagr M.; Salle, B. Biomed. Mass. Spectrom., 1980, 7, 189-192. Jeffcoat, A. R.; Perez-Reyes, M.; Hill, J. M.; Sadler, B. M.; Cook, C. E. Drug Metab. Dispos., 1989, 17, 153-159. Jindal, S. P; Lutz, T. J. Pharm. Sci., 1989, 78, 1009-1014. Zhang, J. Y; Foltz, R. L. ,/. Anal. Toxicol., 1990, 14, 201-205. Roberts, S. M.; Munson, J. W.; James, R. C.; Harbison, R. D. Anal. Biochem., 1992, 202, 256-26 I. Sukbuntherng, J.; Martin, D. K.; Pak, Y.; Mayersohn, M. d. Pharmaceut. Sci., 1996, 85, 567-571. Brzezinski, M. R.; Abraham, T. L.; Stone, C. L.; Dean, R. A.; Bosron, W. F. Biochem. Pharmacol.,1994, 8, 1747-1755. Dean, R. A.; Zhang, J.; Brzezinski, M. R.; Boston, W. F. J. Pharmacol. Expt. Therap., 1995, 275, 965-971. Gatley, S. J. Biochem. Pharmacol., 1991, 41, 1249-1254. Katz, J. L.; Terry, P.; Witkin, J. M. Life Sci., 1992, 50, 1351- 1361. Boyer, C. S; Petersen, D. R. J. PharmacoL Exper. Therap.,1992, 260, 939-946. Xu, Y.-Q.; Crumb, W. J.; Jr.; Clarkson, C. W. J. Pharmacol. Expt. Therap., 1994, 270, 319-325.
38
[40] [41]
[42] [43] [44] [45] [46] [47]
[48] [49]
[50] [51] [52] [531 [54]
[551 [56]
[57]
[58] [59]
[6o] [61]
[62] [63]
[64] [65] [66] [67] [68] [69]
T. ROBINSON
McCance, E. F.; Price, L. H.; Kosten, T. R.; Jatlow, P. I. d. Pharmacol. Expt. Therap., 1995, 274, 215-223. Landry, D. W.; Zhao, K.; Yang, G. X.-Q.; Glickman, M.; Georgiadis, T. M. Science, 1993, 259, 1899-1901. Dalvi, R. R; Jones, R. H. Current Sci., 1986, 55, 558-561. Boelsterli, U. A.; Lanzotti, A.; GSldlin, C.; Oertle, M. Drug Metab. Dispos., 1992, 29, 96-101. Pellinen, P; Honkakoski, P.; Stenback, F.; Niemitz, M.; Alhava, E.; Pelkonen, O.; Lang, M. A.; Pasanen, M. Europ. J. Pharmacol., 1994, 270, 35-43. Elaev, N. R.; Evdokimov, L. D.; Ryzhakov, A. V. Biochemistry USSR 1986, 51, 561-565. Van der Meer, M. J.; Hundt, H. K. L.; MUller, F. O. d. Pharm. Pharmacol., 1986, 38, 781-784. Werner, G; Brehmer, G. Z. Physiol. Chem.,1967, 348, 1640-1642. Werner, G; Schmidt, K. H. Z. Physiol. Chem.,1968, 349, 677-691. Tateishi, T.; Soucek, P.; Caraco, Y.; Guengerich, F. P.; Wood, A. J. J. Biochem. Pharmacol., 1997, 53, 11 l-116. Suh, D.-Y.; Kim, Y. C.; Kang, Y.-H.; Han, Y. N.; Han, B. H. J. Nat. Prod., 1997, 60, 265-269. Paoletti, C.; Lecointe, P.; Lesca, P.; Cros, S.; Mansuy, D.; Datxuong, N. Biochimie, 1978, 60, 1003-1009. Lecointe, P; Puget, A. C. R. Acad. Sci.; Ser. III 1983, 296, 279-282. Braham, Y.; Meunier, G.; Meunier, B. C. R. Acad. Sci. Set. III, 1987, 304, 30 l- 306. Douyette, A. Biomed. Environ. Mass Spec., 1988, 15, 243-247. Gouyette, A.; Auclair, C.; Paoletti, C. Biochem. Biophys. Res. Commun., 1985, 131, 614-619. Raisanen, M.J. Life Sci. 1984, 34, 2041-2045. Rosazza, J. P. N.; Duffel, M. W.; EI-Marakby, S.; Ahn, S. H. J. Nat. Prod., 1992, 55, 269-284. Elmarakby, S. A.; Duffel, M. W.; Rosazza, J. P. N. J. Med. Chem., 1989, 32, 2158- 2162. Le Verge, R; Le Con'e, P.; Chevanne, F.; De Maindreville, M. D.; Royer, D.; Levy, J. J. Chromatog., 1992, 574, 283-292. Oguri, K.; Tanimoto, Y.; Mishima, M.; Yoshimura, H. Xenobiotica, 1989, 19, 171- 178. Tweedie, D. J.; Prough, R. A.; Burke, M. D. Xenobiotica, 1988, 18, 785-796. Canfell, P. C.; Castagnoli, N.; Jr.; Fahey, M. R.; Hennis, P. J.; Miller, R. D. Drug Metab. Dispos., 1986, 14, 703-708. Naoi, M.; Matsuura, S.; Takahashi, T.; Nagatsu, T. Biochem. Biophys. Res. Commun., 1989, 161, 1213-1219. Sekine, Y.; Creveling, C.; Bell, M.; Brossi, A. Helv. Chim. Acta,1990, 73, 426432. He, X; Tadi'c, D.; Brzostowska, M.; Brossi, A.; Bell, M.; Creveling, C. Helv. Chim. Acta, 1991, 74, 1399-141 I. Benyhe, S. Life Sci., 1994, 55, 969-979. Liu, Y.; Bilfinger, T. V.; Stefano, G. B. Life Sci., 1997, 60, 237-243. Marrazzi, M. A.; Luby, E. D.; Kinzie, J.; Munjal, I. D.; Spector, S. Life ScL, 1997, 60, 1741-1747. Amann, T; Zenk, M. H. Tetrahedron Lett., 1991, 32, 3675-3678.
ALKALOIDSIN ANIMALS
[70] [71] [72] [73] [74] [75] [761 [77] [78] [79]
[8o1 [81] [82] [83] [84]
[85] [86] [87]
[88] [89] [90] [911 [92] [93] [94] [95] [96] [97]
39
Ishida, T.; Yano, M.; Toki, S. Drug Metab. Dispos., 1991, 19, 895-899. Xu, B. Q.; Aasmundstad, T. A.; Bjomeboe, A.; Christophersen, A. S.; M~rland, J. Biochem. Pharmacol., 1995, 49, 453-460. Krowech, G.; Caldera-Munoz, P. S.; Straub, K.; Castagnoli, N.; Jr.; Correia, M. A. Chem.-Biol. Interactions, 1986, 58, 29-40. N agamatsu, K; Hasegawa, A. Biochem. Pharmacol., 1992, 43, 2631-2635. Skoulis, N. P.; James, R. C.; Harbison, R. D.; Roberts, S. M. Toxicology, 1989, 5 7, 287-302. Chen, Z. R.; Irvine, R. J.; Bochner, F.; Somogyi, A. A. Life Sci., 1990, 446, 1067- 1074. Chen, Z. R.; Somogyi, A. A.; Bochner, F. Lancet, 1988, H, 914-915. Mikus, G.; Somogyi, A. A.; Bochner, F.; Eichelbaum, M. Biochem. Pharmacol., 1991, 41, 757-762. Dayer, P.; Desmeules, J.; Leemann, T.; Stribemi, R. Biochem. Biophys. Res. Commun., 1988, 152, 411-416. Pawula, M.; Shaw, P. N.; Barrett, D. A. J. Chromatog. B, 1994, 653, 106-111. Aasmundstad, T. A.; Ripel, A.; Bodd, E.; Bjomeboe, A.; M~rland, J. Biochem. Pharmacol., 1993, 46, 961-968. Rane, A.; Gawronska-Szklarz, B.; Svensson, J.-O. J. Pharmacol. Exp. Therap., 1985, 234, 761-765. Aasmundtstad, T. A.; Lillekjendlie, B.; Morland, J. Pharmacol. Toxicol., 1996, 79, 114-119. Coughtrie, M. W. H.; Ask, B.; Rane, A.; Burchell, B.; Hume. R. Biochem. Pharmacol., 1989, 38, 3273-3280. Puig, J. F; Tephly, T. R. Molec. Pharmacol., 1986, 30, 558-565. Kumagai, Y.; Todaka, T.; Toki, S. J. Pharmacol. Exper. Therap., 1990, 255, 504- 510 Persson, K.; LindstrSm, B.; Spalding, D.; WahlstrOm, A.; Rane, A. J. Chromatog., 1989, 491, 473-480. Petersen, D. R.; Norris, K. J.; Thompson, J. A. Drug Metab. Dispos., 1984, 12, 725-731. Langone, J. J.; Franke, J.; VanVanakis, H. Arch. Biochem. Biophys., 1974. 164. 536-543. Kyerematen, G. A.; Owens, G. F.; Chattopadhyay, J. D.; deBethizy, J. D.; Vesell, E. S. Drug Metab. Dispos., 1988, 16, 823-828. Jacob, P.; II|, Shulgin, A. T.; Yu, L.; Benowitz, N. L. J. Chromatog., 1992, 583, 145-154. Kyerematen, G. A.; Taylor, L. H.; deBethizy, J. D.; Vesell, E. S. J. Chromatog., 1987, 419, 191-203. Rop, P. P.; Grimaldi, F.; Oddoze, C.; Viala, A. J. Chromatog., 1993, 612, 302309. Seaton, M. J.; Veell. E. S.; Luo, H.; Hawes, E. M. J. Chromatog., 1993, 621, 49-53. Zuccaro, P; Altieri, I.; Rosa, M.; Passa, A. R.; Pichini, S.; Ricciarello, G.; Pacifici, R. J. Chromatog., 1993, 621, 257-261. Benowitz, N. L.; Jacob, P.; III, Fong, I.; Gupta, S. J. Pharmacol. Exp. Therap., 1994, 268, 296-303. Rtldell, U.; Foth, H.; Kahl, G. F. Biochem. Biophys. Res. Commun., 1987, 148, 192-198. McCoy, G. D; DeMarco, G. J.Biochem. Pharmacol., 1986, 35, 4590-4592.
40
1". ROBINSON
[98]
McCoy, G. D.; DeMarco, G. J.; Koop, D. R.Biochem. Pharmacol., 1989, 38,
[99]
Nwosu, C. G; Crooks, P. A. Xenobiotica, 1988, 18, 1361-1372. Jacob, P.; III, Benowitz, N. L.; Copeland, J. R.; Risner, M. E.; Cone, E. J. J. Pharm. Sci., 1988, 77, 396-400. Pool, W. F.; Houdi, A. A.; Damani, L. A.; Layton, W. J.; Crooks, P. A. Drug Metab. Dispos., 1986, 14, 574-579. Nakayama, H.; Okuda, H.; Nakashima, T.; lmaoka, S.; Funae, Y. Biochem. Pharmacol., 1993, 5, 2554-2556. Peterson, L. A.; Trevor, A.; Castagnoli, N.; Jr. J. Med. Chem., 1987, 30, 249254. Obach, R. S; Van Vunakis, H. Biochem. Pharmacol., 1990, 39, R1-R4. Damani, L. A.; Pool, W. F.; Crooks, P. A.; Kaderlik, R. K.; Ziegler, D. M. Molec. Pharmacol., 1988, 33, 702-705. Williams, D. E.; Shigenaga, M. K.; Castagnoli, N.; Jr. Drug Metab. Dispos., 1990, 18, 418-428. Crooks, P. A; Godin, C. S. J. Pharm. Pharmacol., 1988, 40, 153-154. Oldendorf, W. H.; Stoller, B. E.; Harris, F. L. Proc. Nat. Acad. Sci. U. S., 1993, 90, 307-311. Patterson, T. A; Kosh, J. W. Gen. Pharmacol., 1993, 24, 641-647. Winter, C. K.; Segall, H. J.; Jones, A. D. Biomed. Environ. Mass Spec., 1988, 15, 265-273. Mattocks, A. R; Driver, H. E. Chem.-Biol. Interactions, 1987, 63, 91-104. Lam6, M. W.; Jones, A. D.; Morin, D.; Segall, H. J. Drug Metab. Dispos., 1991, 19, 516-524. Couet, C. E.; Hopley, J.; Hanley, A. B. Toxicon, 1996, 34, 1058-1061. Mattocks, A. R.; Jukes, R. Chem. Biol Interactions, 1990, 75, 225-239. Nigra, L; Huxtable, R. J. Toxicon, 1992, 30, 1195-1202. Mattocks, A. R.; Croswell, S.; Jukes, R.; Huxtable, R. J. Toxicon, 1991, 29, 409- 415. Niwa, H.; Ogawa, T.; Okamoto, O.; Yamada, K. Tetrahedron Lett., 1991, 32, 927- 930. Yan, C. C; Huxtable, R. J. Biochem. Pharmacol., 1996, 51, 321-329, 375-379. Williams, D. E.; Reed, R. L.; Kedzierski, B.; Ziegler, D. M.; Buhler, D. R. Drug Metab. Dispos., 1989, 17, 380-386. Miranda, C; Chung, W.; Reed, R. E.; Zhao, X.; Henderson, M. C.; Wang, J.L.; Williams, D. E.; Buhler, D. R. Biochem. Biophys. Res. Commun., 1991, 178, 546-552. Nishida, R.; Kim, C.; Fukami, H.; Irie, R. Agric. Biol. Chem. Tokyo, 1991, 55, 1787-1792. Nickisch-Rosenegk, E. v.; Schneider, D.; Wink, M. Z. Naturforsch., 1990, 45c, 881-894. Klier, B.; Eilert, U.; Schimmer, O. Planta Med., 1991, 57, A I03. Hoyer, G. L.; Clawson, D. C.; Brookshier, L. A.; Nolan, P. E.; Marcus, F. I. J. Chromatog., 1991, 572, 159-169. Guengerich, F. P. J. Med. Chem., 1984, 27, 1101-1103. Horai, Y; lshizaki, T.; Eichelbaum, M.; Hashimoto, K.; Chiba, K.; Dengler, H.J. Xenobiotica, 1988, 18, 1077-1084. Lennard, M. S. Pharmacol. Toxicol., 1990, 67, 273-283. Pospisil, J.; Patzelova, V.; M,'tca, B. Drug Metab. Dispos., 1992, 29, 330-332.
1185- 1188.
[~001 [loi]
[~02] [103]
[104] [1051 [106]
[107] [IOS] [109] [110]
[Ill] [112] [ll3] Ill4] [115] Ill6] [ll7] [ll8] [119] [120] [121] [122] [123] [124]
[125] [126] [127] [128]
ALKALOIDSIN ANIMALS
41
[129] Ebner, T.; Eichelbaum, M.; Fischer, P.; Meese, C. O. Arch. Pharm., 1989, 322, 399-403. [130] Mathys, K; Brenneisen, R..1. Chromatog., 1992, 593, 79-85. Watanabe, K.; Kayano, Y.; Matsunaga, T.; Yamamoto, I.; Yoshimura, H. Biol. Pharm. Bull. Tokyo, 1995, 18, 696-699. [1321 Rein, R.; Golombek, A., Eds. Computer-Assisted Modeling of Receptor-Ligand Interactions, A. R. Liss: New York, 1989. [133] Blum, M.; Ed. The Toxic Action of Marine and Terrestrial Alkaloids, Alaken: Fort Collins, CO1995. [134] Schmeller, T.; Latz-Br0ning, B.; Wink, M. Phytochemistry, 1997, 44, 257-266. [135] Alkadhi, K. A. Europ. J. Pharmacol., 1987, 138, 257-264. [136] Rand, M. J; Thurau, K., Eds. The Pharmacology of Nicotine, Oxford U. Press,1988. [137] Badio, B.; Shi, D.; Shin, Y.; Hutchinson, K. D.; Padgett, W. L.; Daly, J. W. Biochem. Pharmacol., 1996, 52, 933-939. [138] Brioni, J. D.; Kim, D. J. B.; O'Neill, A. B. Europ. J. Pharmacol., 1996, 301, 1-5. [139] Flores, C. M.; Rogers, S. W.; Pabreza, L. A.; Wolfe, B. B.; Kellar, K. J. Molec. Pharmacol., 1992, 41, 31-37. [140] Pedersen, S. E; Cohen, J. B. Proc. Nat. Acad Sci. U. S., 1990, 87, 2785-2789. [141] Martino-Barrows, A. M; Kellar, K. J. Molec. Pharmacol., 1987, 31, 169-174. [142] Rapier, C.; Lunt, G. G.; Wonnacott, S.J. Neurochem., 1988, 50, 1123-1130. [143] Rozental, R; Aracava, Y.; Scoble, G. T.; Swanson, K. L.; Wonnacott, S.; Albuquerque, E. X..1. Pharmacol. Exp. Therap., 1989, 251, 395-404. [144] Funayama, N.; Shinkai, M.; Takayanagi, I. Gen. Pharmacol., 1995, 26, 977981. [145] Barlow, R. B; Johnson, O. Brit. J. Pharmacol., 1989, 98, 799-808. [146] Flesher, J. E.; Scheffel, U.; London, E. D.; Frost, J. J. Life Sci. 1994, 54, 18831890. [147] Wolf-Pflugmann, M.; Lambrecht, G.; Wess, J.; Mutschler, E. Arzneim.-Forsch., 1989, 39, 539-544. [148] Okonjo, K. O.; Kuhlmann, J.; Maelicke, A. Europ. J. Biochem., 1991, 200, 671677. [149] Galzi, J.-L.; Revah, F.; Bessis, A.; Changeux, J.-P. Ann. Rev. Pharmacol. Toxicol., 1991, 31, 37-72. [150] Pedersen, S. E; Papineni, R. V. L..1. Biol. Chem., 1996, 270, 31141-31150. [151] Banerjee, B; Ganguly, D. K. Europ. J. Pharmacol., 1996, 310, 13-17. [~52] Lentz, T. Biochemistry, 1995, 34, 1316-1322. [153] Middleton, R. E; Cohen, J. B. Biochemistry, 1991, 30, 6987-6997. [154] Manners, G. D.; Panter, K. E.; Pelletier, S. W..I. Nat. Prod, 1995, 58, 863869. [155] Jennings, K. R.; Brown, D. G.; Wright, D. P.; Jr. Experientia, 1986, 42, 611613. [156] Mori, T.; Murayama, M.; Bando, H.; Kawahara, N. Chem. Pharm. Bull. Tokyo, 1991, 39, 379-383. [157] Voitenko, S; Purnin, S.; Omel'chenko, I.; Dyadyusha, G. G.; Zhorov, B.; Brovtsina, N.; Skok, V. Molec. Pharmacol., 1991, 40, 180-185. [~58] Fisher, M.; Huangfu, D.; Shen, T. Y.; Guyenet, P. G..1. Pharmacol. Expt. Therap., 1994, 270, 702-707. [159] Lerner-Marmarosh, N.; Carroll, F. I.; Abood, L. G. 1995, Life ScL 56, PL67-70.
42
T. ROBINSON
[160] Dar, D. E; Zinder, O. Naunyn-Schmiedeberg's Arch. Pharmacol., 1995, 352, 1116. [161] Storch, A; 13 Others Europ. J. Pharmacol., 1995, 290, 207-219. [162] Crooks, P. A; Dwoskin, L. P. Biochem. Pharmacol., 1997, 54, 743-753. [163] Schneider, A. S.; Nagel, J. E.; Mah, S. J. Europ. J. Pharmacol., 1996, 317, R IR2. [~64] Benwell, M. E. M.; Holtom, P. E.; Moran, R. J.; Balfour, D. J. K. Brit. 3,. Pharmacol., 1996, 117, 743-749. [165] Kuijpers, G. A. J.; Vegara, L. A.; Calvo, S.; Yadid, G. Brit..I. Pharmacol., 1994, 113, 471-478. [166] Hulme, E. C.; Kurtenbach, E.; Curtis, C. A. M. Biochem. Soc. Trans.., 1991, 9, 133-138. [167] Richards, M. H. Biochem. Pharmacol., 1991, 42, 1645-1653. [168] Brann, M. R.; Klimkowski, V. J.; Ellis, J. Life Sci., 1993, 52, 405-412. [169] Burgen, A. S. V. Life Sci., 1995, 56, 801-806. [170] Ross, E. M. 3'. Chem. Educ., 1988, 65, 937-942. [171] Dwoskin, L. P.; Teng, L.; Buxton, S. T.; Ravard, A.; Deo, N.; Crooks, P. A. Europ. ,I. Pharmacol., 1995, 276, 195-199. [172] Forsberg, E. J.; Rojas, E.; Pollard, H. B. J. Biol. Chem., 1986, 261, 4915-4920. [173] Drake, M. V.; O'Donnell, J. J.; Polansky, J. R. J. Pharm. Sci., 1986, 75, 278279. [174] Eglen, R. M; Harris, G. C.; Cox, H.; Sullivan, A. O.; Stefanich, E.; Whiting, R. L. Naunyn-Schmiedeberg's Arch. Pharmacol., 1992, 346, 144-151. [175] Gilani, A. H.; Shaheen, F.; Christopoulos, A.; Mitchelson, F. Life Sci., 1997, 60, 535-544. [1761 Waelbroeck, M.; De Neef, P.; Roberecht, P.; Christophe, J. Life Sci., 1984, 35, 1069-1076. [177] Kurachi, Y.; Nakajima, T.; Sugimoto, T. Naunyn-Schmiedeberg's Arch. Pharmacol., 1987, 335, 216-218. [178] Mack, J. E; Matthews, J. C. Biochem. Pharmacol., 1987, 36, 991-994. [179] Sharkey, J.; Ritz, M. C.; Schenden, J. A.; Hanson, R. C.; Kuhar, M. J. J. Pharmacol. Exp. Therap., 1988, 246, 1048-1052. [180] Smith, T. D.; Annis, S. J.; Ehlert, F. J.; Leslie, F. M. J. Pharmacol. Exp. Therap., 1991, 256, 1173-1181. [~81] Schmeller, T.; Sporer, F.; Sauerwein, M.; Wink, M. Pharmazie, 1995, 50, 493495. [182] Matesic, D. F; Luthin, G. R. FEBS Lett., 1991, 284, 184-186. [183] Xu, H; Gintzler, A. R. Proc. Nat. Acad. Sci. U. S., 1992, 89, 1978-1982. [184] Zimanyi, I.; Lajtha, A.; Vizi, E. S.; Reith, M. E. A. Biochem. Pharmacol., 1988, 3 7, 641-645.
[185] Venter, J. C.; Fraser, C. M.; Kerlavage, A. R.; Buck, M. A. Biochem. Pharmacol., 1989, 38, 1197-1208. [186] Harrison, J. K.; Pearson, W. R.; Lynch, K. R. Trends Pharmacol. Sci., 1991, 12, 62-67. [187] Bylund, D. B; Eikenberg, D. C.; Hieble, J. P.; Langer, S. Z.; Lefkowitz, R. J.; Minneman, K. P.; Molinoff, P. B.; Ruffolo, R. R., Jr.; Trendelenburg. U. Pharmacol. Rev., 1994, 6, 121-136. [~88] Levitzki, A. Science, 1988, 241, 800-806. [189] Kobilka, B. K; Kobilka, T. S.; Daniel, K.; Regan, J. W.; Caron, M. G.; Lefkowitz, R. J. Science, 1988, 240, 1310-1316.
ALKALOIDS IN ANIMALS
43
[190] Strosberg, A. D. Protein Sci., 1993, 2, 1198-1209. [1911 Convents, A.; Convents, D.; De Backer, J.-P.; De Keyser, J.; Vauquelin, G. Biochem. Pharmacol., 1989, 38, 455-463. [1921 Yu, S.-M; Ko, F. N.; Chueh, S. C.; Chen, J.; Chen, S. C.; Chen, C. C.; Teng, C. M. Europ. J. Pharmacol., 1994, 252, 29-34. [1931 Chuli~i S.; Noguera, M. A.; Ivorra, M. D.; Cortes, D.; D-Oc6n, M. P. Pharmacology, 1995, 50, 380-387. [1941 Chuli6, S.; Moreau, J.; Naline, E.; Noguera, M. A.; Ivorra, M. D.; D'Oc6n, M. P.; Advenier, C. Brit. J. Pharmacol., 1996, 119, 1305-1312. [195] lto, Y.; Yano, S.; Watanabe, K.; Yamanaka, E.; Aimi, N.; Sakai, S. Chem. Pharm. Bull Tokyo, 1990, 38, 1702-1706. [1961 Hui, K. K.; Yu, J. L.; Chan, W. F. A.; Tse, E. Life ScL 1991, 49, 315-324. [1971 Teng, C.-M; Yu, S. M.; Ko, F. N.; Chen, C. C.; Huang, Y. L.; Huang, T. F. Brit. J. Pharmacol., 1991, 104, 651-656. [198] Kwan, C. Y.; Chen, Y. Y.; Ma, M. F.; Daniel, E. E.; Hui, S. C. G. Life Sci. 1996, 59, PL359-364. [1991 Madden, J. A.; Konkol, R. J.;Keller, P. A.; Alvarez, T. A. Life Sci., 1995, 56, 679-686. [2001 Burnstock, G. Nucleosides & Nucleotides, 1991, 10, 917-930. [2011 Stone, T. W. Gen. Pharmacol., 1991, 22, 25-31. [202] Fredholm, B. B; Abbracchio, M. P.; Bumstock, G.; Daly, J. W.; Harden, T. K.; Jacobson, K. A.; Left, P.; Williams, M. Pharmacol. Rev., 1994, 46, 143-156. [203] Palmer, T. M; Stiles, G.L. Neuropharmacology, 1995, 34, 683-694. [2041 Clark, M; Post, R. M. Europ. d. Pharmacol., 1989, 164, 399-401. [2051 Sebasti~io, A. M; Ribeiro, J. A. Brit. J. Pharmacol., 1989, 96, 211-219. [206] Peet, N. P; Lentz, N. L.; Meng, E. C.; Dudley, M. W.; Ogden, A. M. L.; Demeter, D. A.; Weintraub, H. J. R.; Bey, P. d. Med. Chem., 1990, 33, 31273130. [207] Biaggioni, I.; Paul, S.; Puckett, A.; Arzubiaga, C. d. Pharmacol. Exp. Therap., 1991, 258, 588-593. [208] Rainnie, D. G.; Grunze, H. C. R.; McCarley, R. W.; Greene, R. W. Science, 1994, 263, 689-692. [209] Cooper, R. A; De Freitas, C.; Porreca, F., Eisenhour, C. M.; Lukas, R.; Huxtable, R. J. Toxicon, 1995, 33, 1025-1031. [2]o] Stiles, G. L. d. Biol. Chem., 1992, 267, 6451-6454. [2111 Ammon, H. P. T. Arch. Pharm., 1991, 324, 261-267. [212] Brown, G. R.; Sayers, L. G.; Kirk, C. J.; Michell, R. H.; Michelangeli, F. Biochem. J., 1992, 282, 309-312. [2131 Hirose, K.; lino, M; Endo, M. Biochem. Biophys. Res. Commun., 1993, 194, 726- 732. [214] Carter, A. J.; O'Connor, W. T.; Carter, M. J.; Ungerstedt, U. d. Pharmacol. Expt. Therap., 1995, 273, 637-642. [215] Porkka-Heiskanen, T; Strecker, R. E.; Thakar, M.; BjBrkum, A. A.; Greene, R. W.; McCarley, R. W.Science, 1997, 276, 1265-1268. [216] Shih, J. C.; Yang, W.; Chen, K.; Gallaher, T. Pharmacol. Biochem. Behavior, 1991, 40, 1053-1058,. [217] Hoyer, D; Clarke, D. E.; Fozard, J. R.; Hartig, P. R.; Martin, G. R.; Mylecharane, E. J.; Saxena, P. R.; Humphrey, P. P. A. Pharmacol. Rev., 1994, 46, 157-203. [218] Martin, G. R; Humphrey, P. P. A. Neuropharmacology, 1994, 33, 261-273.
44
T. ROBINSON
[219] Boess, F. G; Martin, I. L. Neuropharmacology, 1994, 33, 275-317. [220] Boess, F. G.; Monsma, F. J.; Jr.; Meyer, V.; Zwingelstein, C.; Sleight, A. J. Molec. Pharmacol., 1997, 52, 515-523. [221] Dyer, D. C. Life Sci., 1993, 53, PL223-PL228. [222] Eich, E.; Pertz, H. Pharmazie, 1994, 49, 867-877. [223] Ward, J. S; 6 Others ,I. Med. Chem., 1988, 31, 1512-1519. [224] Kidric, J.; Kocjan, D.; Hadzi, D. Experientia, 1986, 42, 327-328. [225] De Vos, H.; Czerwiec, E.; De Backer, J.-P.; De Potter, W.; Vauquelin, G. Europ. ,I. Pharmacol., 1991, 207, 1-8. [226] Winter, J. C; Rabin, R. A. ,I. Pharmacol. Exp. Therap., 1992, 263, 682-689. [227] Arthur, J. M.; Casaftas, S. J.; Raymond, J. R. Biochem. Pharmacol., 1993, 45, 2337-2341. [228] Shoji, N; Umeyama, A.;Saito, N.; luchi, A.; Takemoto, T.; Kajiwara, A.; Ohizumi, Y. J. Nat. Prod., 1987, 50, 773-774. [229] Uchimura, N; North, R. A. Brit. ,I. Pharmacol., 1990, 99, 736-740. [230] Hoffman B. J.; Mezey, E.; Brownstein, M. J. Science, 1991, 254, 578-579. [231] Hoffman, B. J.; Mezey, E.; Brownstein, M. J. Science, 1991, 254, 579-580. [232] Erickson, J. D.; Eiden, L. E.; Hoffman, B. J. Proc. Nat. ,4cad. Sci. U. S., 1992, 89, 10993-10997. [233] Izenwasser, S.; Rosenberger, J. G.; Cox, B. M. Life Sci., 1992, 50, 541-547. [234] Brossi, A. Planta Med., 1991, 57, $93-S100. [235] d'Ischia, M.; Constantini, C.; Prota, G. Tetrahedron Lett., 1993, 34, 3921-3924. [236] Baum, S. S; Rommelspacher, H. J. Chromatog. B 1994, 660, 235-241. [237] Caine, S. B; Koob, G. F. Science, 1993, 260, 1814-1816. [238] Civelli, O.; Bunzow, J. R.; Grandy, D. K.; Zhou, Q.-Y.; Van Tol, H. H. M. Europ. J. Pharmacol., 1991, 207, 277-286. [239] Civelli, O.; Bunzow, J. R.; Grandy, D. K. Ann. Rev. Pharmacol. Toxicol., 1993, 33, 281-307. [24o] Gingrich, J. A.; Jarvie, K.; Tiberi, M.; Fremeau, R. T.; Jr.; Caron, M. G. Dupont Biotech Update 1992, 7, 105, 108, 112, 118. [241] Blum, K.; Cull, J. G.; Braverman, E. R.; Comings, D. E. Am. Scientist, 1996, 84, 132-145. [242l Cortes, D.; Figadere, B.; Saez, J.; Protais, P. J. Nat. Prod., 1992, 55, 12811286. [2431 Guo-Zhang, J. Trends Pharmacol. Sci., 1987, 8, 81-82. [244] Chueh, F. Y.; Hsieh, M. T.; Chen, C. F.; Lin, M. T. Pharmacology, 1995, 51, 237-244. [2451 Schaus, J. M.; Titus, R. D.; Foreman, M. M.; Mason, N. R.; Truex, L. L. J. Med. Chem., 1990, 33, 600-607. [246] Spano, P. F; 7 Others Aging 23, Aging Brain and Ergot Alkaloids, Raven Press: NY,1983. [247] Schuldiner, S.; Liu, Y.; Edwards, R. H. J. Biol. Chem., 1993, 268, 29-34. [248] Giros, B; Wang, Y. M.; Suter, S.; McLeskey, S. B.; Pifl, C.; Caron, M. G. J. Biol. Chem., 1994, 269, 15985-15988. [249] Shimada, S.; Kitayama, S.; Lin, C.-L.; Patel, A.; Nanthakumar, E.; Gregor, P.; Kuhar, M.; Uhl, G. Science, 1991, 254, 576-578. [250] Reith, M. E. A.; Xu, C.; Chen, N.-H. Europ. J. Pharmacol., 1997, 324, 1-10. [251] Usdin, T. B.; Mezey, E.; Chen, C.; Brownstein, M. J.; Hoffman, B. J. Proc. Nat. Acad. Sci. U. S., 1991, 88, 11168-11171. [2521 Gawin, F. H. Science, 1991, 251, 1580-1586.
ALKALOIDSIN ANIMALS
45
[253] Faraj, B.A.; Olkowski, Z. L.; Jackson, R. T. Biochem. Pharmacol., 1995, 50, 1007-1014. [254] Imperato, A.; Obinu, M. C.; Demontis, M. V.; Gessa, G. L. Europ. J. Pharmacol., 1992, 229, 265-267. [255] Izenwasser, S.; Newman, A. H.; Katz, J. L. Europ. J. Pharmacol., 1993, 243, 201-205. [256] Self, D. W.; Barnhart, W.; Lehman, D. A.; Nestler, E. J. Science, 1996, 271, 1586-1589. [257] Carroll, F. I; Lewin, A. H.; Abraham, P.; Parham, K.; Boja, J. W.; Kuhar, M. J. J. Med. Chem., 1991, 34, 883-886. [258] H6ron, C.; Costentin, J.; Bonnet, J.-J. Europ. J. Pharmacol., 1994, 264, 391398. [259] Reith, M. E. A.; de Costa, B.; Rice, K. C.; Jacobson, A. E. Europ. J. Pharmacol., 1992, 22 7, 417-425. [260] Ritz, M. C.; Mantione, C. R.; London, E. D. Psychopharmacology, 1994, 114, 47-52. [261] Saadouni, S.; Refahi-Lyamani, F.; Costentin, J.; Bonnet, J.-J. Europ. J. Pharmacol., 1994, 268, 187-197. [2621 Cao, C. J.; Shamoo, A. E.;; Eldefrawi, M. E. Biochem. Pharmacol., 1990, 39, R9- RI4. [263] Rudnick, G.; Steiner-Mordoch, S. S.; Fishkes, H.; Stem-Bach, Y.; Schuldiner, S. Biochemistry, 1990, 29, 603-608. [264] Kalix, P. Neuropharmacology, 1986, 25, 499-501. [265] Dwoskin, L. P.; Leibee, L. L.; Jewell, A. L.; Fang, Z.-X.; Crooks, P. A. Life Sci., 1992, 50, PL233-PL237. [266] Yamashita, H; Kitayama, S.; Zhang, Y.-X.; Takahashi, T.; Dohi, T.; Nakamura, S. Biochem. Pharmacol., 1995, 49, 742-745. [267] Pontieri, F. E.; Tanda, G.; Orzi, F.; Di Chiara, G. Nature, 1996, 382, 255-257. [268] Nakamura, S.; Goshima, Y.; Yue, J.-L.; Miyamae, T.; Misu, Y. Europ. J. Pharmacol., 1992, 222, 75-80. [269] Epple, A.; Nibbio, B.; Spector, S.; Brinn, J. E. Life Sci., 1994, 54, 695-702. [270] Horfik, P.; Haberman, F.; Spector, S. Life Sci., 1993, 52, PL255-PL260. [271] Mignat, C.; Wille, U.; Ziegler, A. Life Sci., 1995, 56, 793-799. [272] Meng, F; Xie, G. X.; Thompson, R. C.; Mansour, A.; Goldstein, A.; Watson, S. J.; Akil, H. Proc. Nat. Acad. Sci. U. S., 1993, 90, 9954-9958. [273] Miyamae, T.; Fukushima, N.; Misu, Y.; Ueda, H. FEBS Lett., 1993, 333, 311314. [274] Ocafla, M.; Del Pozo, E.; Barrios, M.; Baeyens, J. M. Brit. J. Pharmacol., 1995, 114, 1296-1302. [275] Gomathi, K. G; Sharma, S. K. FEBS Lett., 1993, 330, 146-150. [276] Zhu, J.; Chen, C.; Xue, J.-C.; Kunapuli, S.; DeRiel, J. K.; Liu-Chen, L.-Y. Life Sci., 1995, 56, PL201-207. [277] Kristensen, K.; Christensen, C. B.; Christrup, L. L.Life Sci., 1995, 56, PL4550. [278] Young, A. M. J; Key, B. J. Neuropharmacology, 1984, 23, 1347-1350. [279] Adams, R. E; Wooten, G. F. Neurochem. Res., 1993, 18, 1041-1045. [2so] Herz, A. Shippenberg, T. S.; Bals-Kubik, R.; Spanagei, R. Arzneim. Forsch., 1992, 42-1,256-259. [2811 Cabot, P. J.; Dodd, P. R.; Cramond, T.; Smith, M. T. Europ. J. Pharmacol., 1994, 268, 247-255.
46
T. ROBINSON
[2821 Spangler, R.; Ho, A.; Zhou, Y.; Maggos, C. E.; Yuferov, V.; Kreek, M. J. Molec. Brain Res., 1996, 38, 71-76. [2831 Lipkowski, A. W.; Can', D. B.; Langlade, A.; Osgood, P. F.; Szyfelbein, S. K. Life Sci., 1994, 55, 149-154. [284] Bartlett, S. E; Smith, M. T. Life Sci., 1995, 57, 609-615. [285] Huwyler, J.; Drewe, J.; Klusemann, C.; Fricker, G. Brit. J. Pharmacol., 1996, 118, 1879-1885. [286] Airaksinen, M. M.; Saano, V.; Steidel, E.; Juvonen, H.; Huhtikangas, A.; Gynther, J. Acta Pharmacol. Toxicol., 1984, 55, 380-385. [2871 Bowen, W. D.; Vilner, B. J.; Williams, W.; Bertha, C. M.; Kuehne, M. E; Jacobson, A. E. Europ. ,I. Pharmacol., 1995, 279, R l-R3. [288] Watanabe, K.; Yano, S.; Horie, S.; Yamamoto, L. T. Life Sci., 1997, 60, 933942. [2891 Tang, R.; Novas, M. L.; Glavinovir M. I.; Trifaro, J.-M. Brit. J. Pharmacol., 1990, 99, 548-552. [290] Pang, I.-H; Vasko, M. R. Brain Research, 1986, 376, 268-279. [291] Payza, K.; Akar, C. A.; Yang, H.-Y. T. J. Pharmacol. Exp. Therap., 1993, 267, 88- 94. [292] Pellegrini-Giampietro, D. E.; Ruggiero, M.; Gianelli, S.; Chiarugi, V. P.; Moroni, F. Europ. J. Pharmacol., 1988, 149, 297-306. [293] Sergeeva, M. G.; Terentjeva, I. V.; Mevkh, A. T.; Varfolomeev, S. D. FEBS Lett., 1993, 323, 163-165. [294] Hancock, D. L; Coupar, I. M. Gen. Pharmacol., 1997, 28, 709-713. [2951 Harrison, B. L.; Baron, B. M.; Cousino, D. M.; McDonald, I. A. J. Med. Chem., 1990, 33, 3130-3132. [296] McDonald, J. W.; Penney, J. B.; Johnston, M. V.; Young, A. B. Neuroscience, 1990, 35, 653-668. [2971 Bristow, D. R.; Bowery, N. G.; Woodruff, G. N. Europ. J. Pharmacol., 1986, 126, 303-307. [298] Yadid, G.; Maor, G.; Youdim, M. B. H.; Silberman, M.; Zinder, O. Neurochem. Res., 1993, 18, 1051-I055. [299] Grenningloh, G; Pienitz, A.; Schmitt, B.; Methfessel, C.; Zensen, M.; Beyreuther, K.; Gundelfinger, E. D.; Betz, H. Nature, 1988, 328, 215-220. [3001 Ruiz-G6mez, A.; Morato, E.; GarcIa-Calvo, M.; Valdivieso, F.; Mayor, F.; Jr. Biochemistry, 1990, 29, 7033-7040. [3011 Marviz6n, J. C. G; Vfizquez, J.; Calvo, M. G.; Mayor, F., Jr.; G6mez, A. R.; Valdivieso, F.; Benavides, J. Molec. Pharmacol., 1986, 30, 590-597. [302] Marvizon, J. C. G; Skolnick, P. Molec. Pharmacol., 1988, 34, 806-813. [303] Leewanich, P.; Tohda, M.; Matsumoto, K.; Subhadhirasakul, S.; Takayama, H.; Aimi, N.; Watanabe, H. Europ. J. Pharmacol., 1997, 332, 321-326. [3041 Ikegami, F.; Kusama-Eguehi, K.; Sugiyama, E.; Watanabe, K.; Lambein, F.; Murakoshi, I. Biol. Pharm. Bull. Tokyo, 1995, 18, 360-362. [305] Popik, P.; Layer, R. T.; Skolnick, P. Pharmacol. Rev., 1995, 47, 235-253. [306] Layer, R. T.; Skolnick, P.; Bertha, C. M.; Bandarage, U. K.; Kuehne, M. E.; Popik, P. Europ. d. Pharmacol., 1996, 309, 159-165. [307] Carlezon, W. A.; Jr; Boundy, V. A.; Haile, C. N.; Lane, S. B.; Kalb, R. G.; Neve, R. L.; Nestler, E. J. Science, 1997, 277, 812-814. [308] Villar, H. O.; Davies, M. F.; Loew, G. H.; Maguire, P. A. Life Sci., 1991, 48, 593- 602.
ALKALOIDSIN ANIMALS
47
[309] Barolet, A. W.; Li, A.; Liske, S.; Morris, M. E. Can. J. Physiol. Pharmacol., 1985, 63, 1465-1470. [3101 Bartolini, A.; Giotti, A.; Giuliani, S.; Malmberg-Aiello, P.; Patacchini, R. Gen. Pharmacol., 1990, I, 277-284. [311] Beutler, J. A; Karbon, E. W.; Brubaker, E. M.; Malik, R.; Curtis, D. R.; Enna, S. J. Brain Research, 1985, 330, 135-140. [312] Kardos, J.; BlaskJ, G.; Kerekes, P.; Kovacs, I.; Simonyi, M. Biochem. PharmacoL, 1984, 3, 3537-3545. [3131 Jacquet, Y. F.; Saederup, E.; Squires, R. F. Europ. d. Pharmacol., 1987, 138, 285- 288. [314] Uneyama, H.; Harata, N.; Akaike, N. Brit. J. Pharmacol., 1993, 109, 459-465. [315] Barrow, C. J; Sun, H. H. d. Nat. Prod, 1994, 57, 471-476. [316] Granger, I.; Serradeil-le-Gal, C.; Augereau, J. M.; Gleye, J. Planta Med., 1992, 58, 35-38. [317] Gu~ritte-Voegelein, F; Sevenet, T.; Pusset, J.; Adeline, M.-T.; Gillet, B.; Beloeil, J.-C.; Gu6nard, D.; Potier, P. J. Nat. Prod, 1992, 55, 923-930. [318] Rasolonjanahary, R.; S6venet, T.; Voegelein, F. G.; Kordon. C. Europ. J. Pharmacol., 1995, 285, 19-23. [319] Karhap/i/t L; TSrnquist, K. Biochem. Biophys. Res. Commun. 1995, 210, 726732. [320] Debnath, D.; Kumar, G. S.; Nandi, R.; Maiti, M. Ind. J. Biochem. Biophys., 1989, 26, 201-208. [321] Zegar, I.; Gr/islund, A.; Bergman, J.; Eriksson, M.; Nord6n, B. Chem.-Biol. Interactions, 1989, 72, 277-293. [3221 Kumar, G. S.; Debnath, D.; Sen, A.; Maiti, M. Biochem. Pharmacol., 1993, 46, 1665-1667. [323] Malvy, C; Cros, S. 1986, Biochem. Pharmacol.;35, 2264-2267. [3241 Pezzuto, J. M; Che, C.-T.; McPherson, d. D.; Zhu, J.-P.; Topcu, G.; Erdelmeier, C. A. J.; Cordell, G. A. ,/. Nat. Prod, 1991, 54, 1522-1530. [325] Remers, W. A. ,1. Nat. Prod, 1985, 48,173-192. [326] Tornaletti, S.; Russo, P.; Parodi, S.; Pedrini, A. M. Biochim. Biophys. Acta, 1989, 1007, 112-115. [327] Dodin, G.; Schwaller, M.-A.; Aubard, J.; Paoletti, C. Europ. J. Biochem., 1988, 176, 371-376. [328] Monnot, M.; Mauffret, O.; Simon, V.; Lescot, E.; Psaume, B.; Saucier, J.-M.; Belehradek,J.; Jr; Fermandjian, S. FEBS Lett. 1990, 273, 71-74. [329] Schwaller, M.-A.; Aubard, J.; Auclair, C.; Paoletti, C.; Dodin, G. Europ. J. Biochem., 1989, 181, 129-134. [3301 Malvy, C; Bertrand, J. R. FEBS Lett., 1986, 208, 155-157. [331] Caprasse, M; Houssier, C. Biochimie, 1983, 65, 157-167. [3321 Chakraborty, S; Nandi, R.; Maiti, M.; Achari, B.; Saha, C. R.; Pakrashi, S. C. Biochem. Pharmacol., 1989, 38, 3683-3687. [333] Sen, A; Maiti, M. Biochem. Pharmacol., 1994, 48, 2097-2102. [334] Barret, Y; Sauvaire, Y. Phytotherap. Res., 1992, 6, 59-63. [3351 Pezzuto, J. M.; Antosiak, S. K.; Messmer, W. M.; Slaytor, M. B.; Honig, G. R. Chem.-Biol. Interactions, 1983, 43, 323-339. [336] Xu, Y.; Yakushijin, K.; Home, D. A. Tetrahedron Lett., 1992, 33, 4385-4388. [337] Candrian, U.; Ltithy, J.; Schlatter, C. Chem. Biol. Interactions, 1985, 54, 5769.
48
1".ROBINSON
[338] Chan, G. W; Berry, D.; DeBrosse, C. W.; Hemling, M. E.; MacKenzieLoCosto, L.; Often, P. H.; Westley, J. W..1. Nat. Prod., 1993, 56, 708-713. [339] Fukada, M. Biochem. Pharmacol., 1985, 34, 1225-1230. [3401 Fujita, H; Kakishima, H. Chem.-Biol. Interactions, 1989, 72, 105-11 I. [3411 Hudson, J. B.; Graham, E. A.; Fong, R.; Hudson, L. L; Towers, G. H. N. Photochem. Photobiol., 1986, 44, 483-487. [342] Pezzuto, J. M; Mar, W.; Lin, L.-Z.; Cordell, G. A.; Neszm~lyi, A.; Wagner, H. Phytochemistry, 1992, 31, 1795-1800. [343] Kim, Y. C.; Che, Q.-M.; Gunatilaka, A. A. L.; Kingston, D. G. I. J. Nat. Prod., 1996, 59, 283-285. [344] Paulini, H.; Popp, R.; Schimmer, O.; Ratka, O.; R0der, E. Planta Med., 1991, 57, 59-61. [345] Jenison, R. D.; Gill, S. C.; Pardi, A.; Polisky, B. Science, 1994, 263, 14251429. [346] Pratviel, G.; Bemadou, J.; Ha, T.; Meunier, G.; Cros, S.; Meunier, B.; Gillet, B.; Guittet, E. d. Med. Chem., 1986, 29, 1350-1355. [347] Berry, D. E.; MacKenzie, L. J.; Busby, R. W.; Nasuti, C. A. d. Nat. Prod., 1992, 55, 401-413. [348] Froelich-Ammon, S. J; Osheroff, N. d. Biol. Chem., 1995, 270, 21429-21432. [3491 Auclair, C. Arch. Biochem. Biophys., 1987, 259, 1-14. [350] Hsiang, Y.-H. Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem., 1985, 260, 14873-14878. [351] Leteurtre, F.; Fesen, M.; Kohlhagen, G.; Kohn, K. W.; Pommier, Y. Biochemistry, 1993, 32, 8955-8962. [3521 Kjeldsen, E; Mollerup, S.; Thomson, B.; Bonven, B. J.; Bolund, L.; Westergaard, O. J. Molec. Biol., 1988, 202, 333-342. [353] Knab, A. M.; Fertala, J.; Bjomsti, M.-A. J. Biol. Chem., 1993, 268, 2232222330. [354] Larsen, A. K; Grondard, L.; Couprie, J.; Desoize, B.; Comoe, L.; Jardillier, J. C.; Riou, J. F. Biochem. Pharmacol., 1993, 46, 1403-1412. [3551 Mahmood, K; PaYs, M.; Fontaine, C.; Ali, H. M.; Haai, A. H. A.; Guittet, E. Tetrahedron Lett., 1993, 34, 1795-1796. [3561 Kakiuchi, N.; Hattori, M.; Ishii, H.; Namba, T. Planta Med., 1987, 53, 22-27. [357] Tan, G. T.; Pezzuto, J. M.; Kinghom, A. D.; Hughes, S. H. J. Nat. Prod., 1991, 54, 143-154. [358] Kulp, K. S; Vulliet, P. R. Toxicol. ,4ppl. Pharmacol., 1996, 139, 356-364. [3591 Lin, H; Falchetto, R.; Mosca, P. J.; Shabanowitz, J.; Hunt, D. F.; Hamlin, J. L J. Biol. Chem., 1996, 2 71, 2548-2556. [3601 Magnus, P.; Ladlow, M.; Elliott, J. J. Am. Chem. Soc., 1987, 109, 7929-7930. [361] Krayevsky, A. A. Bioorg. Chem. USSR 1978, 4, 853-878. [362] Leatherman, D. L; Middlebrook, J. L. J. Pharmacol. Exp. Therap., 1993, 266, 732-740, 741-748. [3631 Tujebajeva, R. M.; Gaifer, D. M.; Karpova, G. G; Ajtkhozhina, N. A. FEBS Lett., 1989, 257, 254-256. [364] Vrijsen, R.; Vanden Berghe, D. A.; Vlietinck, A. J.; Boey~, A. J. Biol. Chem., 1986, 261, 505-507. [365] Jimenez, J.; Sanchez, L.; Vazquez, D. FEBS Lett., 1975, 55, 53-56. [366] Jeven, M.; van den Berghe, D. A.; Vlietinck, A. J. Planta Med., 1983, 49, 109114.
ALKALOIDS IN ANIMALS
49
[367] Datta, R. K.; Ghosh, J. A.; Antopol, W. Biochem. Pharmacol. 1974, 23, 16871692 [368] Chakrabarti, S.; Kumar, S.; Shankar, R. Biochem. Pharmacol., 1986, 35, 16111613. [369] Lombardini, J. B; Props, C. Biochem. Pharmacol., 1996, 51, 151-157. [370] Uezono, Y; Yanagihara, N.; Wada, A.; Koda, Y.; Yokota, K.; Kobayashi, H.; Izumi, F. Naunyn-Schmiedeberg's Arch. Pharmacol., 1989, 339, 653-659. [3711 Lombardini, J. B.Biochem. Pharmacol., 1996, 52, 253-257. [372] Gong6ra, J. L.; Sierra, A.; Mariscal, S.; Aceves, J. Europ. J. Pharmacol., 1988, 155, 49-55. [373] Brossi, A.; Sch0nenberger, B.; Clark, O. E.; Ray, R. FEBS Lett., 1986, 201, 190- 192. [374] Orgell, W. H. Lloydia, 1963, 26, 36-43, 59-66. [375] Mroue, M. A.; Euler, K. L.; Ghuman, M. A.; Alam, M. J. Nat. Prod., 1996, 59, 890-893. [376] Harvey, A. L. Pharmacol. Therap., 1995, 68, 113-128. [377] Kozikowski, A. P; Xia, Y.; Reddy, E. R.; Ttlekmantel, W.; Hanin, I.; Tang, Y. C. d. Org. Chem., 1991, 56, 4636-4645. [378] McKinney, M.; Miller, J. H.; Yamada, F.; Tuckmantel, W.; Kozikowski, A. P. Europ. J. Pharmacol., 1991, 203, 303-305. [379] Ashani, Y.; Peggins, J. O.; III; Doctor, B. P. Biochem. Biophys. Res. Commun. 1992, 184, 719-726. [380] Zorko, M; Pavlie, M. R. Biochem. Pharmacol., 1986, 35, 2287-2296. [381] Stojan, J; Pavlie, M. R. Biochim. Biophys. Acta 1991, 1079, 96-102. [382] Sepci'c, K; Guella, G.; Mancini, I.; Pietra, F.; Dalla Serra, M.; Menestrina, G.; Tubbs, K.; Macek, P.; Turk, T. J. Nat. Prod, 1997, 60, 991-996. [383] Dougherty, D.A. Science, 1996, 271. 163 [384] Elbein, A. D. Ann. Rev. Biochem., 1987, 56, 497-534. [385] Elbein, A. D. Plant Physiol., 1988, 87, 291-295. [386] James, L. F.; Elbein, A. D.; Molyneux, R. J.; Warren, C. D., Eds., Swainsonine and Related Glycosidase lnhibitors, Iowa S. U. Press: Ames, 1989 [387] Molyneux, R. J.; Benson, M.; Wong, R. Y.; Tropea, J. E.; Elbein, A. D. or. Nat. Prod, 1988, 51, 1198-1206. [388] Asano, N; Nishida, M.; Kizu, H.; Matsui, K.; Watson, A. A.; Nash, R. J. J. Nat. Prod, 1997, 60, 98-101. [389] Molyneux, R. J.; Pan, Y. T.; Goldmann, A.; Tepfer, D.; Elbein, A. D. Arch. Biochem. Biophys., 1993, 304, 81-88. [390] Nash, R. J.; Rothschild, M.; Porter, E. A.; Watson, A. A.; Waigh, R. D.; Waterman, P. G. Phytochemistry, 1993, 34, 1281-1283. [391] Bowlin, T. L; Sunkara, P. S. Biochem. Biophys. Res. Commun. 1988, 151, 859864. [392] Tulsiani, D. R. P.; Broquist, H. P.; James, L. F.; Touster, O. Arch. Biochem. Biophys., 1988, 264, 607-617. [393] Pan, Y. T.; Ghidoni, J.; Elbein, A. D. Arch. Biochem. Biophys., 1993, 303, 134- 144. [394] Koley, A. P.; Robinson, R. C.; Markowitz, A.; Friedman, F. K. Biochem. Pharmacol., 1997, 53, 455-460. [395] Mitsui, N.; Noro, T.; Kuroyanagi, M.; Miyase, T.; Umehara, K.; Ueno, A. Chem. Pharm. Bull. Tokyo, 1989, 37, 363-366. [396] Shute, J. K; Smith, M. E. Biochem. Pharmacol., 1985, 34, 2471-2475.
50
T. ROBINSON
[397] Toescu, E. C.; O'Neill, S. C.; Petersen, O. H; Eisner, D. A. ,1. Biol. Chem., 1992, 267, 23467-23470. [398] Ukena, D.; Schudt, C.; Sybrecht, G. W. Biochem. Pharmacol., 1993, 45, 847851. [3991 Prabhakaram, M; Singh, S. N. Biochem. Intern., 1989, 18, 217-225. [400] Eguchi, N.; Watanabe, Y.; Kawanishi, K.; Hashimoto, Y.; Hayaishi, O. Arch. Biochem. Biophys., 1984, 232, 602-609. [401] Maguire, M. H; Csonka-Khalifah, L. Biochim. Biophys. ,4cta, 1987, 921, 426436. [4021 Son, J.-K.; Rosazza, J. P. N.; Duffel, M. W. J. Med. Chem., 1990, 33, 18451848. [403] Mimaki, Y.; Takaashi, Y.; Kuroda, Y.; Sahida, Y.; Nikaido, T. Phytochemistry, 1996, 42, 1609-1615. [404] Herbert, J. M.; Augereau, J. M.; Gleye, J.; Maffrand, J. P. Biochem. Biophys. Res. Commun., 1990, 172, 993-999. [4051 Kemmerling, W. Z. Naturforsch., 1996, 51c, 59-64. [406] Nakai, N.; Fujii, Y.; Kobashi, K.; Nomura, K. Arch. Biochem. Biophys., 1985, 239, 491-496. [407] Trugman, G.; Rousset, M.; Zweibaum, A. FEBS Lett., 1986, 95, 28-32. [408] Xu, Y.; Liu, J.; Zhang, S.; Liu, L. Biochem. J., 1987, 248, 985-988. [409] Abu-Ghalyun, Y, Masaalmeh, A.; AI-Khalil, S. Gen. Pharmacol., 1997, 29, 621- 623. [41o] Meikle, A. W.; Liu, X. H.; Taylor, G. N.; Stringham, J. D. Life Sci., 1988, 43, 1845-1850. [411] de la Lande, I. S.; Parker, D. A. S.; Proctor, C. H.; Marino, V.; Mackay-Sim, A. Life Sci., 1987, 4 I, 2463-2468. [4121 Chan, G. W. J. Org. Chem., 1993, 58, 2544-2546. [4131 Chang, C.-J; Geahlin, R. L. d. Nat. Prod., 1992, 55, 1529-1560. [414] Fuller, R. W.; Wong, C. J.; Hemrick-Luecke, S. K.Life Sci., 1986, 38, 409-412. [415] Jaspars, M; Rali, T.; Laney, M.; Schatzman, R. C.; Diaz, M. C.; Sehmitz, F. J.; Pordesimo, E. O.; Crews, P. Tetrahedron, 1994, 50, 7367-7374. [416] Lee, M. K; Kim, H. S. Planta Med., 1996, 62, 31-34. [417] Neal, J. J. Phytochemistry, 1989, 28, 451-453. [418] Ori, K.; Mimaki, Y.; Sahida, Y.; Nikaido, T.; Ohmoto, T. Phytochemistry, 1992, 31, 3605-3607, 4337-4341. [419] Tanaka, T; Metori, K.; Mineo, S.; Hirotani, M.; Furuya, T.; Kobayashi, S. Planta Med., 1993, 59, 200-202. [420] Wade, T. G; Whitely, C. G. Biochem. Intern., 1981, 14, 189-197. [421] Keukens, E. A. J; de Vrije, T.; Jansen, L. A. M.; de Boer, H.; Janssen, M.; de Kron, A. J. P. M.; Jongen, W. M. F.; de Kruijff, B. Biochim. Biophys. Acta, 1996, 1279, 243-250. [4221 Keukens, E. A. J; de Vrije, T.; van den Boom, C.; de Waard, P.; Plasman, H. H.; Thiel, F.; Chupin, V.; Jongen, W. M. F.; de Kruijff, B. Biochim. Biophys. Acta, 1995, 1240, 216-228. [4231 Keukens, E. A. J.; de Vrije, T.; Fabrie, C. H. J. P.; Demel, R. A.; Jongen, W. M. F.; de Kruijff, B. Biochim. Biophys. Acta, 1992, 1110, 127-136. [4241 Surewicz, W. K. Biochim. Biophys. Acta, 1982, 692, 315-318. [4251 Roddick, J. G.; Rijnenberg, A. L.; Weissenberg, M. Phytochemistry, 1990, 29, 1513-1518.
A L K A L O I D S IN ANIMALS
51
[426] Arnason, J. T.; Guerin, B.; Kraml, M. M.; Mehta, B.; Redmond, R. W.; Scaiano, J. C Photochem. Photobiol., 1992, 55, 35-38. [4271 Tuveson, R. W.; Larson, R. A.; Marley, K. A.; Wang, G.-R.; Berenbaum, M. R. Photochem. Photobiol., 1989, 50, 733-738. [428] Oubrahim, H.; Richard, J.-M.; Cantin-Esnault, D.; Seigle-Murandi, F.; Tr6court, F.J. Chromatog. A 1997, 758, 145-157. [429] Speisky, H.; Cassels, B. K.; Lissi, E. A.; Videla, L. A. Biochem. Pharmacol., 1991, 41, 1575-1581. [430] Mourelle, M.; Fraginals, R.; RodrIguez, L.; Favari, L.; P~rez-Alvarez, V.Life Sci., 1989, 45, 891-900. [4311 Banerjee, A; Luduena, R. F. J. Biol. Chem., 1991, 266, 1689-1691. [432] Banerjee, A; Luduena, R. F. J. Biol. Chem., 1992, 267, 13335-13339. [433] SackeR, D. L; Varma, J. K. Biochemistry, 1993, 32, 13560-13565. [434] Floyd, L. J.; Barnes, L. D.; Williams, R. F. Biochemistry, 1989, 28, 85158525. [435] Medrano, F. J.; Andreu, J. M.; Gorbunoff, M. J.; Timasheff, S. N. Biochemistry, 1989, 28, 5589-5599. [436] Brossi, A. J. Med. Chem., 1990, 33, 2311-2319. [4371 Hamel, E.; Ho, H. H.; Kang, G.-J.; Lin, C. M. Biochem. Pharmacol., 1988, 37, 2445-2449. [438] Skoufias, D. A; Wilson, L. Biochemistry, 1992, 31, 738-746. [439] Engelborghs, Y.; Dumortier, C.; D'Hoore, A.; Vandecandelaere, A.; Fitzgerald, T. J. J. Biol. Chem., 1993, 268, 107-112. [440] Malty, S. N; Ray, K.; Banerjee, A.; Mukhopadhyay, K.; Roychowdhury, S.; Chaudhuri, G. G.; Biswas, B. B.; Bhattacharyya, B. Ind. J. Biochem. Biophys., 1988, 25, 585-589. [441] Diaz, J. F; Andreu, J. M. J. Biol. Chem., 1991, 266, 2890-2896. [442] Shearwin, K. E; Timasheff, S. N. Biochemistry, 1994, 33, 894-90 I. [443] Uppuluri, S.; Knipling, L.; SackeR, D. L.; Wolff, J. Proc. Nat. Acad. Sci. U. S., 1993, 90, 11598- l 1602. [444] Bums, R. G. FEBS Lett., 1992, 297, 205-208. [445] Rai, S. S; Wolff, J. J. Biol. Chem., 1996, 271, 14707-14711. [446] Hastir S. B.; MacDonald, T. L. Biochem. Pharmacol., 1990, 39, 12271-1276. [447] Raxworthy, M. J. Biochem. Educ., 1988, 16, 2-9. [448] Liliom, K.; Lehotzky, A.; Moln,Sr, A.; Ov,'tdi, J. Anal. Biochem., 1995, 228, 1826. [449] Smith, C. D; Zhang, X. J. Biol. Chem., 1996, 271, 6192-6198. [450] Lobert, S.; Vulevic, B.; Correia, J. J. Biochemistry, 1996, 35, 6806-6814. [451] Prakash, V; Timasheff, S. N. Biochemistry, 1991, 30, 873-880. [452] Jordan, M.A.; Margolis, R. L.; Himes, R. H.; Wilson, L. at. Molec. Biol., 1986, 187, 61-73. [453] Singer, W. D.; Jordan, M. A.; Wilson, L.; Himes, R. H. Molec. Pharmacol., 1989, 36, 366-370. [454] Borman, L. S; Kuehne, M. E. Biochem. Pharmacol., 1989, 38, 715-724. [455] Na, G. C; Timasheff, S. N. Biochemistry, 1986, 25, 6214-6222, 6222-6228. [456] Bowman, L. C.; Houghton, J. A.; Houghton, P. J. Biochem. Biophys. Res. Commun., 1986, 135, 695-700. [4571 SackeR, D. L. Biochemistry, 1995, 34, 7010-7019. [458] Huang, A. B.; Lin, C. M.; Hamel, E. Biochem. Biophys. Res. Commun., 1985, 128, 1239-1246.
52
T. ROBINSON
[459] Takahashi, M; Iwasaki, S.; Kobayashi, H.; Okuda, S.; Murai, T.; Sato, Y. Biochim. Biophys. Acta, 1987, 926, 215-223. [460] Horwitz, S. B. Trends Pharmacol. Sci., 1992, 13, 134-136. [461] Ohmi, K; Nakamura, S. Europ. J. Pharmacol., 1991, 207, 305-310. [462] Cheek, T. R; Burgoyne, R. D. ,1. Biol. Chem., 1987, 262, 11663-11666. [463] Sorrentino, V; Volpe, P. Trends Pharmacol. Sci., 1993, 14, 98-103. [454] McGrew, S. G.; Wolleben, C.; Siegl, P.; Inui, M.; Fleischer, S. Biochemistry, 1989, 28, 1686-169 I. [4651 Pessah, I. N; Zimanyi, I. Molec. Pharmacol., 1991, 39, 679-689. [466] Michalak, M.; Dupraz, P.; Shoshan-Barmatz, V. Biochim. Biophys. Acta, 1988, 939, 587-594. [467] Pessah, I. N.; Francini, A. O.; Scales, D. J.; Waterhouse, A. L.; Casida, J. E. J. Biol. Chem., 1986, 61, 8643-8648. [468] Campbell, K. P; Knudson, C. M.; Imagawa, T., Leung, A. T.; Sutko, J. L.; Kahl, S. D.; Raab, C. R.; Madson, L. J. Biol. Chem., 1987, 262, 6460-6463. [469] Inui, M.; Saito, A.; Fleischer, S. J. Biol. Chem., 1987, 262, 1740-1747. [4701 Takeshima, H; l0 Others Nature, 1989, 339, 439-445. [471] Marks, A. R; Tempst, P.; Hwang, K. S.; Taubman, M. B.; Inui, M.; Chadwick, C.; Fleischer, S.; NadaI-Ginard, B.; Proc. Nat. Acad. Sci. U. S., 1989, 86, 86838687. [472] McPherson, P. S; Campbell, K. P. J. Biol. Chem., 1993, 268, 13765-13768. [473] Hymel, L.; Inui, M.; Fleischer, S.; Schindler, H. Proc. Nat. Acad. Sci. U. S., 1988, 85, 441-445. [474] Hamilton, S. L; Alvarez, R. M.; Fill, M.; Hawkes, M. J.; Brush, K. L.; Schilling, W. P.; Stefani, E. Anal. Biochem., 1989, 183, 31-41. [4751 Lai, F. A.; Misra, M.; Xu, L.; Smith, H. A.; Meissner, G. J. Biol. Chem., 1989, 264, 16776-16785. [4761 Marty, I.; Villaz, M.; Arlaud, G.; Bally, I.; Ronjat, M. Biochem. J., 1994, 298, 743-749. [477] Hasselbaeh, W; Migala, A. Z. Naturforsch., 1992, 47c, 136-147. [478] Shoshan-Barmatz, V; Zarka, A. J. Biol. Chem., 1988, 263, 16772-16779. [479] Waterhouse, A. L.; Pessah, I. N.; Francini, A. O.; Casida, J. E. J. Med. Chem., 1987, 30, 710-716. [480] Welch, W.; Sutko, J. L.; Mitchell, K. E.; Airey, J.; Ruest, L. Biochemistry, 1996, 35, 7165-7173. [481] Zarka, A; Shoshan-Barmatz, V. Biochim. Biophys. Acta, 1992, 1108, 13-20. [4821 Hasselbaeh, W; Migala, A. Z. Naturforsch., 1988, 43c, 140-148. [4831 Barry, V. A; Cheek, T. R. Biochem. J., 1994, 300, 589-597. [484] Komori, S.; Itagaki, M.; Unno, T.; Ohashi, H. Europ. J. Pharmacol., 1995, 277, 173-180. [485] Chavis, P.; Fagni, L.; Lansman, J. B.; Bockaert, J. Nature, 1996, 382, 719-722. [4861 Pessah, I. N.; Stambuk, R. A.; Casida, J. E. Molec. Pharmacol., 1987, 31, 232238. [4871 Lindsay, A. R. G; Williams, A. J. Biochim. Biophys. Acta, 1991, 1064, 89-102. [488] Zhang, Z.-D.; Kwan, C.-Y.; Daniel, E. E. Biochem. Biophys. Res. Commun., 1993, 194, 1242-1247. [489] McPherson, P. S.; Campbell, K. P. J. Biol. Chem., 1993, 268, 19785-19790. [4901 Nakazato, Y.; Hayashi, H.; Teraoka, H. Brit. J. Pharmacol., 1992, 105, 597602.
ALKALOIDSIN ANIMALS
53
[491] Shoshan-Barmatz, V.; Pressley, T. A.; Higham, S.; Kraus-Friedmann, N. Biochem. J., 1991, 276, 41-46. [4921 Kunz, W. S.; Kuznetsov, A. V.; Gellerich, F. N.FEBS Lett., 1993, 323, 188190. [493] Khuchua, Z.; Belikova, Y.; Kuznetsov, A. V.; Gellerich, F. N.; Schild, L.; Neumann, H. W.; Kunz, W. S. Biochim. Biophys. Acta, 1994, 1188, 373-379. [494] Islam, M. S.; Larsson, O.; Niisson, T.; Berggren, P.-O. Biochem. J., 1995, 306, 679-686. [495] Paolisso, G; Nenquin, M.; Schmeer, W.; Mathot, F.; Meissner, H. P.; Henquin, J. C. Biochem. Pharmacol., 1985, 34, 2355-2361. [4961 McNulty, T. J; Taylor, C. W. Biochem. J., 1993, 291, 799-801. [497] Griffin, D. S; Segall, H. J. Biochem. l~armacol., 1989, 38, 391-397. [498] Mancilla, E; Rojas, E. FEBS Lett., 1990, 260, 105-108. [499] Freedman, J. E; Weight, F. F. Europ. J. Pharmacol., 1989, 164, 341-346. [5OOl Clark, R. B.; Sanchez-Chapula, J.; Salinas-Stefanon, E.; Duff, H. J; Giles, W. R. Brit. J. Pharmacol., 1995, 115, 335-343. [5011 G6gelein, H; Capek, K. Biochim. Biophys. Acta, 1990, 1027, 191-198. [5021 Sabath, D. E.; Monos, D. S.; Lee, S. C.; Deutsch, C.; Prystowsky, M. B. Proc. Nat. Acad. Sci. U. S., 1986, 83, 4739-4743. [5031 Pe|assy, C; Aussei, C. Pharmacology, 1993, 47, 28-35. [504] Wang, G; Lemos, J. R. Life Sci., 1995, 56, 295-306. [5051 Needham, L.; Dodd, N. J. F.; Houslay, M. D. Biochim. Biophys. Acta, 1987, 899, 44-50. [5061 Suwalsky, M.; Villena, F.; Bagnara, M.; Sotomayor, C. P. Z. Naturforsch., 1995, 50c, 527-534. [507] D'Oc6n, P.; Bl~zquez, M. A.; Bermejo, A.; Anselmi, E. J. Pharm. Pharmacol., 1992, 44, 579-582. [508] Low, A. M.; Berdik, M.; Sormaz, L.; Gataiance, S.; Buchanan, M. R.; Kwan, C. Y.; Daniel, E. E. Life Sci., 1996, 58, 2327-2335. [509] Xu, Y; Zhang, S. Biochem. Biophys. Res. Commun., 1986, 140, 461-467. [51o] Michalska, L.; Nagel, G.; Swiniarski, E.; Zydowo, M. M. Gen. Pharmacol., 1985, 16, 69-70. [5111 Knaus, H.-G; 11 Others Biochemistry, 1994, 33, 5819-5828. [512] Yano, S; Horiuchi, H.; Horie, S.; Aimi, N.; Sakai, S.; Watanabe, K. Planta Med., 1991, 57, 403-405. [513] Plant, T. D; Henquin, J. C. Brit. J. Pharmacol., 1990, 101, 115-120. [514] Wang, Y.-X.; Zheng, Y.-M.; Zhou, X.-B. Europ. J. Pharmacol., 1996, 316, 307- 315. [5151 Berlinck, R. G. S; Braekman, J. C.; Daloze, D.; Bruno, I.; Riccio, R.; Ferri, S.; Spampinato, S.; Speroni, E. J. Nat. Prod., 1993, 56, 1007-1015. [516] Rando, T. A.; Wang, G. K.; Strichartz, G. R. Molec. Pharmacol., 1986, 29, 467- 477. [517] Corbett, A. M; Vander Kok, M. A. Biochem. Biophys. Res. Commun., 1994, 199, 1305-1312. [518] Dumbacher, J. P.; Beehler, B. M.; Spandr T. F.; Garraffo, H. M.; Daly, J. W. Science, 1992, 258, 799-801. [519] Elenu, N.; Botana, L.; Espinosa, J. Biochem. Pharmacol., 1988, 3 7, 3523-3525. [520] Iwata, H.; Ohta, A.; Baba, A. Jap. J. Pharmacol., 1986, 41,293-297. [521] Kusaka, M; Sperelakis, N. Europ. J. Pharmacol., 1994, 271, 387-393.
54
T. ROBINSON
[522] Pauwels, P. J.; Van Assouw, H. P.; Peeters, L.; Leysen, J. E. J. Pharmacol. Exp. Therap., 1990, 255, 1117-1122. [523] Jacobson, K. A; Guay-Broder, C.; van galen, P. J. M.; Gallo-Rodriguez, C.; Melman, N.; Jacobson, M. A.; Eidelman, O.; Pollard, H. B. Biochemistry, 1995, 34, 9088-9094. [524] Koob, G. F; Bloom, F. E. Science, 1988, 242, 715-723. [525] Altura, B. M.; Zhang, A.; Cheng, T. P.-O.; Altura, B. T. Europ. J. Pharmacol., 1993, 246, 299-301. [526] He, G.-Q.; Zhang, A.; Altura, B. T.; Altura, B. M. d. Pharmacol. Exper. Therap., 1994, 268, 1532-1539. [527] Crumb, W. J.; Jr; Clarkson, C. W. d. Pharmacol. Expt. Therap., 1995, 274, 1228- 1237. [528] Kaminsky, S. M.; Levy, O.; Garry, M. T.; Carrasco, N. Europ. d. Biochem., 1991, 200, 203-207. [529] Young, J. D.; Mason, D. K.; Fincham, D. A. d. Biol. Chem., 1988, 263, 140143. [5301 Steinfelder, H. J; PethO-Schramm, S. Biochem. Pharmacol., 1990, 40, 11541157. [531] Blankemeyer, J. T.; Atherton, T.; Friedman, M. d. Agric. Food Chem., 1995, 43, 636-639. [532] Blankemeyer, J. T.; Stringer, B. K.; Rayburn, J. R.; Bantle, J. A.; Friedman, M. d. Agric. Food Chem., 1992, 40, 2022-2025.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22
55
9 2000 Elsevier Science B.V. All rights reserved
USING CHEMICAL ECOLOGY TO LOCATE NEW ANTIFUNGAL NATURAL PRODUCTS S T E P H A N I E J. E C K E R M A N and K A T E J. G R A H A M *
Chemistry Department College of St. Benedict~St. John's University 3 7 S. College Avenue St. Joseph, MN 56374, USA ABSTRACT: The quest for new antifungal drugs is critical for several reasons. Immune suppression causes susceptibility to fungal infections. The number of immune-suppressed individuals continues to rise as society is faced with an aging population, an increase in AIDS infected patients, and medical advances. Most drugs used to treat mycological infections have low bioavailability or are too toxic for prolonged use. Also, many new fungal strains are emerging with drug resistance as fungal pathogens are exposed to extended pharmaceutical treatment. As the need for new antifungal drugs continues to rise, chemical ecology appears to be an attractive tool for identification of such compounds. In competitive ecosystems, it is generally accepted that many organisms thrive because they produce secondary metabolites providing a selective advantage over competing organisms. Biorational criteria, in this context, meansusing the ecology of natural systems to reveal organic chemicals with specific bioactivities. By employing biorational criteria in selecting sources, potential drugs can be more effectively located. Therefore, biorationale predicts organisms encountering fungal competitors or pathogens will be a good source of fungistatic or fungicidal chemicals. Ecological clues point to a variety of sources which are expected to produce fungistatic secondary metabolites. Examples of such sources include antagonistic fungi, plants with fungal pathogens, and mycoparasites. Studies of antagonistic species can provide useful information for scientists interested in chemical ecology, but can also serve as a valuable complement to random, high-throughput screening for new bioactive compounds. THE N E E D F O R N E W A N T I F U N G A L D R U G S With the advent o f the antibiotic era, the possibility was raised that infectious diseases would be eradicated altogether. However, infections still remain the leading cause o f death worldwide [1]. N e w infectious diseases are continually being identified and, in addition, many k n o w n pathogens which were under control are again becoming health problems [1]. Elimination o f these infectious diseases has been hindered for several reasons [1 ]: 1) new human pathogens are being discovered and transmitted worldwide; 2) known microbes are mutating to form new, more virulent forms; 3) microbes are able to develop resistance to antibiotics; 4) new, stronger pharmaceutical agents have the side effect of decreasing the host's resistance; 5) the life o f an individual suffering from a chronic disease can
56
ECKERMAN and GRAHAM
be extended resulting in a suppressed immune system and 6) there is a lack of effective, non-toxic pharmaceutical treatments. Of particular interest are the last three reasons; the side effect of these aspects of modern medicine is a group of individuals who are more susceptible to infections. These individuals are usually immunocompromised and, thus, are susceptible to pathogens and even to microorganisms that would not typically attack a healthy individual. Microbes that are innocuous in the immunologically intact host can cause devastating infections in patients with compromised immune systems; organisms causing these infections are termed opportunists [ 1]. Opportunistic infections can be caused by all major groups of microbes, but opportunistic fungal infections assume particular medical significance because they are difficult to treat and can sometimes be fatal. The majority of mycoses in healthy individuals are infections of the skin and adjacent mucous membranes. Topical fungal infections are extremely common, highly refractory to therapy and tend to relapse, but these infections are rarely life-threatening. However, systemic opportunistic infections can be severe and life-threatening situations. These systemic opportunistic infections are becoming increasingly more frequent and, unfortunately, are difficult to treat due to a paucity of available antifungal drugs [2]. Many situations can lead to the development of an opportunistic fungal infection. Continued treatment with broad-spectrum antibiotics or hormones commonly causes an imbalance in the microecology of the respiratory and gastrointestinal tracts leading to fungal colonization. Immune suppression also causes susceptibility to fungal infections. The number of immune-suppressed individuals continues to rise as society is faced with an aging population, an increase in AIDS infected patients, and medical advances including tumor treatments and organ transplants. Cytotoxic therapies used in the treatment of malignant diseases have improved the prognosis of such patients; however, they have led to an increase in the frequency of invasive fungal infections by decreasing the host's resistance. In a traumatic accident resulting in an open wound or bum, the patient often has a reduced immune response and can develop a fungal infection. In addition, patients requiring treatment involving catheters, prostheses or other invasive devices are prone to fungal infections. These clinical situations have been reported throughout the medical literature [3-15]. Some of the causal agents of these opportunistic infections are wellknown opportunistic mycoses. However, an increasing number of mycoses are being caused by fungi not previously known in human medicine. These organisms include soil fungi, plant and insect pathogens, and saprophytic fungi. In fact, it appears that virtually any fungus can infect an individual with a compromised immune system. The numbers and types of opportunistic fungi isolated from patients are increasing
ANTIFUNGAL NATURAL PRODUCTS
57
rapidly. It is likely that this trend will continue as long as the numbers of immunocompromised patients continues to grow [ 16]. The most common fungus causing opportunistic infections is the yeast Candida [1,17,18]. The yeast's commensualistic relationship with humans enable it to multiply and replace much of the normal flora when environmental conditions are favorable. Yeast infections by Candida species have been increasing dramatically ill the last decade and there are now as many as seventeen different species known to cause infections in humans [17,18]. The most frequently isolated species are C. albicans, C. tropicalis, C. glabrata (formerly known as Torulopsis glabrata), C. parapsilosis, C. guilliermondii, and C. krusei [ 17-20]. Cryptococcus is the most common systemic fungal infection in AIDS patients and is usually in the form of meningitis due to C. neoformans. Cryptococcal meningitis, if untreated, progresses rapidly to death. Even with treatment, mortality rates remain unacceptably high at-~50% [21 ]. Other significant, but less frequent, opportunistic fungal pathogens that cause life-threatening disseminated mycoses include Aspergillus, Histoplasma, Coccidioides, and Fusarium. Aspergillosis can occur as a non-invasive infection of the respiratory tract in immunocompetent hosts; however, in immunosuppressed individuals, it can become an invasive disseminated infection which is often lethal. Histoplasma capsulatum and Coccidioides immitis are pathogens of immunocompetent hosts as well. Again, in immunosuppressed hosts, these pathogens can cause deadly systemic infections. Fusarium has only recently been noted as an opportunistic infection in immunosuppressed individuals and appears to have an intrinsic resistance to many antifungal agents [21 ]. In addition, the number of new pathogens and previously rare organisms being isolated from patients is increasing rapidly [17]. These organisms can vary greatly in pathology and in their response to current antifungal therapy. This situation poses considerable difficulty to medical practitioners who have had little training in mycology. As more eases are reported, physicians and the clinical microbiology laboratory must become better prepared to identify and treat fungal infections from a variety of new and emerging yeasts. Treatment of systemic mycoses is further complicated because of the discrepancy between the variety and severity of infections and availability of possible therapeutics. Until recently, there was a perception that lifethreatening fungal infections were too rare to require the attention of pharmaceutical companies. While this view has slowly been discounted, the number of antifungal drugs on the market remains limited. The development of antifungal drugs has also been hampered by the fact that fungi are eukaryotes, making it more difficult to develop drugs that inhibit fungal growth but are not toxic to the human patient. While the number of pathogens has been steadily increasing, there are still only four major classes of antifungal drugs currently available"
58
ECKERMAN and GRAHAM
polyene macrolides, azoles, fluoropyrimidines and allylamines [21-25]. The mode of action of these antifungal agents has been reviewed by Kerridge [26]. These classes of drugs are described in more detail below.
Polyene Macrolides The polyene macrolides, including Amphotericin B, 1, and Nystatin, 2, are fungicidal agents. These drugs have worked well for systemic mycoses, OH
s,,...~,~
~
HO~...,,,
OH
OH
OH
O
OH
H
OH
H2~
%, H O-
) ._
O
OH
OH
OH
OH
H3C--k QH
.o--X-x-A:y\ i H2N~
CO2H
'
but extensive attempts at chemical modification of the drug have been unable to reduce the nephrotoxicity of the polyene macrolides. There are over 200 antifungal agents which belong to this chemical class; all are structurally similar and have a common mechanism of action. Polyene antibiotics bind sterols in the fungal cell membrane resulting in disruption of cellular integrity [21]. Due to the severe toxicity of Amphotericin B, efforts have been made to utilize this drug in combination with other antifungal agents [21,22]. While these synergistic drug interactions are documented, so are many contraindications with other pharmaceutical agents such as cyclosporin, aminoglycoside antibiotics, digitalis glycosides, and neuromuscular blockers. Increased bioavailability and
ANTIFUNGALNATURALPRODUCTS
59
decreased toxicity of drugs of this class have also been achieved with either liposomal preparations or colloidal suspensions of the drug complexed with other constituents of cell membranes, thereby allowing intravenous administration in tolerable therapeutic doses. Unfortunately, new data suggests that resistance to Amphotericin B among new and emerging pathogens is becoming significant [23, 24]. Azoles
The synthetic imidazoles were introduced more than ten years ago as broad-spectrum antifungal agents for topical use [21-23]. The drugs of this class work by inhibition of the cytochrome P450-1inked monooxygenase component of C-14 lanosterol demethylase which catalyzes a key step in the biosynthesis of ergosterol, the main sterol in fungi. Ergosterol is an irreplaceable constituent in fungal cellular membrans and is, therefore, required for cell proliferation. Unfortunately, an isozyme of the cytochrome P450 is also a prerequisite in the synthesis of cholesterol, the main sterol in mammalian cells.
clO CI
i 3
S > OOCH3
0
\
60
ECKERMAN and GRAHAM
Ketoconazole, 3, is a member of the azole class of antifungals and was the first orally absorbed antifungal agent for treatment of mycoses. However, it was found to be mostly ineffective for disseminated mycoses in immunocompromised individuals. Structure-activity relationship studies led to the development of the newer, less toxic triazoles. The newer triazoles have a higher specificity for the fungal enzyme than the related human enzyme which leads to lower toxicity. In addition, these compounds have been designed to have a higher bioavailability. Fluconazole, 4, has been introduced on the US market for a number of years and is indicated for dermal and vaginal mycoses, cryptococcal meningitis and mucocutaneous candidiasis. Given orally, it exhibits little toxicity and is generally well tolerated. A large number of related triazoles are in various stages of development or clinical use. Itraconazole, 5, is the most recently approved azole derivative in clinical use and appears to be active against a number of disseminated mycoses for which fluconazole has had limited success such as aspergillosis, histoplasmosis, blastomycosis, coccidiodomycosis and sporotrichosis [27-29]. In addition, there are at least two new azole derivatives currently in clinical trials; Voriconizole, made by Pfizer, and Schering-Plough's SCH 56592 are both reported to be active against fluconazole-resistant infections.
5-fluoropyrimidines Flueytosine (5-fluoroeytosine), 6, is a synthetic nueleoside that is converted intraeellularly to 5-fluorouraeil which, consequently, interferes with protein synthesis [22]. Although this drug is indicated for disseminated cryptococcosis and disseminated candidiases, flucytosine is rarely used alone due to substantial resistance developed by many opportunistic fungal pathogens. It also has the side effect of suppressing bone marrow production which is particularly problematic in AIDS patients. Flucytosine is sometimes used in combination with amphotericin B to suppress the rapid development of resistance to the flueytosine, but the toxicity appears to increase dramatically in these circumstances [21 ].
O
F NH 2
ANTIFUNGAL NATURAL PRODUCTS
61
Allylamines One of the first allylamine antimycotics reported, naftifine, 7, was shown to be a broad-spectrum antifungal agent. The allylamines inhibit the enzyme squalene epoxidase which catalyzes a key step in the ergosterol biosynthesis pathway. SARs of naftifine led to the development of terbinafine, 8 [22]. Terbinafine has found widespread clinical use in both oral and topical therapy of fungal infections of the skin, nails and hair. Terbinafine is active against a wide range of fungal pathogens, but is exceptionally potent against dermatophytes including B. dermatitidis, H. capsulatum, S. schenkii, and T. mentagrophytes [22]. In contrast, C. albicans is much less susceptible to in vitro doses of terbinafine. Terbinafine has generally shown poor activity for systemic mycoses, although there is some evidence that this drug may have application for treatment of some systemic mycoses [25]. Efficacy has been observed in sporotrichosis and there are some reports of successful therapy in cases of aspergillosis. An additional potential clinical use is in combination with other mycotics such as the azoles [25].
7
B
The increasing reports of fungal resistance to the available antifungal drugs described above is particularly troublesome in light of the variety and growing incidents of opportunistic infections [30-37]. With only a limited number of effective antifungal drugs to treat such infections, microbes are able to develop resistance mechanisms which undermine the utility of the currently available drugs. Recent studies have found examples of fungal resistance to agents such as flucytosine, amphotericin B and many of the azoles [30-37]. One current area of research is the investigation of fungal resistance mechanisms [30-32]. Several pharmaceutical companies are attempting to circumvent the resistance mechanisms by developing new agents with different modes of action.
62
ECKERMAN and
GRAHAM
DEVELOPMENT OF NEW ANTIFUNGAL DRUGS Progress is continuing in the study of existing agents and the development of new potential drugs. Further research in the biochemistry of fungi will provide novel molecular targets in the fungal cell and molecular bioassays for the identification of new lead compounds. One strategy used to develop new agents is to inhibit processes or enzymes unique to the fungus. Koltin [23] has reviewed some potential future antifungal targets. In addition, many pharmaceutical companies are exploring compounds that target enzymes and structures needed to make or maintain the fungal cell wall [29]. Pneumocandins and echinocandins compose a new class of cyclic lipopeptide antifungal compounds [38]. Echinocandin B, 9, inhibits 1,3-[3D-glucan synthase in C. albicans, a critical enzyme in the production of cell well components. Two semi-synthetic derivatives have entered clinical trials. Merck's L-724,872 has been well tolerated in phase I clinical studies and is currently in phase II trials in patients with HIV and candidiasis. However, it is not absorbed orally and must be delivered intravenously. Lilly's LY303366 is also currently in clinical trials with people with HIV and candidiasis. These compounds appear to have fungicidal activity yet are not as toxic as Amphotericin B [21, 29, 30].
H OH
.Q [i
H HH~.'~
/
/ - - \/
HO ~
OH ."
~r'-'- 0
, ~ ' ~ , , r I O "Ltn~176
3t::~0 H/
o.
,.Me
IT "X,,"X"OH -.
/ HO 9
Benanomicin A, 10, and B, 11, and Pradimicin A, 12, have recently been isolated from soil bacteria [39]. These compounds appear to bind calcium-dependent mannan causing disruption of the cell membrane.
ANTIFUNGAL NATURAL PRODUCTS
63
Bristol-Meyers has developed 200 analogs of compounds in this class. Due to potential liver toxicity, the compounds have not yet entered clinical trials [21, 29, 30]. 3)CO2H
H I0 Benanomicin A I I Benanomicln B 12 P r a d i m i c l n A OH
R = OH R = NH 2 R = NHCH 3
OH
O
O
OH
O
The polyoxins and nikkomycins are products of soil Streptomycetes and appear to inhibit chitin synthetases 1 and 2 [40]. Chitin is an important structure in the fungal cell wall. Shaman Pharmaceuticals is currently exploring the development of Nikkomycin Z and related analogs, 13-15, [29]. Polyoxins and nikkomycins have not yet proven to be clinically useful drugs because the compounds are unable to penetrate fungi in the environment of the human body; however, structure-activity relationship studies of these classes of compounds or new inhibitors of chitin synthetase may still yield a successful antifungal drug. HO
OH
HNL
NH 2 HO
OH _CHO
R=
HN
HN
HN j
or
HO
OH
m
13 Nikkomycin Z
14 Nikkomycin X
1 5 Polyoxln C
64
ECKERMAN and GRAHAM
The overall situation is consequemly far from favorable, and there is an urgent need for further development of more classes of antifungal drugs. Obviously, these new drugs could reduce the incidence of resistance and provide feasible options for instances when such resistance arises. The ultimate goal is to develop non-toxic, well-tolerated antifungal medications with a high efficacy rate and no resistance. Moreover, a strong point should be made that efficacy and tolerance correlate inversely with the breadth of the spectrum of activity [2]. However, pharmaceutical companies appear to ignore this relationship and continue to develop antifungal compounds with as broad a spectrum of activity as possible so as to increase the use and money generated by their drug. Such a demand also ignores practical medical necessities, as mixed infections do not usually occur in systemic mycoses. The trend of future development in the field of antifungal agents should lead in the direction of narrow spectrum antifungal agents. SEARCH FOR NEW POTENTIAL ANTIFUNGAL LEADS Past and current research to develop new, clinically effective antifungal drugs has focused on four approaches [21 ]" 1) structure-activity relationship studies of known antifungal agents; 2) combination therapy of existing drugs; 3) delivery systems for existing drugs and 4) discovery of new prototype antibiotics. Only the last strategy is a potential source of new lead compounds. Given the need to identify new leads for effective antifungal drug development, natural products are a prime source for the discovery of new lead compounds [41]. It has been estimated that only 5-15% of higher plants have been systematically investigated for the presence of bioactive compounds, while even fewer marine and fungal sources have been explored [42, 43, 44]. A recent review article [41 ] estimated that the new approved antibacterial and anti-infective drugs reported between 1983 and 1994 are predominantly from natural sources or are modeled on a natural product prototype. The data reveal the essential role played by natural products in the discovery of drugs for a variety of purposes; discovery of novel antifungal compounds from natural sources is clearly a feasible and productive approach. In addition, natural products present a virtually limitless potential for secondary metabolic diversity. Several strategies and philosophies are available for the identification of bioactive compounds from natural sources. Different aspects of these strategies have been reviewed [44-47]. Several such approaches have been used previously for this purpose" 1) ethnobotany, 2) serendipity, 3) random sampling, and 4) exploitation of chemotaxonomic knowledge. More than 80% of the world's population use plants as their primary source of medicinal agents [45], leading to a well-established system of traditional medicine. Some of these remedies are well documented and are
ANTIFUNGAL NATURAL PRODUCTS
65
either commercially available as the traditional preparation or have been exploited in the development of a pharmaceutical drug [41]. Recent interest has been shown in the preservation and collection of traditional medicinal lore, in light of the increasing loss of biodiversity and native cultures. The area of science which has been involved in the discovery of biologically active natural products from plants used in traditional medicine is ethnobotany. At the outset, ethnobotany requires intensive studies in epidemiology, traditional medicine, language, culture and ecology of a people and their environment. Then, botanists and medical doctors must work together in presenting specific disease descriptions and identifying the traditional healers' treatments. Once a plant and its medicinal preparation have been identified, a sustainable supply and policies for economic development of the potential drug must be established. Finally, the isolation and structure determination work can be done, followed by the clinical evaluation of the biologically active component [45, 47]. The ethnobotanieal strategy for uncovering bioactive compounds may eventually yield a novel antifungal compound, but the attempt can be a difficult and expensive endeavor. Some natural product chemists are interested in the isolation and characterization of novel compounds without any realistic expectation that the compounds will be developed for drug use. This approach, "serendipity", involves the selection of organisms to be analyzed by their potential to yield interesting chemicals; whether or not the organisms have known bioactivity is of little concern [46]. Typically, an exhaustive analysis of a plant sample for all types of secondary metabolites is performed. The result of this method is many new compounds with no known bioactivity. Biological activity is sometimes subsequently discovered in these libraries of purified chemicals. Many pharmaceutical companies resort to random sampling in the search for new structural types with potential biological activity [48]. This approach simply involves screening as many organisms as possible through a wide variety of biological assays. The goal is to acquire biodiversity in order to broaden the chemical diversity. For most pharmaceutical companies, the source organism is irrelevant until a lead has been identified. High-throughput screening can be a rapid and effective method for lead development for large companies that have automated systems and the ability to purchase thousands of samples on a regular basis. Typically, the rate of discovery of potential leads is very low so this method is not feasible for smaller labs. Once a chemical structural type has been identified with a specific biological activity, the compound must be isolated from the source or synthesized for further studies. If the lead compound is not available due to an ineffective synthesis or a limited source, then a search for alternative source organisms can be initiated. Organisms of similar taxa often produce a similar distribution of natural products. Exploiting this chemotaxonomic
66
ECKERMAN and GRAIIAM
knowledge can aid in the search for new sources of known compounds or in the attempt to augment the collection of structures that are currently available for analysis. However, in order for exploitation of chemotaxonomic knowledge to be effective, immediate access to several plant species is imperative [45, 46]. These approaches for identifying novel drug leads have all been shown to be effective. These efforts should and will continue. However, there are some benefits to be gained from the expansion of these approaches to include ecology-based searches for efficient location of biologically active molecules. CHEMICAL ECOLOGY As the need for novel antifungal compounds continues to rise, ecologybased strategies are becoming more prevalent. Chemical ecology is the study of chemical interactions between organisms and their environment, including other organisms. In competitive ecosystems, it is generally accepted that many organisms thrive because they produce chemicals which provide a selective advantage over competing organisms. Secondary metabolites are used by all species for such uses as deterring enemies, fending off pathogens, competing for nutrients, protecting against physical hazards of the environment and sexual selection. Chemical ecology is a multidisciplinary field that involves both isolation and characterization of chemicals that mediate environmental interactions and an understanding of the biological mechanisms for chemical signal recognition and transduction. Major progress has been made in chemical ecology in recent decades and ecology-based approaches have recently received attention as a potential strategy to search for biologically active compounds [49]. This reflects the highly improved chemical techniques for isolating and characterizing very small quantities. The increased sensitivity is particularly important because of the very low levels of secondary metabolites produced by most organisms. In addition, progress in sociobiology, ecology, and evolutionary biology has helped to provide new understandings of interspecies interactions. For example, biological monitoring of ecological systems has made it easier to study variation in source organisms. Given that chemical activities are often seasonal and sporadic, an understanding of an ecosystem would allow organisms to be chosen for chemical study under the correct circumstances and season. This approach should result in a more efficient survey of the potential secondary metabolites than that of a random screening program. For example, the leafcutter ant, Atta cephalotes, cultivates a fungus for food. The leafcutter ant has evolved to avoid leaves of Hymenaea courbaril, which produces a terpenoid that is toxic to the fungus [50]. However, the plant shows a dramatic decline in the production of this terpenoid during the latter half of the wet season just
ANTIFUNGAL NATURAL PRODUCTS
67
before the dry season. This decline is probably due to a reduction in the synthesis of antifungal compounds in the dry season, when the risk of fungal attack is low. Random screening may not have led to the discovery of this potential antifungal agent, but an understanding of the ecology of the ant and its feeding patterns would have clearly indicated the season for collecting leaves that would contain reasonable quantities of antifungal compounds [50-53]. New advances in biochemistry and molecular biology may also promote the use of ecology-based searches for medicinal agents. Understanding the biochemical basis of an ecological interaction can lead to drug development as evidenced in an excellent review by Caporale [48]. For example, medicinal chemists are interested in locating inhibitors of the enzyme hydroxymethylglutaryl (HMG)-CoA reductase as inhibitors can lead to lower plasma cholesterol levels. HMG-CoA reductase catalyzes the conversion of acetyl-CoA to mevalonate, a key step in sterol biosynthesis. It is also known that mevalonate can overcome catabolite repression of gibberellin synthesis. Gibberellins are plant growth hormones produced by a phytopathogenic fungus, Gibberella fujikuroi. Thus, an inhibitor of HMG-CoA reductase may be involved in the regulation of the biosynthetic pathway of gibberellins. A chemical ecologist might have suggested screening such phytopathogenic fungi to locate inhibitors of HMG-CoA reductase [48,54]. Drug discovery targets can also be suggested by the study of organisms that have evolved the ability to regulate our biochemistry. Parasites and viruses often manipulate regulatory steps in host defenses [48, 55]. For example, some viruses are able to override cell death programs that otherwise eliminate virus-infected cells [48]. Understanding the biochemical control mechanisms of cell death can lead to the ability to save neurons in neurodegenerative diseases or kill cells in a tumor. Other organisms, such as leeches and ticks, are good sources of agents that selectively block the action of coagulation proteases [48]. Random screening may locate a compound, such as a coagulation protease inhibitor or an inhibitor of cell death, which has evolved to interact with the target of the screen. However, since diverse organisms share a great deal of biochemical structures and pathways, it is not surprising that small molecules from species as distant as plants, fungi, and bacteria can interact with macromolecules in humans. It may be that an interaction is coincidental; however, a bioactive natural product that interacts with a human protein may have evolved to interact with a homologous protein domain or active site in the organism in which it was made or an organism with which it is interacting. In this case, the definition of chemical ecology can be extended to include any study of molecular information transfer in biological systems. In an article on signal transduction by Clardy [56], it is argued that chemical ecology can include the effect that extracellular molecules can have on intracellular processes.
68
ECKERMAN and GRAHAM
Similarity in signaling pathways in different biological systems allows for the use of the secondary metabolites to probe cellular signaling. These studies can subsequently lead to the development of pharmaceutical agents [56]. For example, tetrandrine is a human L-type calcium channel blocker used to treat angina and hypertension isolated from a Chinese herb, Stephania tetrandra [57]. Evidence suggests that tetrandrine was produced to control the calcium channels within the original plant [48, 58]. The apparent homology of the receptors in animal and plant membranes suggests that knowledge of substrate-receptor interactions in other biological systems could lead to the development of new pharmaceuticals. Studying the diverse interspecies interactions revealed by chemical ecology may lead to the identification of key points of biochemical regulation. Molecular mechanisms have evolved that enlist and modify useful structures for new purposes. A clear understanding of the structure of a drug discovery target and its relationship to other proteins will lead to a more efficient search for potent and selective drugs. This understanding will come only from the detailed study of organisms in complex environmental settings and an increased attention to identifying the potential use of secondary metabolites as drugs. ECOLOGY-BASED
SEARCHES TO LOCATE SOURCES OF
ANTIFUNGAL AGENTS Evidence suggests that most secondary metabolites exist to protect an organism from pathogens or predators [47]. Thus, an understanding of chemical ecology appears to be a reasonable and efficient approach, particularly in the discovery of anti-infective or antifeedant compounds. Biorational criteria, using the ecology of natural systems to reveal organic compounds with antifungal bioactivities, is the underlying theme of this approach [55]. Ecological clues point to a variety of sources which are expected to produce fungistatic secondary metabolites and become an obvious target for antifungal research. Therefore, biorationale predicts organisms encountering fungal competitors or pathogens will be a good source of fungistatic or fungicidal chemicals. Studies of antagonistic species can provide useful information for scientists interested in chemical ecology, but can also serve as a valuable complement to random, high-throughput screening for new antifimgal compounds. NEW ANTIFUNGAL COMPOUNDS LOCATED BY CHEMICAL ECOLOGY
Several antifungal compounds which are capable of producing the above effects have been isolated and characterized using an ecology-based
ANTIFUNGAL NATURAL PRODUCTS
69
approach. The following section is not intended to be an exhaustive list of antifungal drugs from natural sources but is simply a general overview of the applications of chemical ecology in the search for antimycotics.
Fungi Research has shown fungi to be excellent sources of novel bioactive metabolites which have been insufficiently explored [59, 43, 60]. Mechanisms of fungal antagonism and defense often include the production of biologically active metabolites by one species that exert effects on potential competitors and/or predators. Fungi chosen for investigation based on ecological considerations, such as evidence of interspecies antagonism, will likely be good sources of fungistatic or fungicidal chemicals [61-64]. In fact, the pneumocandins discussed earlier were isolated from the fungus Zalerion arboricola [38, 65]. These compounds appear to be the causative agents of antagonistic effects of Zalerion arboricola against a competing fungus on its natural substrate. Examples of antagonistic fungi include coprophilous [66, 67], aquatic [6870], lignicolous [71, 72], and saprophytic fungi [73, 74]. H CH
HO,,
HO HO
~
O
H
H3C
0". H. - 0
O
16
17
HO
v
18
H
HO
OH
H
OH 19
COOH OH NH2
E C K E R M A N and G R A H A M
70
Several reviews on the use of coprophilous fungi from competitive ecosystems as sources of antifungal natural products have been written by Gloer [61-63]. Coprophilous fungi are those which colonize the dung of herbivorous vertebrates. Many of these fungi are known to produce antifungal agents that inhibit the growth of competitors. Merck has reported several broad-spectrum antifungal agents from common coprophilous fungal species: australifungin, 16, from Sporormiella australis [75]; the sonomilides, 17, [76]; the zaragozic acids (e.g. 18), isolates from Sporormiella intermedia [77] and the sphingofungins, 19, from Paecilomyces variotti [78, 79]. Gloer's research group has also described several novel secondary metabolites isolated from coprophilous fungi with antifungal activity (e.g. 20-32) [80-89]. Given the high incidence of antimycotic activity in this category of fungi, it seems likely that this is a reasonable source of antifungal lead compounds. In fact, many of the compounds isolated from these fungi have shown potent effects against other coprophilous fungi, but only a few have shown activity against medically relevant fungi. i
H Oso. ~
~H..
~
21
20
.... t,
.OH
o,,:::
I
22 m u
OH
J'"
n ~
23
0
J "eH
,,iT' 24 25
ANTIFUNGAL NATURAL PRODUCTS
71
OH
QH
"
oi~
26
27
Q....{ ~..~o 7;
~ CH3
|
~ CH3
28
~
H3
k o
OH H
OCH3
29
ao ~ OH .COOH
o ~.,."%~
o#'-,o. ~
31
ECKERMAN and GRAllAM
72
( /
T-o
0 31
OH m
OH
32 stachybotrin A 33 stachybotrin B
R = OH R = H
Studies of antagonistic aquatic fungi have also been revealed as a potential source of antifungal lead compounds. Review articles [68-70] discussing aquatic fungi indicate that very little research has been done on this category of fungi. Gloer has isolated a number of antifungal compounds from antagonistic aquatic fungi including stachybotrin A and B (32 and 33) [90], kirschsteinin, 34, [91] and anguillosporal, 35, [92]. The sesquiterpenedione culmorin, 36, [68] was isolated from the lignicolous marine fungus Leptosphaeria oraemaris and was identified as an agent responsible for interference competition between different marine fungi. A review by Shearer suggests that other lignicolous freshwater fungi could be sources of antifungal compounds [71 ].
ANTIFUNGAL NATURAL PRODUCTS
73
OCH a
OH
0
OH
0
34
HO
H
HO-
OH
35
36
Antagonistic interactions between wood-rotting fungi have been studied because fungal decay can drastically reduce the market value of lumber. Fungitoxic compounds produced by antagonistic lignicolous fungi were isolated by Ayer , 37, [92, 93] and Strunz, 38, [94]. While these compounds are of interest for production of lumber, there is little evidence as yet that these compounds will be active against medically relevant fimgi. H
H
O
H3C OH 37' Trichodermal
38
Scytalidin
74
ECKERMAN and GRAHAM
Mycoparasitism involves the invasion of hyphae or sclerotia of one fungal species by another. As part of this process, some mycoparasites produce antifungal compounds which damage host cells. In particular, mycoparasites of the pathogenic fungus, Aspergillus sp. would be predicted to be potential sources of antifungal drugs [96]. Recent investigations of mycoparasitic fungi by Gloer have resulted in several known antifungal metabolites as well as two new tetraketides, three new monocillin analogs and some new substituted benzoate esters [96, 97]. In terms of saprophytic fungi, Choudhury [98] reported the isolation of two new secondary metabolites, 39 and 40, from Sporothrixflocculosa and Sporothrix rugulosa, fungicolous hyphomycetes found on sporocarps of other fungi. Other examples of antagonism between saprophytes and pathogens in the phyllosphere has been reviewed by Blakeman [99]. It appears that the antagonistic interactions of saprophytes and plant pathogens are usually based on competition for nutrients.
39
0 OH
Chemical ecology of dimorphic fungi also promises to be a source of potential antifungal lead compounds. A common feature of the majority of human pathogenic fungi is that they exhibit dimorphism. Pathogenicity in C. albicans appears to be linked to its ability to switch rapidly between morphological forms: yeast cells (single celled) and mycelia [100]. This ability of C. albicans to alter its phenotype rapidly appears to allow the fungus to adapt to different host environments [101-105]. Because an understanding of the biochemical regulation would be helpful in the design of new antifungal drugs, the molecular mechanism for control of filament formation is being studied. A genetic pathway that regulates morphological transition between blastospore and filamentous forms in C. albicans has been discovered [106-108]. Recently, a new model has been developed whereby a transcriptional repressor, TUP1, represses the genes responsible for initiating filamentous growth. This repression is lifted under inducing environmental conditions [ 109, 110]. Many environmental variables such as temperature, media and ratio of CO2 to 02 are known to influence the morphological state of C. albicans; however, no extracellular signal for the control of reversion has been identified [100]. Our preliminary data [111] indicate that a compound secreted by the
ANTIFUNGAL NATURAL PRODUCTS
75
dimorphic fungus, Candida albicans, can trigger the dimorphic switch. These data suggest that biochemical control of fungal morphology is initiated by an extracellular signal as in the bacterial systems. An extracellular signaling compound responsible for biochemical regulation of dimorphism has been partially purified. Further purification and characterization is in progress. This finding could potentially lead to new antifungal drugs, and is a starting point for deeper understanding of the intracellular mechanism of dimorphism. This is a clear example of the response of an organism to chemical signals in the environment which could lead to the development of a potential pharmaceutical agent. Fungi have been shown to produce the requisite chemical diversity needed in the search for new drug leads. Other types of antagonistic fungi may well prove to be sources of drug leads [64, 112, 113]. More basic research in the area of ftmgal ecology and fungal biochemistry will clearly enable researchers to more effectively locate potential sources of new antifungal compounds. In addition, biorationale predicts fungal sources to be potential sources of other biologically active compounds such as insecticides, herbicides, pharmaceuticals from [61-63, 113]. Plants
Phytochemistry has proven to be fertile ground for antifungal drug discovery [42] and promises to continue to do so given the inherent chemical diversity and relevance of the chemical defense mechanisms in plants in response to fungal infections [114]. An overview of the research in this area is presented as much of this work has been reviewed many times. Plants synthesize and accumulate a broad variety of secondary metabolites to protect themselves from herbivores and infection from microbial pathogens [115, 116]. Compounds which inhibit the development of fungi and bacteria such as alkaloids, phenolics, or terpenoids are often accumulated on the external surface in leaf wax or trichomes [ 115, 116]. Cell walls of some plants have also been shown to contain antimicrobial proteins, such as thionins and defensins (see discussion later). Pathogens which do manage to penetrate these external barriers may encounter further deterrents, such as enzymes which can hydrolyze pathogen walls or biopolymers which restrict further pathogen development [ 115, 116]. It has been suggested that these enzymes, such as chitinases and glucanases, could be medically useful antifimgal drugs [117]. In addition, certain plant cells maintain high levels of antimicrobial secondary metabolites (constitutive chemicals) such as various phenols, flavenoids, alkaloids, coumarins, or cyanogenic glycosides [ 116, 118-120]. These products are often found in strategically located sites such as epidermal tissues or latex. Some of these products are activated by
76
ECKERMAN and GRAHAM
wounding; these compounds are often quickly oxidized to highly reactive antimicrobial phenols, quinones, and free radicals. After this rapid constitutive response to a pathogen, many plants begin to biosynthesize and accumulate low molecular weight, antimicrobial, lipophilic compounds (phytoalexins) in and around the site of infection. Screening different genotypes of one plant species with different degrees of resistance to a particular fungal pathogen has established a relationship between phytoalexin quantity and the extent of resistance [116, 121,122]. There are over 300 known phytoalexins with a striking amount of chemical diversity; phenolic acids, pyrones, flavenoids, pterocarpans, terpenoids and coumarins have all been reported as antimicrobial phytoalexins [118120]. In general, a plant may produce several phytoalexins in response to infection by a pathogen and many of these compounds are not otherwise found in the plant. Phytoalexins are not transported, so their protective effect is limited to the region of the infection and their synthesis is strictly regulated by the plant. Once the pathogen has been deterred, the phytoalexins are degraded; these compounds are thus transient and often do not show up in random screening [ 121,122]. Research on the chemical ecology of secondary metabolites in plants is highly relevant to the utilization of some of these substances in medicine. Over 250 antifungal metabolites originating from plants have been characterized since 1982 [118]. About half of these defense compounds are constitutive agents, while the other half are inducible phytoalexins. Antifungal compounds isolated from higher plants represent a wide variety of chemical classes including terpenoids, aromatics, aliphatics and alkaloids [118-120]. Since plants are known producers of antifungal compounds and because biorationale predicts that plants will produce defensive compounds against plant pathogens, continuing research on the characterization of phytoalexins produced in response to a fungal infection is likely to produce many novel antifungal compounds [123]. However, the success of drug discovery based on direct extraction of vegetative plant material is impacted by the availability of the plant source and the variability in phytochemistry due to genetic and/or metabolic regulation of secondary metabolism reflecting environmental conditions. The use of plant cell culture can address these issues by providing access to a broader array of phytochemicals from each plant species and creating a library of plant cell culture which can be grown and scaled up under controlled conditions whenever needed [124]. Genetic or environmental manipulation, hormones, or pathogen infection can be applied to the cell culture to induce phytochemical expression in the extract [125]. Recent developments in plant cell technology has allowed for the production of a number of plant products through cell culture. Shikonin, a red pigment produced by Mitsui and Co. from Lithosperm~m cells is a well known example of an industrial application of this approach [126].
ANTIFUNGAL NATURAL PRODUCTS
77
In particular, Phytcra, Inc. has worked on developing protocols to establish and manipulate plant cell suspension cultures derived from native plants to locate antifungal drug leads. This work has led to the characterization of several compounds, 41-45, [127] with antifungal activity produced by from plant cell culture. In addition, this same company recently reported the characterization of sunillin, an antifungal compound which has never been isolated from the native plant [128].
oX
H a C O J J ~j~ OCH 3
OCHa 42 Chelerythrine
41 Chelfl
)
,~o macarpme
HO
i (HOH2C • ~
~
HO"
]
OH
O v ~ ~ O H
44 Verbascoslde
OH OH H~ H(>
OH OH
OH OH
45 Myricoside
78
ECKERMAN and GRAHAM
Sunilllin was isolated from an extract of a plant cell culture in which the host defense pathways had been triggered [128]. The development of sunillin as a marketable drug is currently being explored. Plants have been shown to be producers of antifungal compounds as a chemical defense system against fungal pathogens. In particular, exploring the chemical response of a plant (either the native plant or the plant cell culture) to a pathogen or fungal cell compounds is an ecology-based approach for location of transient, plant-derived antifungal compounds.
Marine Invertebrates and Microorganisms Marine natural products research has resulted in the isolation of numerous diverse and novel chemical structures with potent biological activities [44, 68, 69, 128]. Extensive screening of marine invertebrates for antifungal activity has been carried out over the last three decades [129-130]. More than 100 antifungal metabolites have been isolated from marine invertebrates and some have been developed for clinical use. While biorationale would predict that some of the soft-bodied invertebrates such as sponges need chemical defenses against predators [131 ] and microbial pathogens, most of the known antifungal compounds were discovered using high-throughput screening. Very little work has been done using chemical ecology of the marine organisms in their environment to locate antifungal drugs. In fact, it should be emphasized that some of the compounds isolated from marine invertebrates are believed to be produced by symbiotic microorganisms such as blue-green algae, bacteria or fungi [ 129]. Recent research in marine microbiology has focused on a search for pharmacologically active compounds [132], but these efforts have been hindered by a lack of understanding of the ecology of these complex systems. Current research on the chemical ecology of marine macroorganisms and associated microorganisms will increase the understanding of the role of antifungal compounds and facilitate the search for new antifungal agents [69, 13 l, 132]. In a review by Paul and Fenical, the chemical ecology of tropical marine algae was examined [133]. Consideration of the chemical ecology led to the use of ecologically relevant bioassays. In particular, antimicrobial assays were performed against known strains of marine bacteria and fungi as well as the usual terrestrial pathogenic microorganisms. The results of this study indicate that many of the algal metabolites have antifungal activity, and some are active against medically relevant fungi [133]. In particular, the sesquiterpenoid, 46, from Penicillus capitatus and two metabolites of Halimeda, 47 and 48, showed activity against Candida albicans. This approach should provide more information about the possible antimicrobial roles of secondary metabolites in the marine environment.
ANTIFUNGAL NATURAL PRODUCTS
46
79
"OAc
Ac
Ac
OAc ~
~
CHO CHO~. CHO
H~ 48
Probably one of the most striking examples of the identification of antifungal compounds based on chemical ecology involves epibiosis. Marine microorganisms living on the surface of other organisms are called epibionts and play a significant role in the ecosystem [69]. Embryos of the shrimp Palaemon macrodactylus are resistant to infection by the fungus Lagenidium callinectes, a known pathogen of crustaceans. This resistance is due to the antifungal compounds 2,3-indolinone, 49, and tyrosol, 50 produced by bacteria isolated from the surface of the embryos [69, 134]. This observation suggest that aquatic plants and animals may be protected from pathogenic fungi by epibiontic bacteria. O
H
49
50
More research on the detailed interspecies interactions of marine and aquatic ecosystems are needed to improve our understanding of underwater habitats and to protect these ecosystems [135]. Recently, the coral reefs near Jamaica have been replaced by regions dominated by algae.
80
ECKERMAN and GRAIIAM
This change appears to be due to an imbalance of the ecosystem which allowed a pathogenic fungus to invade the natural reef population [135]. Somehow the natural antifungal protection was lost; the details of this development are unclear. Perhaps this protection was conferred by an epibiontic bacteria, although this explanation is speculative at this point. Only by preserving these ecosystems and this biodiversity can questions like this one be investigated. Bacteria
One of the first antifungal compounds to be used clinically was nystatin, 2 [ 136]. Nystatin was isolated by Brown and Hazen from the soil bacteria Streptomyces noursei. Since that time, more than 100 polyene macrolide antibiotics have been isolated from Streptomycetes, including Amphotericin B, 1, [136]. In addition, numerous other antifungal compounds have been discovered from soil bacteria and actinomycetes. For example, griseofulvin, 51, from Penicillium griseofulvum is used for treatment of topical mycoses. Cycloheximide, 52, is a powerful fungicide isolated from Streptomyces griseus which is too toxic for clinical use but has found utility as an agricultural fungicide. These bacteria continue to be valuable sources of chemically diverse antifungal agents, and major pharmaceutical companies continue to screen these bacteria as potential sources of antimycotics [137-141 ]. According to one study, at least one quarter of all actinomycetes produce antimicrobial metabolites [142]; it appears that the antagonistic interactions of the actinomycetes with soil fungi are usually based on competition for nutrients. Soil bacteria have been and continue to be among the most productive sources of diverse antifungal lead compounds, a fact which is not surprising given that an ecological niche must be shared with fungal competitors.
o.) . o
r 12
51
0
52
ANTIFUNGAL NATURAL PRODUCTS
81
In a system similar to the marine epibiontic bacteria, rhizobacteria appear to confer disease resistance to the host plants. For example, treatment of sugar beets with the rhizobacterium Pseudomonas in nonsterile soil has been shown to result in enhanced plant growth and severely reduced fungal root colonization (up to 62%) [143]. Rhizobacteria clearly may be sources of antifungal compounds for pharmaceutical or agricultural use.
Insects
Insects are a largely unexplored source of pharmaceutical agents. However, biorationale predicts that many insects could benefit from antibacterial and antifungal compounds in order to protect food stores as well as eggs and larvae. Myrmicacin, 53, isolated from the metathoric glands of leaf-cutting ants Atta sexdens and Atta cephalotes, prevents sprouting of intruding fungal spores in the nest. This is an important strategy because these ants cultivate a particular strain of fungus as a food source for larvae, and therefore need to maintain a pure fungal culture. The same compound is apparently used to protect seed stores by the harvest ant Messor barbarus [ 144].
~COOH 53
Leaf-cutting ants cultivate fungi by storing leaves in the nest on which the fungus will grow. Consequently, these ants present interesting probes for broad-spectrum antifungal compounds because they will avoid food sources which may endanger their fungus culture [50-53]. There are strong indications that epiphylls which deter ,4tta cephalotes from feeding on their host plants actually do so by producing antimycotics which could endanger the ants' fungus garden [145]. Another instance in which an insect species potentially employs antifungal compounds is illustrated by the American burying beetle, Nicrophorus americanus. As the name suggests, burying beetles inter small animal or bird carcasses in the soil to feed their larvae, and the carcass is coated with secretions to retard decay [ 146]. Presumably these secretions contain antibacterial and antifungal compounds. Currently research on this insect and the secretions it employs is hindered by the
82
ECKERMAN and GRAHAM
beetle's endangered status, but this point underscores the need to preserve biodiversity if we are to exploit the vast advantage evolution has had in developing antifungal compounds over millions of years. Antifungal compounds in insects are not limited to small molecules. For example, the larvae of many insects have been found to contain antimicrobial proteins, notably in cecropia moths [147]. Because these types of compounds have been shown to be ubiquitous, occurring in vertebrates as well as plants and insects, their discussion is relegated to a subsequent section. Vertebrates
In the past decade a number of vertebrate species have been shown to produce antifungal compounds. The dogfish shark, Squalus acanthius, faces potential problems from fungal infections of its reproductive system because sharks lack placentas and must flush their wombs with microbeladen sea water to remove fetal waste. Research into this system revealed that the shark produces a potent antimicrobial steroid, squalamine, 54, which is highly effective against Candida [148]. Sharks also produce a limited number of antibodies when compared to other vertebrates, and it may be that this deficit is alleviated through the use of antimicrobials such as squalamine.
CH, CH
""oH
H2 54
Amphibians generally require a moist environment, as do fungi, and consequently many amphibian species may benefit from antifungal compounds. The observation that African Clawed Frogs with wounds rarely became infected, despite an environment conducive to microbial propagation, led to the discovery of the magainans [149-152]. Magainans are polypeptide antimicrobial compounds which have since been observed in a wide variety of frog skins [153, 154].
ANTIFUNGAL NATURAL PRODUCTS
83
Antimicrobial Peptides There has been an explosion of recent interest in antimicrobial peptides, which have been shown to be ubiquitous in nature as evidenced in numerous review articles [155-159]. Frequently, these peptides have been located where microbial control is important, such as in mammalian intestines or trachea, in seminal fluid, on amphibian skin, as plant defense compounds or on insect larvae. Hence, such peptide antibiotics can be seen as a response to ecological conditions, specifically the propensity of microorganisms to thrive wherever they can gain access to a macroorganlsm. Antimicrobial peptides can be relatively low molecular weight compounds (3000 to 5000) such as cecropins, defensins and magainins, or higher molecular weight proteins such as lysozyme and serprocidins. Several of these compounds, including magainins, defensins, and the serprocidin, proteinase 3, have demonstrated potent antifungal activity. The diversity of these families of antimicrobials, which may stem from a need to overcome pathogen resistance, makes them a particularly bountiful source of pharmaceutical lead compounds. This area of research has garnered intense interest recently, with literally hundreds of articles being devoted to antimicrobial peptides in the last five years. CONCLUSION While chemical ecology will not replace other methods for locating sources of antifungal compounds, it is nevertheless an important and effective technique. Consideration of situations in which an organism would have a particular need to defend itself from fungi is a logical starting point from which to find leads for antimycotics. In several instances, questions about how an organism would cope with its environment have led directly to the discovery of compounds with antifungal activity which play a significant role in preserving the organism within its ecosystem. In this manner, the advantage that evolution has used in developing bioactive compounds can be exploited. REFERENCES [l] [2] [3]
DuPont, P., "Candida albicans, the Opportunist: a Cellular and Molecular Perspective", d. Am. Pod. Med. Assoc., 1995, 85(2) 104-115. Male, O., "The Significance of Mycology in Medicine", In Frontiers in Mycology, D.L. Hawksworth, Ed.; CAB International: Regensburg, 1991, Chapter 7. McCullough, M.; Ross, B.; Reade, P.; "Oral Candida albicans from patients infected with the human immunodeficiency virus and characterization of a
84
ECKERMANand GRAtlAM
genetically distinct subgroup of Candida albicans", Austr. Dent. J., 1995, 40(2) 91-97. [4] Cater, R.; "Chronic Intestinal Candidiasis as a Possible Etiological Factor in chronic Fatigue Syndrome", Med. Hypotheses, 1995, 44, 507-515. [5] Still, J.; Law, E.; Belcher, K.; Spencer, S.; "A Comparison of Susceptibility to Five Antifungal Agents of Yeast Cultures from Bum Patients", in Burns, Elsevier Science, Ltd.: Great Britain, 1995, 21(3) 167-170. [6] Burger, S.; Classen, D.; Burke, J.; Blatter, D.; "Candidal Brain Abscess Associated with Vascular Invasion", Clin. Infect. Diseases, 1995, 21,202-205. McCullough, M.; Ross, B.; Dwyer, B.; Reade, P.; "Genotype and phenotype of [7] oral Candida albicans from patients infected with the human immunodeficiency virus", Microbiology, 1994, 140, 1195-1202. Lim, E.; Stem, P.; "Candida Infection after Implant Arthroplasty", J. Bone Jt. [8] Surg., 1994, 68-,4(1), 143-145. Bozzette, S.; Gordon, R.; Yen, A.; Rinaldi, M.; Ito, M.; Fierer, J., "Biliary [9] Concentrations of Fluconazole in a Patient with Candidal Cholecystitis", Clin. Infect. Dis., 1992, 15, 701-703. [lo] Cooley, D.; Burnett, C.; "Fungal Infection in a Dissecting Aneurysm of the Thoracic Aorta", Texas Heart Institute Journal, 1993, 20 (1), 51-54. [11] Wessel, A.; Simon, C.; Regensburger, D.; "Bacterial and fungal infections after cardiac surgery in children", Eur. J. Pediatr., 1987, 146, 31-33. [12] Barber, G.; Miransky, J.; Brown, A.; Colt, D.; Lewis, F.; Thaler, H.; Kiehn, T.; Armstrong, D.; "Direct Observations of Surgical Wound Infections at a Comprehensive Cancer Center", Arch. Surg., 1995, 130(10), 1042-1047. [131 Lopez, P.; Bastida, M.; Martinez, J.; Ribas, J.; Jove, N.; "Acute Cholecystitis and Wound Infection due to Candida albicans", Eur. J. Clin. Microbiol. Infect. Dis., 1995, 14 (3) 253. [14] Docimo, S.; Kang, J.; Rukstalis, D.; Cotton, D.; Rukstalis, M.; DeWolf, W.; "Candida Epididymitis", Urology, 1993, 41 (3) 280-282. [15] Smith, L.; "Right Upper Quadrant Pain in Diabetic", Patient Care, 1994, 28 (12), 154. [16] Rinaldi, M.; "The Escalating Significance of Fungal Infections and the Need for New Antifungal Agents", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, NJ; Strategic Research Institute: New York, New York, 1995. [17] Hazen, K.; "New and Emerging Yeast Pathogens", Clin. Microbiol. Rev., 1995, 8(4), 462-478. Odds, F.; "Candida Species and Virulence", Am. Soc. Microbiol. News, 1994, 60(6), 313-318. [19] Banerjee, S.; Emori, T.; Culver, D.; Gaynes, R.; Jarvis, W.; Horan, T.; Edwards, J.; Tolson, J.; Henderson, T.; Marton, W.; "Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989", Am. J. Med., 1991, 91(Suppl. 3B) 86S-89S. [20] Badenhorst, L.; Botha, P.; Van Rensburg, M.; "The incidence of hospital fungal infections -- yeast fungaemia", South Afr. Med. J., 1991, 79, 302-303. [21] Clark, A.; "The Need for New Antifungal Drugs", In New Approaches for Antifungal Drugs, P. Fernandes, Ed., Birkhauser: Boston, 1992, 1-19. [22] Richardson, K.; Marriott, M.; "Antifungal Agents", Ann. Rep. Med. Chem., Academic Press, Inc., 1987, Vol. 22, Chapter 16, 159-167.
ANTIFUNGALNATURALPRODUCTS
85
Koltin, Y., "Targets for Antifungal Drug Discovery", Ann. Rep. Med. Chem., Academic Press, Inc., 1989, Vol. 25, Chapter 15, 141-148. [24] Cossum, P.; "Preclinical Evaluation of a Liposomal Nystatin Formulation", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, N J; Strategic Research Institute: New York, New York, 1995. [25] Ryder, N.; "Recent Advances with the Allylamine Antimycotic, Terbinafine", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, NJ; Strategic Research Institute: New York, New York, 1995. [26] Kerridge, D.; "Modes of Action of Antifungal Agents", Adv. Microb. Physiol., 1986, 27, 1. [27] Powderly, W.; "Fungal Infections", Journal of Physicians in AIDS Care, 1996, 2(10). [28] MacDougall, D.; "Opportunistic Infections: The Growing Challenge", Journal of Physicians in AIDS Care, 1996, 2(10). [29] Smart, T.; "ICAAC's Future Fungal Fighters", Gay Men's Health Crisis, 1996, 10(10), 10-12. [301 Stephenson, J., "Investigators Seeking New Ways to Stem Rising Tide of Resistant Fungi", d. Am. Med. Assoc., 1997, 277(1), 5-6. [31] Gallagher, P.; Bennet, D.; Henman, M.; Russell, R.; Flint, S.; Shanley, D.; Coleman, D.; "Reduced Azole Susceptibility of Oral Isolates of Candida albicans from HIV-positive Patients", d. Gen. MicrobioL, 1992, 138, 1901-1911. [32] Nguyen, M.; Morris, A.; Tanner, D.; Nguyen, M. ; Snydman, D. Wagener, M.; Rinaldi, M.; Yu, V.; "The Changing Face of Candidemia: Emergence of NonCandida albicans Species and Antifungal Resistance", Am. J. Med., 1996, 100. 617-623. [33] Kerridge, Marco and Wayman, "Drug Resistance in Candida albicans and Candida glabrata", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 245. [34] Scherer, S.; Magee, P.; "Genetics of Candida albicans", Microbiol. Rev., 1990, 54(3), 226-241. [35] Chakrabarti, A.; Ghosh, A.; Batra, R.; Kaushal, A.; Roy, P.; Singh, H.; "Antifungal Susceptibility Pattern of non-albicans Candida species and Distribution of Species Isolated from Candidaemia Cases", Indian J. Med. Res., 1996, 104, 171-176. [36] Hitchcock, C.; "Resistance of Candida Species to Azole Antifungal Agents", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, N J; Strategic Research Institute: New York, New York, 1995. [37] Sanglard, D. 1997. Cloning of Candida albicans Genes Conferring Resistance to Azole Antifungal Agents: Characterization of CDR2, a New Multi-drug ABC Transporter Gene", Microbiology, 1997, 143, 405-416. [38] Gordee, R.; Debono, M.; Parr, T.; "The Fungal Cell Wall-- A Target For Lipopeptide Antifungal Agents", In New Approaches for Antifungal Drugs, P. Fernandes, Ed., Birkhauser: Boston, 1992, 46-63. [39] Oki, T.; "Pradimicin, A Novel Antifungal Agent", In New Approaches for Antifungal Drugs, P. Femandes, Ed., Birkhauser: Boston, 1992, 64-87. [40] McCullough, J.; "Importance of Chitin Synthesis for Fungal Growth and a s a Target for Antifungal Agents", In New Approaches for Antifungal Drugs, P. Fernandes, Ed., Birkhauser: Boston, 1992, 32-45. [41] Cragg, G. M.; Newman, D. J.; Snader, K. M.; "Natural Products in Drug Discovery and Development", J. Nat. Prod, 1997, 60(1), 52-60. [23]
86
[42] [43]
[44] [45] [46] [47] [48] [49]
[50] [51]
[52] [53] [541
[55] [56] [57]
[581 [591
[60]
ECKERMAN and GRAHAM
Balandrin, M.; Kinghom, A.; Famsworth, N; In Human Medicinal Agents from Plants, Kinghom and Balandrin, Eds., ACS Symposium Series 534, American Chemical Society: Washington, D.C., 1993, 2-12. Hawksworth, D.; "The Fungal Dimension of Biodiversity: Magnitude, Significance, and Conservation", Mycol. Res., 1990, 95, 641. Rinehart, K; "Screening to Detect Biological Activity", In Biomedical Importance of Marine Organisms, Fautin, Ed., California Academy of Sciences: San Francisco, 1988, 13-22. Cordell, G.; "Changing Strategies in Natural Products Chemistry", Phytochemistry, 1995, 40(6), 1585-1612. Waterman, P.; "Searching for Bioactive Compounds: Various Strategies", J. Nat. Prod, 1990, 53(1), 13-22. Rouhi, M.; "Seeking Drugs in Natural Products", Chem. Eng. News, 1997, 75(14), 14-29. Caporale, L.; "Chemical Ecology: A View from the Pharmaceutical Industry", Proc. Natl. Acad. ScL, USA, 1995, 92, 75-82. Eisner, T.; Meinwald, J.; In Chemical Ecology: The Chemistry of Biotic Interaction, T. Eisner and J. Meinwald, Eds., National Academy Press: Washington, D.C., 1995. Hubbell, S.; Wiemer, D.; Adejare, A.; "An antifungal terpenoid defends a neotropical tree against attack by fungus-growing ants", Oecologia, 1983, 60, 321-327. Hubbell, S.; Howard, J.; Wiemer, D.; "Chemical Leaf Repellency to an Attine Ant" Seasonal Distribution Among Potential Host Plant Species", Ecology, 1984, 65(4), 1067-1076. Rockwood, L.; Hubbell, S.; "Host-plant selection, diet diversity, and optimal foraging in a tropical leaf-cutting ant", Oecologia, 1987, 74, 55-61. Rockwood, L; "Plant Selection and Foraging Patterns in Two Species of Leafcutting Ants", Ecology, 1976, 57, 48-61. Bruckner, B.; In Secondary Metabolites: Their Function and Evolution, Ciba Foundation Symposium 171, D. Chadwick and J. Whelan, Eds., Wiley: Chichester, U.K., 1992, 129-143. Eisner, T.; "Prospecting for Nature's Chemical Riches", Chemoecology, 1990, 1, 38-40. Clardy, J.; "The Chemistry of Signal Transduction", In Chemical Ecology: The Chemistry of Biotic Interaction, T. Eisner and J. Meinwald, Eds., National Academy Press: Washington, D.C., 1995. King, V.; Garcia, M.; Himmel, D.; Reuben, J.; Lam, Y.; Pan, J.; Han, G.; Kaczorowski, G.; "Interaction of Tetrandrine with Slowly Inactivating Calcium Channels", J. Biol. Chem., 1988, 263, 2238-2244. Graziana, A.; Fosset, M.; Ranjeva, R.; Hetherinton, A.; Lazdunski, M.; "Ca+2 Channel lnhibitors that Bind to Plant Cell Membranes Block Ca+2 Entry into Protoplasts", Biochemistry, 1988, 27, 764-768. Dreyfuss, M.M.; Chappella, I.; "Potential of fungi in the discovery of novel, low -molecular weight pharmaceuticals", In The Discovery of Natural Products with Therapeutic Potential V.P. Gullo, Ed., Butterworth-Heinemann: Boston, 1994, 49-80. Crittenden, P.; Porter, N.; "Lichen-forming fungi: potential sources of novel metabolites", TIB Tech., 1991, 9, 409-414.
ANTIFUNGAL NATURAL PRODUCTS
[61] [62] [63]
[64] [65]
[66] [67]
[68] [69] [70] [71] [72] [73] [74]
[75]
[76]
[77]
87
Gloer, J., "The Chemistry of Fungal Antagonism and Defense", Can. J. Bot., 1995, 73(1), S 1265-S 1274. Gloer, J.; "New Antifungal and Anti-insectan Natural Products from Fungi: An Ecology-Based Approach", Rev. Latinoam. Quire., 1996, 24, 191-208. Gloer, J.; "Fungi from Competitive Ecosystems as Sources of Novel Bioactive Natural Products", Dev. Ind. Microbiol., 1993, 33, 1-11. Hussain, S.; Lane, S.; "Fungi .vs Fungi: A Potential Application of Antagonistic Properties", Mycologist, 1992, 6, 29-30. Schwartz, R.; Sesin, D.; Joshua, H.; Wilson, K.; Kerripf, A.; Goklen, K.; Kuehner, D.; Gaillot, P.; Gleason, C.; White, R.; Inamine, E.; Bills, G.; Salmon, P.; Zitano, L.; "Pneumocandins from Zalerion arboricola: Discovery and isolation", J. Antibiot., 1992, 45, 1853-1866. Webster, J.; "Coprophilous Fungi", Trans. Br. Mycol. Soc., 1970, 54, 161-180. Wicklow, D.; Hirschfield, B.; "Evidence of a competitive hierarchy among coprophilous fungal populations", Can. J. Microbiol., 1979, 25, 855-858. Liberra, K.; Lindequist, U.; "Marine Fungi -- A Prolific Resource of Biologically Active Natural Products?", Pharmazie, 1995, 50, 583-588. Konig, G.; Wright, A.; "Marine Natural Products Research" Current Directions and Future Potential", Planta Med., 1996, 62, 193-211. Shearer, C.; Zare-Maivan, H.; "In vitro hyphal interactions among wood- and leafinhabiting ascomycetes and fungi imperfecti from freshwater habitats", Mycologia, 1988, 80, 31-37. Strongman, D.; Miller, J.; Calhoun, L.; Findlay, J. ; Whitney, N.; " The biochemical basis for interference competition among some lignicolous marine fungi", Bot. Mar., 1987, 30, 21-26. Ginns, J.; Lefebvre, M.; Lignicolous Corticoid Fungi of North America: Systematics, Distribution, and Ecology, American Phytopathology Society: St. Paul, MN, 1993. Gains, W.; "The analysis of communities of saprophytic microfungi with special reference to soil fungi, In Fungi in Vegetation Science, W. Winterhoff, Ed., Norwell-Kluwer Academic Publishers, 1992, 183-223. Fokkema, N.; "Antagonism between fungal saprophytes and pathogens of aerial plant surfaces, In Microbiology of aerial plant surfaces, C. Dickinson, T. preece, Eds., Academic Press: New York, 1976, 487-506. Mandala, S.M.; Thornton, R.A.; Frommer, B.R.; Curotto, J.E.; Rozdilsky, W.; Kurtz, M.B.; Giacobbe, R.A.; Bills. G.F.; Cabeilo, M.A.; Martin, I.; Pelaez, F.; Harris, G.H.; "The Discovery of Australifungin, a Novel Inhibitor of Sphinganine N-acetyltransferase from Sporomiella australis I. Producing organism, fermentation, isolation and biological activity", d. Antibiot., 1995, 48(5), 349356. Morris, S.A.; Curotto, J.E.; Zink, D.L.; Dreikom, S.; Jenkins, R.; Bills, G.F.; Thompson, J.R., Vicente, F.; Basilio, A.; Liesch, J.M.; Schwartz, R.E.; "Sonomolides A and B: New, broad-spectrum antifungal agents isolated from a coprophilous fungus", Tetrahedron Lett., 1995, 36(50), 9101-9104. Bills, G.; Pelaez, F.; Polishook, J.; Diez-Matas, M.; Harris, G.; Clapp, W.; Dufresne, C.; Byme, K.; Nallin-Omstead, M.; Jenkins, R.; Mojena, M.; Huang, L.; "Distribution of Zaragozic Acids among filamentous ascomycetes", Mycol. Res., 1994, 98, 733-739.
88
[78]
[79]
ECKERMAN and GRAHAM
Horn, W.S.; Smith, J.L.; Bills, G.F.; Raghoobar, S.L.; Helms, G.L; Kurtz, M.B.; Marrinan, J.A.; Frommer, B.R.; Thornton, R.A.; Mandala, S.M.; "Sphingofungins E and F: novel serine palmitoyl inhibitors from Paecilomyces variotii", J. Antibiot., 1992, 45(10), 1692-1696. VanMiddlesworth, F.; Giacobbe, R.; Lopez, M.; Garrity, G.; Bland, J.; Bartizal, K.; Fromtling, R.; Polishook, J.; Zweerink, M.; Edison, A.; Rozdilski, W.; Wilson, K.; Monaghan, R.; Sphmgofungins A, B, C, and D; a new family of antifungal agents I. Fermentation, isolation and biological activity", J. Antibiot., 1992, 45, 861-867. Wang, Y.; Gloer, J.B.; Scott, J.A.; Malloeh, D.; "Appenolides A-C: three new antifungal furanones from the coprophilous Podospora appendiculata", J. Nat. Prod., 1993, 56(3), 341-344. Wang, Y.; Gloer, J.B.; Scott, J.A.; Malloch, D.; "Terezines A-D: new amino acid-derived bioactive metabolites from the coprophilous fungus Sporormiella teretispora", J. Nat. Prod., 1995, 58(1), 93-99. AIfatafta, A.A.; GIoer, J.B.; Scott, J.A.; Malloch, D.; "Apiosporamide, a new antifungal agent form the coprophilous fungus Apiospora montagnei", J. Nat. Prod., 1994, 57(12), 1696-1702. Weber, H.A.; GIoer, J.B.; "Interference competition among natural fungal competitors: an antifungal metabolite from the coprophilous fungus Pr'eussia fleischhakii", J. Nat. Prod., 1988, 51(5), 879-883. Weber, H.A.; Baenziger, N.C.; Gloer, J.B.; "Podosporin A" a novel antifungal metabolite from the coprophilous fungus Podospora decipiens (Wint.) Niessl.", J. Org. Chem., 1988, 53, 4567-4569. Weber, H.A.; Baenziger, N.C.; Gloer, J.B.; "Structure of Preussomerin A: an unusual new antifungal metabolite from the coprophilous fungus Preussia isomera",J. Am. Chem. Soc., 1990, 112, 6718-6719. Weber, H.A.; Swenson, D.C.; Gloer, J.B.; Malloch, D.; "Similins A and B: new antifungal metabolites from the coprophilous fungus Sporormiella similis", Tetrahedron Lett., 1992, 33(9), 1157-1160. Weber, H.A.; Gloer, J.B.; "The Preussomerins: novel antifungal metabolites from the coprophilous fungus Preussia isomera Cain", dr. Org. Chem., 1991, 56(14), 4355-4360. Lee, K.K.; GIoer, J.B.; Scott, J.A.; Malloch, D.; "Petriellin A" A novel antifungal depsipeptide from the coprophilous fungus Petriella sordida", J. Org. Chem., 1995, 60(17), 5384-5385. Whyte, A.; Gloer, K.; Gloer, J.; Koster, B.; Malloch, D.; "New antifungal metabolites from the coprophilous fungus Cercophora sordarioides", Can. J. Chem., 1997, 75(6), 768-772. Xu, X.; de Guzman, F.S.; Gloer, J.B." Shearer, C.A.; "Stachybotrins A and B" Novel bioactive metabolites from a brackish water isolate of the fungus Stachybotrys", J. Org. Chem., 1992, 57, 6700-6703. Poch, G.; GIoer, J.; Shearer, C.; "New bioactive metabolites from a freshwater isolate of the fungus Kirschsteiniothelia sp.", J. Nat. Prod., 1992, 55, 10901099. Harrigan, G.G.; Armentrout, B.L.; Gloer, J.; "Anguillosporal, a new antibacterial and antifungal metabolite from the freshwater fungus Anguillospora longissima", J. Nat. Prod., 1995, 58(9), 1467-1468. 9!
[80] [81]
IS2] [83] [84]
[85] [86]
[871 [88] [89]
[90] [91] [92]
9
ANTIFUNGALNATURALPRODUCTS
89
[93] Ayer, W.; Miao, S.; "Secondary metabolites of the aspen fungus, Stachybotrys cylindrospora", Can. J. Chem., 1992, 71, 487-493. [94] Ayer, W.; Kawahara, N. "Lecythophorin, a potent inhibitor of blue-stain fungi, from the hyphomycetous fungus Lecythophora hoffmannii", Tetrahedron Lett., 1995, 36, 7953-7956. [95] Strunz, G.; Kakushima, M.; Stillwell, M.; "Scytalidin: A New Fungitoxic Metabolite produced by a Scytalidium Species", J. Chem. Soc. Perkin Trans. I, 1972, 2280-2283. [96] Joshi, B.; Gloer, J.B.; "Antifungal Metabolites from Mycoparasites of Aspergillus flavus Sclerotia", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy" Iowa City, Iowa, 1997. [97] Soman, A.G.; Gloer, J.B.; "New Bioactive Metabolites from the Mycoparasitic Fungi Mortierella vinaceae and Neocosmospora vasinfecta", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy: Iowa City, Iowa, 1997. [98] Choudhury, S.R.; Traquair, J.A.; Jarvis, W.R.; "4-methyl-7,1 l-heptadecadienal and 4-methyl-7,1 l-heptadecandienoic acid: new antibiotics from Sporothrix flocculosa and Sporothrix rugulosa", J. Nat. Prod., 1994, 57(6), 700-704. [99] Blakeman, J.; "Ecological succession of leaf surface microorganisms in relation to biological control", In Biological Control on the Phylloplane, C. Windels, S. Lindow, Eds., American Phytopathological Society: St. Paul, MN, 1985, 6-30. [100] Shepherd; "Pathogenicity of Morphological and Auxotropic Mutants of Candida albicans in Experimental Infections", Infect. Immun., 1985, ~.O.,54 I. [101] Mathews; "Pathogenicity determinants of Candida albicans: potential targets for immunotherapy?", Microbiology, 1994, 140, 1505. [102] Gow; Crombie; Gooday; "Polarized Morphogenesis of Candida albicans: Cytology, Induction and Control", in Candida and Candidamycosis, Tumbay, Ed., Plenum, New York, 1991, 13. [103] Shepherd; "Morphogenetic Transformation of Fungi", in Current Topics in Medical Mycology, McGinnis, Ed., Springer-Verlag, New York, 1985, ~., 278. [104] Scherer; Magee; "Genetics of Candida albicans", Microbiol. Rev., 1990, 54. 3, 226. [105] Odds, F.; "Candida Species and Virulence", Am. Soc. Microbiol. News, 1994, 6._0_0,313-318. [106] Liu; Kohler; Fink; "Suppression of Hyphal Formation in Candida albicans by Mutation of a STEl2 Homolog", Science, 1994, 266, 1723. [107] Kohler; Fink; "Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signalling components have defects in hyphal development", Proc. Natl. ,4cad. Sci. U.S.A., 1996, 93, 13223. [108] Leberer; "Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus, Candida albicans", Proc. Natl. Acad. Sci. U.S.A.,.1996, 93, 132217. [109] Braun; Johnson; "Control of Filament Formation in Candida albicans by the Transcriptional Repressor TUPI", Science, 1997, 277, 105. [110] Magee, P.T.; "Which Came First, the Hyphae or the Yeast?", Science, 1997, 277., 52. [l l l] Tasto, J.J.; Jensen, E.C.; and Graham, K.J., "Isolation of a Compound Responsible for the Biochemical Control of Dimorphism in Candida Albicans",
90
[112] [113] [114] [115] [116] [117] [118] [119] [120] [121]
[122] [123] [124] [125]
[126] [127]
[128]
ECKERMAN and GRAHAM
Annual Proceedings of the National Conference on Undergraduate Research, 1997, 5, 1704. Buczacki, S.T.; "A microecological approach to larch canker biology", Trans. Br. Mycol. Soc., 1973, 61, 315-329. Miller, J.D.; "Mycology, Mycologists and Biotechnology", In Frontiers in Mycology, D.L. Hawksworth, Ed., CAB International: Regensburg, 1991, Chapter 11,225-240. Harborne, J.; "Recent Advances in Chemical Ecology", Nat. Prod Rep., 1996, 83-98. Harbome, J.B.; "Role of secondary metabolites in chemical defense mechanisms in plants", In Bioactive Compounds from Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 126-139. Kuc, J.; "Compounds from plants that regulate or participate in disease resistance", In Bioactive Compounds from Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 213-229. Roberts, W.K.; Laue, B.E.; Selitrennikoff, C.P.; "Antifungal Proteins from Plants", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 141-151. Grayer, R.J.; Harborne, J.B. Harborne, "Anti fungal Compounds from Higher Plants", Phytochemistry, 1994, 37, 19. Clark, A.M.; Hufford, C.D.; "Antifungal Alkaloids", In The Alkaloids, Academic Press, 1992, Vol. 42, Chapter 2, 117-150. Wink, M.; "Allelochemical Properties or the Raison D'Etre of Alkaloids", In The Alkaloids, Academic Press, 1993, Vol. 43, Chapter 1, 1-118. Barz, W.; Bless, W.; Borger-Papendorf, G.; Gunia, W.; Mackenbrock, U.; Meier, D.; Otto, C.; Super, E.; "Phytoalexins as part of induced defence reactions in plants: their elicitation, function and metabolism", In Bioactive Compoundsfrom Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 140-156. Collinge, D.B.; Slusarenko, A.J.; "Plant gene expression in response to pathogens", Plant Molec. Biol., 1987, 9, 389-410. Bailey, J.A.; "Plant-pathogen interactions: a target for fungicide development", In Antifungal Agents: Discovery and Mode of Action, G.K. Dixon, L.G. Copping, D. Hollomon, Eds., BIOS Scientific Publishing: Bristol, 1995. Fowler, M.W.; "Industrial applications of plant cell culture", In Plant Cell Culture Technology, M. Yeoman, Ed., Blackwell: Oxford, 1986, 207-227. Fowler, M.W.; Cresswell, R.C.; Stafford, A.M.; "An economic and technical assessment of the use of plant cell cultures for natural product synthesis on an industrial scale", In Bioactive Compounds from Plants, D. Chadwick, J. Marsh, Ed., Wiley: Chichester, 1990, 157-174. Fujita, Y.; "Industrial production of shikonin and berberine", In Applications of Plant Cell and Tissue Culture", Wiley: Chichester, 1988, 228-238. Siegel, S.A.; "Accessing and Exploiting Phytochemical Diversity for Antifungal Discovery -- A Natural Approach", Abstracts of Papers, The 1995 Antifungal Drug Discovery Summit, Princeton, NJ; Strategic Research Institute: New York, New York, 1995. Pazoles, C.J.; "Plant Cell Cultures as a Source of Chemical Diversity and Novelty for Drug Discovery", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy" Iowa City, Iowa, 1997.
ANTIFUNGAL NATURAL PRODUCTS
91
[129] Fusetani, N.; "Antifungal Substances from Marine Invertebrates", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences' New York, 1988, 113-127. [1301 Molinski, T.F.; "Antifungal Marine Natural Products and Emerging Resistant Fungal Pathogens", Abstracts of Papers, 38th Annual Meeting of the American Society of Pharmacognosy, American Society of Pharmacognosy: Iowa City, Iowa, 1997. [131] Garson, M.J.; "The biosynthesis of sponge secondary metabolites: Why it is important", In Sponges in Time and Space, R. van Soest, T. van Kempen, J. Braeka, Eds., Balkema: Rotterdam, 1994, 427-440. [132] Fenical, W.; Jensen, P.R.; "Marine Micro-organisms. A New Biomedical Resource" In Marine Biotechnology, D. Attaway, O. Zaborsky, Eds., Plenum Press: New York, 1993, 419-457. [133] Paul, V.J.; Fenical, W.; "Natural Products Chemistry and Chemical Defense in Tropical Marine Algae of the Phylum Chlorophyta", In Bioorganic Marine Chemistry, P. Scheuer, Ed., Springer-Verlag: Berlin, 1987, Vol. 1, 1-29. [1341 GiI-Turnes, M.S.; Hay, M.E.; Fenical, W.; "Symbiotic Marine Bacteria Chemically Defend Crustacean Embryos from a Pathogenic Fungus", Science, 1989, 246, 116-118. [135] Hughes, T.P; "Catastrophes, phase shifts and large-scale degradation of a Carribean reef", Science, 1994, 265, 1547-1551. [136] Omura, S.; Macrolide Antibtiotics: Chemistry, Biology and Practice, Academic Press: Newy York, 1984, Chapters 9-12. [137] Kohno, J.; Nishio, M.; Kawano, K.; Suzuki, S.I.; Komatsubara, S.; "TMC-34, a new macrolide antifungal antibiotic from a Streptomyces sp.", J. Antibiot., 1995, 48(10), 1173-1175. [138] Fujiu, M.; Sawairi, S.; Shimada, H.; Takaya, H.; Aoki, Y.; Okuda, T.; Yokose, K.; "Azoxybacilin, a novel antifungal agent produced by Bacilius cereus NR2991: Production, isolation and structure elucidation", J. Antibiot., 1994, 47(7), 833835. [139] Achenbach, H.; Muhlenfeld, A.; Fauth, U.; Zahner, H.; "The Galbonolides: Novel, Powerful Antifungal Macrolides from Streptomyces galbus ssp. eurythermus", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 128-140. [140] Inouye, Y.; Okada, H.; Nakamura, S.; "Hydroxamic Acid Antimycotic Antibiotics", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 180-182. [141] Kempf, A.J.; Hensens, O.D.; Schwartz, R.E.; Sykes, R.S.; Wilson, K.E.; Wichman, C.F.; Zink, D.L.; Zitano, L.; Mochales, S.; "L-660,631, a New Antifungal Agent", In Antifungal Drugs, St. Georgiev, Ed., New York Academy of Sciences: New York, 1988, 183. [142] Barbier, M.; Introduction to Chemical Ecology, Longman Group Limited: London, 1979. [143] Schroth, M.N.; Hancock, J.G.; "Disease-Suppressive Soil and Root-Colonizing Bacteria", Science, 1982, 216, 1376-1381. [144] Schildknecht, H.; Koob, K.; "Myrmicacin, The First Insect Herbicide", Angew. Chem. Internat. Edit., 1971, 10, 124-125. [145] Mueller, U.; Wolf-Mueller, B.; "Epiphyll Deterrence to the Leafcutter Ant Atta cephalotes", Oecologia, 1991, 59, 615-625.
92
ECKERMAN and GRAHAM
[146] Line, L.; "Microcosmic Captive Breeding Project Offers New Hope for Beleaguered Beetle", New Fork Times, 1996, September 17, BS-B6. [147] Boman, H.G.; "Cell-free immunity in insects", Ann. Rev. Biochem., 1987, 41, 103-126. [148] Stone, R.; "Deja Vu Guides the Way to a New Antimicrobial Steroid", Science, 1993, 259, 1125. [149] Zasloff, M.; "Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial eDNA sequence of precursor", Proc. Natl. ,4cad. Sci. USA, 1987, 84, 5449-5453. [150] Bevins, C.L.; Zasloff, M. "Peptides from frog skin", ,4nnu. Rev. Biochem., 1990, 59, 395-414. [151] Zasloff, M.; Martin, B.; Chert, H-C.; "Antimicrobial activity of synthetic magainin peptides and several analogues", Proc. Natl..4cad. Sci. USA, 1988, 85, 910-913. [152] Bechinger, B.; Zasloff, M.; Opella, S.J.; "Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy", Protein Sci., 1993, 2, 2077-2984. [153] Berkowitz, B.A.; Bevins, C.L.; Zasloff, M.; "Magainins: a new family of membrane-active host defense peptides", Biochera. Pharmacol., 1990, 39, 625629. [154] Barra, D.; Simmaco, M.; "Amphibian skin: a promising resource for antimicrobial peptides", TIB Tech., 1995, 13, 205-209. [155] Gabay, J.E.; "Ubiquitous natural antibiotics", Science, 1994, 264, 373-374. [156] Cociancich, S.: Bulet, P.; Hetru, C.; Hoffmann, J.A.; "The inducible antibacterial peptides of insects", Parasitol. Today, 1994, 10, 132-139. [157] Ganz, T.; Lehrer, R.I.; "Defensins", Pharmacol. ?'her., 1995, 66, 191-205. [158] Genz, T.; Lehrer, R.I.; "Antimicrobial peptides of leukocytes", Curt. Opin. Hematol., 1997, 4, 53-58. [159] Cammue, B.; De Bolle, M.; Schoofs, M.M.; Terra, F.; Thevissen, K. Osborn, R.W.; Rees, S.B.; Broekaert, W.F.; "Gene-encoded antimicrobial peptides from plants", In Antimicrobial peptides, J. Marsh, J.A. Goode, Eds., Ciba Foundation Symposium 186, John Wiley & Son's: Chichester, 1994, 9 l- 106.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistpy, Vol. 22
9:1
9 2000 Elsevier Science B.V. All rights reserved
NATURAL TRITERPENOIDS AS ANTI-INFLAMMATORY AGENTS RIOS, J.L.*; RECIO, M.C.; MAI~IEZ, S. and GINER, R.M.
Departament de Farmacologia, Factdtat de Farmbcia, Universitat de ValEncia, Avda. Vicent Andrds Estellds s/n., 46100 Burjassot (Valbncia),
Spain
ABSTRACT" This chapter reviews the natural triterpenes with anti-inflammatory activity, including the traditional ones and the new compounds isolated over the last six years. Triterpenes are widely distributed in plants, and in many cases are the principles responsible for their anti-inflammatory effects. Many of these compounds are active in different in vivo experimental models such as hind paw edema induced by carrageenan, serotonin and phospholipase A2; ear edema induced by phorbol and daphnane esters, ethylphenylpropiolate, arachidonic acid and capsaicin; adjuvant arthritis and experimental models of allergy. Other effects have been studied in vitro, and some triterpenes are active against inflammatory enzymes like 5-1ipoxygenase, elastase and phospholipase A2. Others inhibit histamine, collagenase and interleukin release, lipid peroxidation and free radical-mediated processes, metabolism of endogenous corticoids, and complement and protein-kinase activities. In certain cases the mechanism of action depends on the skeleton type and/or substituents. For example, 13-boswellie acid (ursane-derived) and derivatives markedly inhibit 5-1ipoxygenase activity, whereas the principal mechanism of 1813-glycyrrhetinic acid (oleanane-derived) is the inhibition of endogenous corticoid metabolism. Some lanostanes are active against phospholipase A2 (e.g. ganoderic and dehydrotumulosic acids), and compounds with highly unsaturated rings can act as anti-peroxidatives (e.g. celastrol, a tetraunsaturated friedooleanane). FOREWORD Although certain kinds of larger molecules originating from the mevalonate synthetic pathway do in fact exist in plants, triterpenes represent the culmination of this metabolic route in the vast majority of botanical taxa. Thanks to the steadily increasing number o f scientific reports on new structures and interesting biological activities of this class of compounds in the recent years, our knowledge o f this area o f Phytochemistry and Pharmacognosy has expanded greatly. As might be expected, a large number of reviews focusing on occurrence and chemical aspects have been published, such as those from Mahato et al. [1-3] and Connolly and Hill [4-12]. The present review differs completely, however, both in aims and content, because it centres on the
94
R[OS et al.
description of the triterpenes for which novel anti-inflammatory properties have been reported from 1992 to 1997 ~ and on additional studies of well-known principles already considered active, such as glycyrrhetinic, ursolic and oleanolic acids, etc. Reports on triterpene glycosides, mostly saponins, are excluded as they are usually studied separately because of their particular physical and biological properties. Nevertheless, their importance is reflected in Pharmacognostic Background section. It should be pointed out that for the sake of coherence, most of the examples illustrating the chemistry section of this chapter are taken from the literature on terpenoids that have effects on the inflammatory process. Additionally, a section on the principles of inflammation and its pharmacology is included before the reports on the activity of triterpenes, which are classified according to the test models and mechanism of action. AN INTRODUCTION TO TRITERPENES
Concept The triterpenoids comprise a large group of diverse C30 natural secondary metabolites having relatively complex cyclic structures, usually tetra or pentacyclic, although acyclic or monocyclic skeletons can also be found. Most of them are alcohols, aldehydes, carboxylic acids or esters, and they are regarded as an important class of compounds in Phytochemistry. Modern isolation and analysis techniques have refined the structural elucidation of many already isolated and also newly found compounds. More than 40 skeletal types arising from the cyclisation and subsequent rearrangements of their biosynthetic precursor squalene can be distinguished. Most of the triterpenes, with the exception of those with hopane and gammacerane skeletons have a 3]3-oxygen function. The attachment of linear or branched sugar moieties to the triterpene framework, usually at the 3-hydroxyl position, results in the formation of a large number of naturally occurring saponins. Disubstitution or, less frequently, trisubstitution of the triterpenoid molecule with sugars is a regular feature for these compounds.
Occurrence Triterpenoids are widely distributed throughout the plant kingdom. Oleananes and ursanes, which often occur together, and lupanes are found *Authors' note: For complementary information see also the review of Safayhi, H.; Sailer, E.-R. Planta Med.,
1997, 63, 487.
NATURAL TRITERPENOIDS
95
in a wide range of families. Lanostanes are common in fungi and marine organisms and also occur in higher plants. The remaining skeletal types are more restricted in their natural occurrence. Cucurbitanes occur in the Cucurbitaceae and they have also been detected occasionally in at least five other families, namely Begoniaceae, Cruciferae, Desfontainiaceae, Elaeocarpaceae and Scrophulariaceae [13]. Dammaranes occur in several plant families, including Anacardiaceae, Rutaceae, Betulaceae and Rubiaceae, friedelanes in Celastraceae and Buxaceae, serratanes in Pinaceae and the Polipodiaceae fern [8], strictanes in lichens and fernanes in ferns. Hopanes are found in lichens, ferns, and certain higher plant families and can also be detected in geochemical samples. They are widespread among the prokaryotes, where they may perform the role of steroids in plants. Gammaceranes are also described in prokaryotes. The quassinoid nortriterpenoids are confined to the Simaroubaceae; the limonoids, by contrast, are found in this family and also, more abundantly, in the three related families, Rutaceae, Meliaceae and Cneoraceae [13].
Biological Significance The physiological role of triterpenoids is not yet wholly understood. However, their function in chemical defence has been established. This class of natural substances is involved in plant-animal and plant-plant interactions that occur in many ecosystems. Cucurbitacins represent the clearest example. These tetracyclie triterpenes of the Cueurbitaeeae normally repel insect feeders, and are therefore sequestered and stored by insects for defensive purposes. Cucurbitacin D, found in some Chrysomelidae species, is a potent kairomone to a phytophagous beetle that feeds specifically on these plants. Storage of this toxin protects against predation by mantids, which, however, have not learnt to avoid the adverse effect of ingesting these beetles. Another example of chemical protection occurs in the paper birch (Betula resinifera). The high content in papyriferie acid of the young internodes and twigs makes them unpalatable and deters grazing by the hare [14]. The tetracyclie triterpenoid quassin is an antifeedant to the aphid Myzus persicae [ 15], and the limonoids azadirachtins from Azadirachta indica are also potent insect antifeedants [ 13]. Ursolic acid has been identified as an allelopathic agent in Ceratiola ericoides and Calamintha ashei on competing sandhill grasses [16]. Lupeol, betulin, betulin aldehyde and betulinir acid from Melilotus messanenis possess potential allelopathic activity on dicotyledon species like Lactuca sativa and Lepidium sativum [17]. As a consequence of plantplant co-evolution, higher plants are able to form haustoria and thus become parasitic on their host plant. The oleanane triterpene soyasapogenol A isolated from the root of Lespedeza sericea, a
96
R|OS et aL
Leguminosae host of Agalinis species, has been described as a haustoriainducing compound [ 16]. Glycinoeclipin A is a pentanortriterpene which stimulates the parasitization of the soybean (Glycine max) by cyst nematodes [ 14]. As a result of plant-microbial parasite interaction, plants elaborate constitutive antifungal agents. Cucurbitacin I protects cucumber from Botrytis cinerea because it inhibits the induction of an enzyme involved in tissue damage and the spread of infection [18].
Pharmacognostic Background An overview on the traditional importance of anti-inflammatory triterpenoids in Pharmacognosy gives rather poor results unless saponins are included. Without them, the list would be reduced to glycyrrhetinic acid from liquorice, papyriogenins from Tetrapanax papyriferum, and triterpenoids from the resins of Commiphora species. However, diverse crude drugs containing triterpenoids in the combined form of saponins have been used extensively for their anti-inflammatory properties, not only in folk medicine but also in modem clinical therapeutics. The most important of these drugs is liquorice root (Glycyrrhiza glabra), which has been used since of the time of the ancient Greeks as an expectorant, antitussive and sweetening agent. In both Chinese and European medicine, liquorice decoctum is employed to treat throat inflammation. It contains about 6-13% glycyrrhizin, a glycoside from glycyrrhetinic acid. The extract and the aglycone have been shown to have corticosteroid-like effects, and are employed for the treatment of rheumatoid arthritis, Addison disease and other inflammatory processes. It has been reported that glycyrrhetinic acid has 1/8 of the anti-inflammatory potency of cortisol, reaching 1/5 of cortisol in the case of carbenoxolone, the sodium salt of glycyrrhetinie acid hemisuccinate [19]. The tincture of Aesculus hippocastanum seeds has been used successfully for haemorrhoids and venous congestion. Aesein, a mixture of oleanane triterpene saponins with a yield of about 13% relative to the crude drug weight, shows anti-inflammatory action, and it is administered orally for clinical use [19]. Among Oriental medicinal remedies there are many herbal drugs, such as ginseng or saiko, which contain triterpene saponins as their principal constituents and the ones that seem to be responsible for their efficacy [20]. Ginseng, the root of Panax ginseng has been well known in East Asian countries since ancient times as a panacea drug that favours longevity. It contains oleanolie acid and dammarane triterpene saponins called ginsenosides with diverse pharmacological properties including antiinflammatory activity [21]. Ginseng and its saponins are drugs that
NATURAL TRITERPENOIDS
97
normalise abnormal behaviour or symptoms caused for the most part by stress, but they cure no specific disease directly. The root of the Bupleurum species has been used in China as a traditional remedy for inflammatory diseases [20]. The main constituents of this drug are oligoglycosides of oleanane triterpenes called saikosaponin a-f. Saikosaponins cause a reduction in histamine secretion and enhance the anti-inflammatory actions of glucocorticoids [22]. Some of them have been shown to inhibit prostaglandin E2 production in the macrophage culture system [23]. It has recently been reported that the saikosaponins present in Heteromorpha trifoliata, which are structurally related to those of Bupleurum falcatum (saiko in Japanese), have anti-inflammatory activity. One in vivo study concluded that the isolated saikosaponins act by a mechanism close to that of steroids, but do not involve the glucocorticoid receptor [24]. Other crude drugs used as anti-inflammatory agents are the stem-bark of Akebia quinata, which provides saponins of oleanolie acid and hederagenin, roots of Astragalus membranaceus, from which the saponin astramembrainnin I has been isolated, and the roots of Platycodon grandiflorum with saponins called platycodins. Anti-inflammatory activity was also established with saponins from extracts of Phytolacca americana, Patrinia scabiosaefolia, Lamium album and Swertia cincta. In addition, saponins isolated from Eryngium planum, Hydrocotyle vulgaris, Polemonium coeruleum, Sanicula europaea and Thea sinensis have been shown to have anti-exudative activity [20,25,26]. Widespread reports indicate that, in addition to their anti-inflammatory role, triterpenes have other activities. Triterpenoids have been found to possess cytotoxic, antimicrobial and interesting effects on metabolism. Triterpenoids with antitumour activity include oleanane, lanostane, lupane, friedelane, hopane and quassionoid types. Glycyrrhetinic acid has been described as an antiviral, hypolipidemic and anti-atherosclerotic agent. Cucurbitacins B and E, and oleanolic acid possess a potent protective action on the liver, and ganoderic acid and its derivatives have been shown to be inhibitors of cholesterol biosynthesis. Lanostane derivatives, like suberosol, have also been found to inhibit HIV replication in H9 lymphocytes [1,3]. CHEMISTRY
Biosynthesis Triterpenes originate from squalene via mevalonic acid, which is formed from sequential condensation of three acetyl-coenzyme A units and subsequent reduction with NADPH to generate (3S)-3-hydroxy-3methylglutaryl-coenzyme A. The next ATP/Mg2+-dependent steps
R|OS et al.
98
convert mevalonate into (R)-5-diphosphomevalonic acid, and the latter into isopentenyl pyrophosphate (IPP), which is isomerised to dimethylallyl pyrophosphate (DMAPP). Both isomers represent the activated monomer building blocks for all terpenoids. Head to tail condensation of IPP with DMAPP generates geranyl diphosphate, and addition of a second IPP unit furnishes farnesyl pyrophosphate (FPP). Tail to tail condensation of two FPP units gives rise to squalene, which is oxydised to (3S)-2,3-oxidosqualene [27]. Oxidosqualene cyclase catalyses the conversion of (3S)-2,3-oxidosqualene to cycloartenol, the first cyclic precursor of tetracyclic triterpenes and phytosterols in higher plants and algae. In animals and fungi, oxidosqualene is cycled into lanosterol, the precursor of sterols. The formation of cycloartenol proceeds via the prec h a i r - b o a t - c h a i r conformation of (3S)-oxidosqualene, yielding a protosteryl C-20 cation that suffers a series of 1,2-methyl and hydride shifts with proton elimination, to give a cycloartene skeleton. Cyclization to pentacyclic triterpenes proceeds from the pre all-chair conformation of the substrate, yielding a tetracyclic dammarenyl C-20 cation. The following rearrangement leads to pentacyclic triterpenes via the baccharenyl, lupenyl and oleanyl cationic intermediates [28]. Squalene can also be cycled directly, without previous oxidation, to pentacyclic 3deoxytriterpenes, thus leading to hopane and gammacerane skeletons, as occurs in some bacteria and protozoa.
Structural Types Recent reviews classify triterpenoids according to chemical characteristics or biogenetic origin [3,11 ], but in the present review the emphasis is on activity and for this reason our classification is based only on antiinflammatory substances. T a b l e 1.
General
Skeletons of Anti-inflammatory Triterpenes Cited in this Review
29
30
28
23
24
~lcmimie
Ursane
NATURAL TRITERPENOIDS
99
(Table 1). contd.....
Taraxastane
Hopane
I"
Lupane
DtUlUIIWanf3
h O S t M C
Cucurbitane
100
R[OS et al.
The main kind of anti-inflammatory triterpenes isolated have oleanane, ursane, taraxastane, lupane and lanostane skeletons (Table 1). Some minor compounds such as hopane are included in other structural groups. Other anti-inflammatory triterpenes like the different cucurbitacins are not included in this review because of their high toxicity.
Oleanane Type Oleanane triterpenoids are the largest group within the triterpenes and encompass a huge number of active compounds. They are structurally classified as olean- 12-ene (Table 2) and 11-keto-olean- 12-ene (Table 3), directly derived from the oleanane skeleton. Other modifications give rise to the D:C-friedooleananes (Table 4), friedelanes (D:A-friedooleananes), 24-nor-D:A-friedooleananes (Table 5) and 24,30-dinor-D:Afriedooleananes (Table 6). The oleananes include glycyrrhetinic acid, probably the most widely studied triterpene. Table 2.
Anti-inflammatory Triterpene$ Derived from Olean-12-ene
128 )
R3 ,
,,,
,
16
RI6
R23
R24
R28
CH3
CH3
,,
p-Amyrin
OH[3
tt
CH3
[i-Amyrin acetate
OAc~
H
CH3
CH3
CH3
Erythrodiol
OHp
H
CH3
CH3
CH2OH
Oleanolic acid
OH~
CH3
CH3
COOH
Hederagenin
OH~
CH2OH
CH3
COOH
,
,
,
r
.,
acid
H
CH3
COOH
M~miladiol
'OH~ "
OHa
OH
CH3
CH3
CH3
Longispinogenin
OH[3
OH
CH3
CH3
CH2OH
.
J
....
.
.
.
.
.
.
CH3 .
NATURAL TRITERPENOIDS
Table 3.
101
Anti-inflammatory Triterpenes Derived from l l-Keto-olean-12-ene
'1'
R3 1813-Glycyrrhetinic acid
OH
.~
coo.
OH
Ha
COOH
HI3
COOEt
,,
,,
18a-Glycyrrhetinic acid .
.
.
.
.
.
.
.
.
.
.
.
.
.
,
H.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Ascorbic acid phosphate Na
Glycyrrhetinic phosphate vitamin C (GEPC) .
Anti-inflammatory Triterpenes Derived from D:C-Friedooleanane
Table 4.
D . . . .
R3
R7
R29
OHI3
H2
COOH
68
OCO(CH2)2cooK
H2
COOK
7-Oxo-isomultiflorenol
A8
OH[I
-O
CH3
3-Epibryononol
A8
OHa
H2
CH2OH
7-Oxo'dihydrokarounidiol
A8
OHa
=0
CH2OH
A7,9( 11)
OHa
H2
cH2oH
82
CH2OH
H2
CH2OH
Bryonolic acid " .
.
.
.
.
Bryonolic acid-3-O-succinate(K +)
.
.
.
.
.
.
.
m
.
Karounidiol Karounidiob3-O-i~enzoate
3-Epik~ounidiol
. . . . A7,9(11)
OcoPha .
A7,9(11) .....
OHIB
.
.
.
.
.
.
.
.
102
RIOS et a/.
Table 5.
Anti-inflammatory Triterpenes Derived from 24-nor-D:A-Friedooleanane
Ill
A
R2
R3 i
i
R6
R29
R7
ill m
Celastrol
A3,5,7,10(1)
Acetilcelastrol
=O
OH
A3,5,7,10(1)
=O
OAc
Pristimerin
A3,5,7,10(1)
=O
OH
Regeol B
A4
OHa
=O
OH~
Regeol C
A2,4,7,10(!)
OH
OH
=O
,
H
i
H
. . . . . . . .
. . . . .
CH3
OH
,,,
i
Anti-inflammatory Friedooleanane
Table 6.
H2
Triterpenes
Derived
H
H
ill
from
24,30-dinor-D:A-
R2
|
i
i
A i
|, ill
,,
R2 i
i,
,
I-'
'
,"
A3,5,7,]0(~)
Tingenin B
A2,4,10(1)
[ Regeoi A , i
'
,
=O OH ,
.....
i,ll
I II
NATURAL TRITERPENOIDS
103
Ursane Type
This structural group is divided into two subgroups, 3]3-hydroxy-urs-12ene (Table 7) and 3tx-hydroxy-urs-12-ene (Table 8). The ursane 13boswellic acid and its derivatives have been studied in vivo and in vitro as
anti-inflammatory agents. Anti-inflammatory Triterpenes Derived from 315-Hydroxy-urs-12-ene
Table 7.
= R20
[28 I
R2
R3
RI6
R20
H
H
|
ct-Amyrin .
.
.
.
R28 i
OH
.... c . ;
.
o~-Amyrin acetate
H
OAc
H
H
CH 3
a-Amyrin-linoleate
H
O-linoleate
H
H
" CH 3
ot-Amyrin-palmitate
H
O-palmitate
H
' CH 3
Uvaol
H .
.
.
Ursolic acid Brein Tormentic acid
.
.
. . . . . . H ....
OH .
.
.
.
H H
OH OH
OH
OH
H .
.
,L
.
.
H .
.
.
.
H OH
H H
'H
OH
CH2OH .
.
,.,
COOH CH 3 COOH
Anti-inflammatory Triterpenes Derived from 3t~-Hydroxy-urs-12-ene
Table 8.
R3~,,""
R3 , .=
,
,,, ,,,,,,
,
RII
,
[i-Boswellic acid Acetyl-I l-keto-lS-b0swellic acid (AKBA) I l-Keto-~-boswellic acid
OH
HE
OAc
-0
OH
=O
'
f04
RIOS et al.
Taraxastane Type The active taraxastane triterpenes are included in two g r o u p s - - t h e taraxast-20-ene, or taraxastene type (Table 9); and its isomer taraxast20(30)-erie, or ~-taraxastene type (Table 10). Table 9.
Anti-inflammatory Taraxastene Triterpenes
i
I
J
,
R ,,
,
,
__L_,
,
,
"
1 Taraxasterol Taraxasterol acetate T a b l e 10.
,,
"
_OH
"
OAc
Anti-inflammatory v-Taraxastene Triterpenes
"'*t R22 :16
R3
RI6
R22
R28
Illlll ,,
v-Taraxasterol
,
OH
H
H
CH3
OAc
H
H
CH 3
OH
OH
H
CH 3
Faradiol-myristate
O-myristate
OH
H
CH 3
Faradiol-palmitate
O-palmitate
OH
H
CH3
OH
OH
OH
CH2OH
Heliantriol C
OH
OH
H
CH3
Amidiol
OH
OH
H
u
acetate
Faradiol
,..
.
.
.
.
.
.
.
.
Heliantriol B0 .
.
.
.
.
_
.
.,
CH3
NATURAL TRITERPENOIDS
105
Lupane and Neolupane Types Betulin and betulinic acid are the best studied lupanes as antiinflammatory derivatives, and a great deal of research on their activity and mechanism of action has been published (Table 11). While the lupanes are widespread in plants, the occurrence of neolupanes is reduced to a small number of compounds and plants. Table 11.
Anti-inflammatory Lupane Triterpenes
28
RaV , , , e ~
~f
R3 I
I
iii
,,,,
RI6
R26
R28
H
CH3
CH3
,,
Lupeol
OH
Betulin
OH
CH3
CH2OH
Betulinie acid
OH
CH3
COOH
Calenduladiol
OH
OH
CH3
CH3
Heliantriol B2
OH
OH
CH2OH
CH3
........
L
Lupeoi-palmitate
O-palmitate
CH3
CH3
Lupeol-linoleate
O-linoleate
CH3
CH3
Lanostane and seco-Lanostane Types The lanostanes are the most relevant group of the tetracyclic triterpenes (Tables 12-13). Many of this type of compounds are described as antiinflammatories and their mechanism of action has been studied frequently. Cycloartenol and some 3,4-seco-lanostane derivatives have been reported to have activity also.
RiOS et al.
106
Anti-inflammatory Lanostane Triterpenes
Table 12.
~
R,,
,,,,-\
_.~R16
... m
R6
A
R6
R3 ,
,
ill,
....
RI6
,
A8
OAcl3
H
OH
Dchydropachymic acid
A7,9(11)
oAcp
H
OH
6a'Hydroxydehydropachymic acid
A7,9(I I)
OAcl] .....
OH
OH
Dehydroeburiconic acid
A7,9( I I)
---O
H
H
l) . . . .acid . . . . . . . . . .A7,9(1 . Dehydrotumulosic
0H~
H
OH
Pachymic acid
,
Anti-inflammatory Lanostane Triterpenes
Table 13.
R22 R21oo_ ~
~ oRI6
%
jR26 R27
~
Rls
i R,
I R, I s", S'6 I S~' ! S2~ I R26 I, Re'
A ,
.
,
,. . . .
i
A7,9(1 l) ' OFI[~
3]]-HYdroxylanosta-7,9(l 1),24trien-2 l-~ic acid .
.
.
.
.
.
3-O-Acetyl-I 6cthydroxytrametenolic acid
16ct-Hydroxytrametenolic acid .
.
.
.
H
H
"COOH
H
CH3
CH3
H
OHa
COOH
H
CH3
CH3
OHtx "'COOH
H
CH3
CH3
.......
oAc~)
AS '
AIi
OH~..........H ..
NATURAL TRITERPENOIDS
107
(Table 13). contd.....
•
,
','I
Masticadienoic acid*
A7
Masticadienolic acid (Schinol)* .
.
.
.
.
.
A7
.
.
.
.
.
.
.
.
H
H
CH3
H
OHo~
H
H
CH3
H
......
.
Pistacigerrimone A .
=O
CH3
COOH
CH3
COOH
.....
=0
A 1,7
H .
.
.
.
.
.
.
.
.
H
CH3
H
CH3
COOH
CH3
H
CH3
COOH
.
Pistacigerrimone D
A 1,5,8
=0
H
H
Ganoderic acid R
A7:9(11),24
OH~
H
H
CH3
OAc COOH
CH3
. . . . . . .
Ganoderic acid S
A7,9(11),24
=0
H
H
CH3
Ganoderic acid T
A7,9(I 1),24
OHo.
OH'
H
CH3
H
COOH
CH3
OAc COOH
CH3
r
*Compounds with 13oq14[~,!7o~-substitution
Hopa n e
Type
Of the hopane type only three compounds have been reported to be active
(Table 14). Table 14.
A n t i - i n f l a m m a t o r y H o p a n e Triterpenes
.....3 . r
R22
R
A Moretenol
A22(29i
Moretenol acetate
A22(29)
R3
CH3 OAc
Tetrahydroxybacterihopane (THBH) . . . . . . .
.
.
.
.
.
.
H2 .
.
R22
.
.
CH3 CH2(CHOH)3CH2OH '"
.
Isolation and Identification
Extraction, Separation and PreliminaryAnalysis Triterpenoids are usually non-polar compounds, but some oxygenated substituents, like earboxyl or hydroxyl, produce a moderate increase in polarity in the molecule. Apolar solvents such as dichloromethane and
108
R[OS eta/.
chloroform are usually needed to obtain an extract containing free triterpenes, but supercritical-fluid extraction (SFE) is being introduced into the isolation process used for natural products, including triterpenes [29,30]. There are different types of tests for detecting the presence of triterpenes in an extract. The Liebermann-Bouchard reagent is the one most frequent used to differentiate triterpenes and steroidal aglycones, whereas Zimmermann's test is appropriate for 3-oxo triterpenoids. Different reagents can be used to detect triterpenes in thin-layer chromatography (TLC) plates. Thionyl chloride, phosphomolibdic acid, silicotungstie acid [31], stannous chloride, arsenic chloride [32], antimonnium chloride, rhodamine 6G, and vanillin phosphoric acid [33] have all been described. It is possible to distinguish some compounds easily by derivatization in the plate. Oleanolic and glycyrrhetinic acids can be transformed into their methyl esters with methyl iodide, o~- and 13amyrin into acetyl derivatives with acetic anhydride/pyridine, and lupeol with acetyl chloride. The products of reaction are less polar and more easily separable from other natural products in TLC [33]. Silicagel impregnated with AgNO3has been used in TLC plates and flash columns to separate triterpenes [34]. Some of the anti-inflammatory triterpenes that have been described are not natural products, but artefacts. Using methanol during the extraction process may transform some kinds of glycosides, like saikosaponins, into 11-methoxy derivatives. Similarly, in the hydrolysis process a large number of glycosides may be transformed when mineral acids, like HCI or H3PO4 are used. This can be avoided by previous permethylation or enzymatic hydrolysis of original compounds. Separation methods vary depending on the type of compound. Free aglycones are easily separated by routine chromatographic systems preparative TLC (p-TLC), conventional column chromatography (CC), flash-CC, medium pressure liquid chromatography (MPLC), vacuum liquid chromatography (VLC), etc.---or by more complex techniques such as gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), droplet counter current chromatography (DCCC) or capillary-CC. Reverse-phase (RP)-HPLC is probably the best system for purifying triterpenoids, principally when mixtures of isomers are present [35]. Gunther and Wagner in 1996 [36] carried out the separation and quantification of active triterpenes from Centella asiatica employing an RP system with acetonitrile-water as mobile phase. Recently, Gaspar et al. [37] described the complete separation of a mixture of triterpenoid isomers from the fruit of A r b u t u s u n e d o by HPLC coupled to a mass spectrophotometer by means of a particle beam interface (HPLC-PBMS). The separation of different quassinoids from crude bark of Quassia amara was developed by Vitanyi et al. [38] using a reverse-phase HPLC-MS
NATURAL TRITERPENOIDS
109
with thermospray ionisation. Application of this technique to the study of triterpenes and their glycosides has been successful [39]. When Heinzen et al. [40] compared thermospray liquid chromatography (TSP-LC) and GCMS with electron impact ionisation (GC-MSEI) techniques to study triterpenes in different samples, greater selectivity and sensitivity were observed with TSP-LC. Determination of glycyrrhizin and glycyrrhetinie acid can be done in a capillary electrophoresis system. The great advantage of this technique is the running time, because the analysis of these two constituents can be completed in 10 min, whereas the HPLC analysis could take about 50 min [41]. Takaishi et al., [42] combined CC (silica gel and sephadex LH-20) and HPLC in the isolation of the anti-inflammatory compounds of Tripterygium wilfordii. Della Loggia et al. [43] isolated the topical antiinflammatory triterpenes mixture from Calendula officinalis using COz SFE, and the extract was purified using VLC and MPLC (silica gel) and HPLC (columns of silica impregnated with AgNO3, diol and RP). Later, Zittlerl-Eglseer et al. [44] isolated the anti-edematous triterpenes faradiol esters and v-taraxasterol from the same source using only silica gel CC and preparative-HPLC. Structural Elucidation
Diverse spectroscopic methods have been employed to characterise triterpenes. Ultraviolet (UV) and infrared (IR) spectroscopy are not very useful techniques in elucidating the structure of triterpenes, but the former gives information about compounds with conjugated double bonds and the latter may provide some information about substituents like the hydroxyl group, ester carbonyl group or t~,13-unsaturate carbonyl. Other physical data may be of interest to characterise new compounds, but the use of modem spectroscopic methods of nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are essential for the structural determination. NMR is the tool most widely used to identify the structure of triterpenes. Different one-dimension and two-dimension techniques are usually used to study the structures of new compounds. Correlation via H-H coupling with square symmetry (~H-~H COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), distortionless enhancement by polarisation transfer (DEPT), incredible natural abundance double quantum transfer experiment (INADEQUATE) and nuclear Overhauser effect spectroscopy (NOESY) allow us to examine the proton and carbon chemical shift, carbon types, coupling constants, carbon-carbon and proton-carbon connectivities, and establish the relative stereochemistry of the chiral centres.
110
RiOS et al.
Two excellent reviews describing the t3C NMR of pentacyclic triterpenes have been published in the last years. In 1992 Agrawal and Jain [45] published a compilation of the spectral data on 456 oleanane triterpenes and Mahato and Kundu [2] reviewed the spectral data of 393 compounds, including 43 types of structural groups. The use of MS together the NMR spectroscopy has been essential for the structural elucidation of triterpenoids. The review by Shiojima et al. [46] covers the mass spectra of saturated and unsaturated triterpenoids and explains each type of compound fragmentation. Bioassay-guided Isolation and Anti-inflammatory Studies
To isolate active compounds it is necessary to monitor the activity during fractionation and isolation. In the case of triterpenes as anti-inflammatory agents, different kinds of tests are employed. Subplantar administration of carrageenan in rats was commonly used in earlier studies to establish the anti-inflammatory activity of orally administered plant extracts or fractions, but a large amount of sample was necessary. Topical application of inflammatory agents is more useful in screening extracts and pure compounds, because the amount of sample is smaller than with oral application and it is easier to track the activity. The introduction of in vitro tests could be an alternative way to monitor the anti-inflammatory activity. Several examples of bioassay-guided isolation are given in the literature. Recio et al. [47] used a topical irritant agent to track the activity during the isolation of the anti-inflammatory triterpenes from Diospyros leucomelas, whereas Cu611ar et al. [48] employed a combined system of in vivo and in vitro experiments, consisting of the inhibition of an enzyme activity "Fig. (1)". Other authors prefer in vitro models. Ammon and Safayhi [49] used the inhibition of leukotriene formation in rat peritoneal neutrophils to isolate the boswellic acids of gum resin exudate of Boswellia serrata, whereas Takaishi et al. [42] studied the inhibition of interleukin-1 secretion, and Jain et al. [50] studied the in vitro inhibition of phospholipase A2 activity. The main results of these studies are summarised in the pharmacological activity section of this chapter. ANTI-INFLAMMATORY ACTIVITY OF TRITERPENES Introduction
Inflammation is characterised by redness, heat, pain, swelling and organ decreased function. This process is complex and involves different mechanisms attributable to a large variety of mediators, which are distributed in three phases.
NATURAL TRITERPENOIDS
111
Poria cocos
[ Concentratlon I.,II[,. ~Maceration: MeOH/H20 (7: in,
~, Extraction EtOAc/H20
t
l EtOAc extraci'I
t
I H2~ extract
I
Phytochemlcal analysis: TLC, HPIA2 {triterpene detection} Bloassay: TPA (acute ear edema}. PLA2 (in vitro Inhibition} _
IIIIII
I
I
II
_
_
IIII
I III
I
i
Active extract: EtOAc
Separation: CC (sephadex LH-20, dlol}, VI~ (SIO2) Bloassay-gulded Isolation: TPA edema and PLA2 activity
Selection of active principles Identification by spectroscopy
Pachymic acid
Dehydrotumuloslc acld
Anti-inflammatory activity: I n vivo a n d in vitro studies Mechanism of action Fig. (1). Scheme of isolation, identification and biological activity study of anti-inflammatory lanostanes from P o r i a c o c o s sclerotia.
The first one is an acute transient phase with local vasodilatation and increased vascular permeability. It is produced by vasoactive amines, such as histamine, which induce vascular permeability by acting on the H l receptors, and also has a variety of actions on inflammatory cells. Serotonin or 5-hydroxytriptamine (5-HT) is released from blood platelets and also contributes to vasopermeability. Plasmatic bradykinin (BK), the most important kinin, is a potent vasodilator and promoter of vascular
112
RIOS eta/,
permeability, and also stimulates the release of histamine from mast cells and the synthesis of prostaglandins (PGs). PGs constitute a group of compounds derived from the C-20 polyunsaturated arachidonic acid (AA), a component of the phospholipids of the cell membrane that is usually released by hydrolysis caused by phospholipase A2 (PLA2). Once AA is hydrolysed, it is converted through the cyclooxygenase (COX) pathway into prostaglandins (PGE2, PGD2 and PGF2,x), prostacyclin (PGI2) and thromboxane B2 (TXB2). PGs are potent vasodilators, with little effect on vascular permeability, and they potentiate the pain-producing effect of other mediators. The lipoxygenase (LOX) pathway produces leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). LTs are active as permeability-enhancing factors, although LTB4 is the most powerful endogenous chemotactic factor yet described "Fig. (2)".
Lyso-PAF
Ca 2+
Vascular Permeability Increase
Fig. (2). Release of lipidic mediators and their role in the inflammation.
A second delayed subacute phase is characterised by infiltration of leukocytes and phagocytic cells. The recruitment of inflammatory cells involves the concerted interaction of several types of chemotactic mediators such as complement factor C5a, platelet activating factor (PAF) and LTB4. Several cytokines, especially interleukin-1 (IL-1) and tumour
NATURAL TRITERPENOIDS
113
necrosis factor (TNF), appear to play an essential role. IL-1 comprises two distinct polypeptides (IL- ltx and IL-1J3) that produce similar biological responses, such as mobilisation and activation of polymorphonuclear leukocytes (PMNL), induction of COX and LOX enzymes, production of other growth factors and cytokines (IL-2, IL-6, IL-8, etc.). At the site of inflammation the stimulated PMNL are capable of producing reactive oxygen species such as the cytotoxic superoxide anion ('O2-) which can react with other molecules to produce the extremely reactive hydroxyl radical ('OH), considered the main initiator of lipid peroxidation. In addition, inflammatory cells are able to release large quantities of hydrolases, such as elastase and collagenase, that catalyse the hydrolysis of tissue components involved in the extracellular proteolysis of rheumatoid arthritis and other inflammatory states "Fig. (3)". If the noxious stimulus persists, the action of all these mediators contributes to the chronic proliferative phase of inflammation. It is in this third and last phase that tissue degeneration and fibrosis occur. Differentiation between the acute and/or chronic inflammation is traditionally based on the kind of inflammatory cells that predominate in the lesions. In fact, in acute injury Mast Cells
VASODILATATION
VASCULAR PERMEABILITY
Q
Cells
Enzymes Free radicals
] Hydrolytic
Complement system
Other Lymphoklnes
Lymphocytes
Fig. (3). Relationships between inflammatory mediators and some relevant cellular events.
I 14
RIOS et at
PMNL predominate, while in chronic lesions mononuclear phagocytic cells are the main ones present. In conclusion, pharmacological control of inflammation can be modulated by antagonising or preventing the release of mediators involved in initiating or amplifying inflammation, or by direct action on inflammatory cells. It is possible to act in four major areas" 1) inflammatory amine release and activity; 2) AA metabolism; 3) phagocytic and inflammatory cell functions, and 4) autoimmune processes. Antiinflammatory agents may therefore inhibit COX and/or 5-LOX pathways, block chemotactic factors and other mediators, inhibit activation of the complement system, oxidative phosphorylation, adhesion to vascular endothelium by leukocytes, scavenge free radicals, stabilise cell membranes, etc. "Fig. (4)". Membrane phosphollplds
I lllll
~I~'VASODILATATI(~N~ I ~ ~ ~ ~
VASCULAR
PERMIEAB!LITY
!
Chemotaxls --I _.'_. ' i
"'-| IL,'XVLt~, n
Mononuc|ear i . Cells n
-"
"-
. .
r--1 ..... ~ .......
_.._ . . . . . . . . . . .
tess " "
.............
i
,,nmune
9 IIII Fig. (4). Main targets of the anti-inflammatory drugs.
General Considerations About Research on New Anti-Inflammatory Drugs
In general, two major classes of antiphlogistic drugs are available for therapy: Non steroidal anti-inflammatory drugs (NSAIDs) which interfere
NATURAL TRITERPENOIDS
115
with the COX system (e.g. salicylates), and steroidal anti-inflammatory drugs such as glucocorticoids, which have the ability to inhibit both the COX and LOX pathways by interfering with the PLA2-reaction. Developing new anti-inflammatory agents requires the use of experimental models of inflammation that reflect, if possible, the whole set of symptoms and mechanisms normally present in the inflamed tissues. In practice, such a model is difficult to find, and it is necessary to combine experimental models in vivo with tests in vitro. Furthermore, the pharmacological response observed in experimental animals such as mouse or rat can not be the same as in humans. In vitro pharmacological studies can serve as an initial screening step in which the presumed effect on a particular aspect of an inflammatory process can be investigated. In vitro techniques are very reproducible and highly sensitive, but the usefulness of the results obtained is limited by the fact that they do not reflect what happens in vivo. They therefore can not replace the in vivo methods, but are a good complement for the conventional procedures. In any case, all potential therapeutics must ultimately be evaluated in in vivo models [51-
531.
The most readily available animal models generally reflect acute or subacute reactions because they are simple, reproducible and provide clues to the biochemical and cellular mechanisms involved in the transition from acute to chronic inflammation. The cardinal signs of inflammation are the end points for the measurement of anti-inflammatory drug action (erythema, hyperemia, edema or exudation).
Screening Methods The carrageenan-induced paw edema in rat or mouse is the method most commonly used to investigate the mechanism of the inflammatory process and to evaluate the anti-inflammatory effect of an agent. Few of the available anti-inflammatory drugs produce more than 60% inhibition of edema. The properties shown by carrageenan include granuloma formation, action on blood coagulation and the kinin system, and immunologic elicitation. The edema is expressed as the difference between the foot volume before and after the injection. On the other hand, paw edemas induced by substances like 5-HT or histamine are insensitive to conventional NSAIDs and could be considered experimental models in the search for anti-allergic compounds [54]. Among the models of inflammation in vivo, those that involve the skin have the particular advantage that the results are immediately and continuously observable. Models of skin inflammation are numerous and varied, ranging from acute and limited to chronic and tissue-destructive. Croton oil, different phorbol esters, principally 12-tetradecanoylphorbol13-acetate (TPA), AA and oxazolone, provide a range of skin inflammation models suitable for the evaluation of both topical and/or
116
RIOS et at
systemic anti-inflammatory drugs. The irritant is usually applied in the right ear while the left ear remains untreated and serves as a control [55]. On the other hand, rat adjuvant-induced arthritis is a model of chronic inflammation frequently reported in screening for anti-arthritic agents. Adult rats are the animals of choice because the disease does not occur in hamsters, mice or guinea pigs. The clinical and pathological changes in adjuvant disease are comparable to those of rheumatic arthritis [56]. Numerous medicinal plants and natural compounds have been tested for anti-inflammatory activities using these models, despite their having the disadvantage of variable biological response. In addition, large quantities of pure compounds must be administered to a large number of animals in order to construct dose response-curves. However, natural products are often isolated in small quantities, which makes it necessary to analyse their activity with in vitro tests, employing cells or enzymatic systems, where only 2-5 mg of the product is necessary. Since rat or human peripheral leukocytes, when stimulated, are able to produce prostanoids, LTs and PAF, they are good cell models for studying the activity of compounds on the synthesis of lipid mediators. Among the enzyme preparations, COX, LOX or complement fractions from different sources are commonly employed. In recent years, interest in PLA2 inhibitors has increased notably because extracellular PLA2s are implicated in the pathogenesis of several important inflammatory diseases. A substantial effort has been made to establish assay protocols for the screening and characterisation of specific competitive PLA2 inhibitors [50,57]. The present-day search for new natural anti-inflammatory drugs is based on the use of these models of inflammation in which products can interfere with any or all the systems involved in the inflammation processes. In the last few years, interest in triterpenes as anti-inflammatory agents and their mechanisms of action has also increased greatly. Oleanolic acid and ursolic acid have been recognised to have anti-inflammatory activity in carrageenan-induced paw edema in rats or mice, adjuvant-induced arthritis in rats, etc. Oleanolic acid, in addition, was able to suppress the delayed hypersensitivity reaction in mice induced by dinitrochlorobenzene (DNCB). These effects are attributable to different mechanisms of action ranging from the inhibition of histamine release to inhibition of complement activity [ 1,58]. The mechanism of action of boswellic acids from crude extracts of B o s w e l l i a serrata has been established [59] and it was reported recently that they inhibit the increased urinary excretion of LTE4 in astrocytoma patients in vivo and block leukotriene biosynthesis ex vivo [60]. Until now only a few natural terpenoids have been recognised as antiP LA2 compounds. However, it seems that acidic triterpenes such
NATURAL TRITERPENOIDS
117
pachymic, dehydrotumulosic, masticadienolic or masticadienoic acids are a new class of PLA2 inhibitors [48,50]. In the next sections the recent advances regarding the anti-inflammatory activity and mechanisms of action of triterpenes are presented and their chemical structure/pharmacological activity relationships are discussed. In vivo Anti-inflammatory Studies
Carrageenan-induced Edema Among the plant terpenoids with a lupane skeleton, lupeol is the simplest one and has recently been reported as the active principle of Crataeva religiosa (Capparidaceae). When it was tested on the habitual models of inflammatory response, it showed moderate activity on the carrageenaninduced rat hind paw and rat pleurisy [61 ]. A compound with a related structure is moretenol acetate, which was the most active of the triterpenes from Pluchea lanceolata (Asteraceae). When administered 50 mg/kg p.o., it inhibited by 55% the edema caused 3.5 h after carrageenan injection into rat hind paw [62]. At 100 mg/kg p.o. neolupenol, from the same plant, reached 55% inhibition at 2.5 h and 70% inhibition at 5 h [63].
9 9 I.,VA
L . t [ ~ l . , J t Jt V A
Taraxasterol acetate from the Ayurvedic drug Echinops echinatus (Asteraceae) reached 63% inhibition after 4 h, but at a high dose regime (200 mg/kg, p.o.), and this activity was not much improved when this product was given i.p. (68% inhibition) [64]. In a study on the anti-inflammatory properties of resins from Burseraceae species, mansumbinoic acid, an octanordammarane isolated from Commiphora incisa, induced, at 0.25 mmol/kg p.o., a 48% decrease in the area under the time-course curve obtained 6 h after induction of edema. This effect proved to be dose dependent, and the calculated ED50 was 0.15 mmol/kg. The closely related ketone mansumbinone was of minor potency
118
RIOS et al.
[65]. The same authors also undertook a search for the anti-inflammatory principles of Polygonum bistorta (Polygonaceae) and obtained positive results with friedelanol and glutin-3-one. Glutin-3-one induced 56% inhibition at 0.12 mmol/kg [66].
HOO
Mansumbinone
Mansumbinoic acid
Friedelanol
Some members of the lanostane class from Pistacia integerrima (Anacardiaceae) galls proved to be very potent. When administered at only 5 mg/kg i.p. they reduced carrageenan inflammation. Pistacigerrimone A (70%) and pistacigerrimone D (62%) were especially effective [67].
/
O/cloartenol
NATURAL TRITERPENOIDS
119
The cycloartenol localised in Crataegus monogyna (Rosaceae) constitutes the main component of a fraction that reduced the 3 h carrageenan paw edema at 40 mg/kg p.o. Moreover, this fraction almost abolished leukocyte infiltration in the mouse peritonitis caused by the same inflammatory agent [68]. In a comparative study on the anti-inflammatory activity of the Diospyros leucomelas (Ebenaceae) principles and other readily available triterpenoids administered at 100 mg/kg p.o., betulinic acid showed the highest activity (45% inhibition) 3h after carrageenan injection [47]. Erythrodiol had a more durable effect, inducing a 40% inhibition after 5 h [69]. After comparing the variable effects observed on this model, it was concluded that the presence of a primary alcohol or carboxylic acid function at C-27 or C-29 is clearly positive. Adjuvant Arthritis Apart from the acute anti-inflammatory activity described in the preceding section, taraxasterol acetate was also tested for chronic activity in the model of Freund's adjuvant-induced rat arthritis. When 80 mg/kg of this compound were administered i.p., a 57% decrease with respect to the control value for the injected limb was observed at 18 h. After 21 days, the secondary reaction, which is measured on the non-injected limb, was reduced by 78%. Given the fact that in this condition the injected limb suffered a comparable reduction of 67%, it seems that taraxasterol acetate did not act by modifying immune mechanisms [64]. Duwiejua et al. [66] also reported appreciable anti-arthritic activity for glutin-3-one: 44% and 74% reductions in the ipsilateral and contralateral paw swelling, respectively, after 10 days. In the last few years, Kweifio-Okai et al. [70-72] have been studying the anti-arthritic activity of some triterpenoids and their natural or semisynthetic fatty acid esters by applying the adjuvant model and evaluating the progression of the disease by means of blood cell counts and microscopic examination of bone joints. These authors established that the effect of orally administered (56 mg/kg) ct-amyrin palmitate on the acute phase (from days 11 to 19 after adjuvant administration) is that it leads to a suppression of the increase in the ipsilateral ankle swelling, o~-Amyrin palmitate reversed the increase in spleen weight by 50% and consistently reduced plasmatic hyaluronate and the number of granulocytes in blood and bone marrow. Histological results showed amelioration of joint damage expressed as synovial proliferation and erosion of subchondral bone [70]. A very close study carried out with esters of lupeol demonstrated that the 3-1inoleyl derivative had the strongest activity, with profiles similar to that reported for o~-amyrin palmitate, but that its effect lasted longer and was observable on day 42 [71,72].
120
RiOS et al.
To expand our understanding of the anti-inflammatory activity of the well-known oleanolic acid isolated from one of its best characterised sources, the seeds of Luffa cylindrica (Cucurbitaceae), a series of experiments were performed to correlate complement activity with the inflammation caused by both carrageenan and Freund's adjuvant, in presence and absence of oleanolic acid and ibuprofen, which were administered at 60 mg/kg i.p. It was concluded that the effect of this triterpenoid must be mediated by its anti-complementary activity [73]. An analogous conclusion was drawn regarding the activity of boswellic acids in the same tests [74]. Serotonin-induced Edema
The models based on intradermal or subcutaneous injections of 5-HT not only provide a good system for measuring the earliest inflammatory phase characterised by a rapid plasma extravasation, but are also utilised as the last step in certain experiments designed to establish whether a drug exerts its anti-inflammatory activity through interactions on glucocorticoid receptor (GCR) and on DNA or protein synthesis [75]. When a series of triterpenoids were studied under this scheme, the lupane derivatives betulin and betulinic acid, administered s.c. at 50 mg/kg, proved to be quite effective against 5-HT-induced edema in mouse hind paw, producing 76% and 63% inhibition, respectively. The effect of betulinic acid was greatly reduced by concomitant progesterone (GCR antagonist), actinomycin D (transcription inhibitor) or cycloheximide (ribosomal peptidyltransferase inhibitor), which suggests that this triterpenoid may act by a steroidal mechanism. Other compounds like ursolic acid and erythrodiol were also active, but to a lower degree, and they were not much affected by these corticoid inactivators [47,69]. In a later study with the two main lanostanes of the fungus Poria cocos (Polyporaceae), both pachymic and dehydrotumulosic acids were effective (62% and 79% inhibition, respectively). Progesterone and actinomycin D substantially reduced the activity of pachymic acid, none of the inhibitors affected the activity of dehydrotumulosic acid. Pachymic acid probably acts by a corticoid-related mechanism, while in the case of dehydrotumulosic acid the inhibition of PLA2 should be the main anti-inflammatory mechanism (see below) [76]. Ph ospholipase A z-induced Edema
The anti-inflammatory effect of pachymic and dehydrotumulosic acids was determined in vivo against mouse PLA2 paw edema. At a dose of 50 mg/kg i.p., both were active (54% and 57% inhibition, respectively). As it has been proved that this model involves a release of histamine and 5-HT, an antagonism with one or both of these amines might occur, although this
NATURAL TRITERPENOIDS
121
effect could arise from the previously known inhibitory effect on PLA2 in vitro [77,48]. In vivo Models o f Allergy
In an attempt to discover triterpenoids which, because of a structural relationship, might share the anti-inflammatory and/or anti-allergic properties of glycyrrhetinic acid without its adverse reactions, Tabata et al. [78] surveyed the activity of bryonolic acid on different models of experimental mouse allergy. This acid is a friedooleanene derivative characteristic of the Cucurbitaceae that was described as a new product in Bryonia dioica and later localised in many genera of the family such as Citrullus, Cucumis, Luffa and others. Bryonolic acid itself showed an IDs0 of 376 mg/kg when given i.p. to mice in order to relieve a passive cutaneous anaphylaxis (type I allergy), but certain semisynthetic derivatives such as 3-phtalate (2 K +) or diol 3,29-diphtalate (2 K +) were more potent, with IDs0 of 34.2 and 41.4 mg/kg, respectively. These esters and other closely related ones also proved to be very effective when given orally. Bryonolic acid-3-succinate (2 K +) was also active against sheep erythrocyte-induced Arthus reaction in mice (type III allergy) and picryl chloride-induced contact dermatitis (type IV allergy) [78]. Influence on ear edema in mice
The triterpene-enriched fraction of the supercritical CO2 extract of the dried flowers of Calendula officinalis (Asteraceae) inhibited the croton oilinduced ear edema in mice. Of the identified compounds, the faradiol monoesters, lupeol, ~F-taraxasterol and a mixture of taraxasterol/13-amyrin were tested for their anti-inflammatory activity. Faradiol, obtained by hydrolysis of the extract, was the most active compound. It showed a dose-dependent effect with a potency that equals that of indomethacin at 0.14 I.tmol/cm 2 (48% and 47% edema inhibition, respectively). The esterification of faradiol resulted in a reduction of more than 50% in the activity (only 31% inhibition was observed at 0.14 Bmol/cm2), whereas Wtaraxasterol, a C-1613 dehydroxylated derivative of faradiol, was less active (47% inhibition at a dose of 0.28 lamol/cm2) [43]. Given that the biological activity of the ester mixture from C. officinalis had already been studied, Zitter-Eglseer et al. [44] isolated, separated and identified the ester components in order to study their topical antiedematous properties using the croton oil-induced inflammation test in mice. The main compounds were identified as faradiol-3-myristic acid ester, faradiol-3-palmitic acid ester and qJ-taraxasterol. The two faradiolesters showed nearly the same dose dependent anti-edematous activity: 50% inhibition at 240 ~tg/cm2 and about 65% when the dose was double. These data confirm the previous observation made by Della Loggia et al.
122
RiOS et al.
[43], that the presence of the OH-group at C-16 enhances the activity while esterifieation at position 3 reduces it [44]. The same type of triperpenes has been found in the flowers of some Asteraceae plants: taraxasterol in Cynara seolymus and faradiol in Chrysanthemum morifolium. Both of these substances were examined for inhibitory activity against TPA-indueed ear edema in mice. The compounds were applied 30 min before TPA treatment and showed a strong inhibition of edema (IDs0 = 0.3 rag/ear for taraxasterol and 0.2 mg/ear for faradiol). In comparison with the standard drugs, the triterpenes were similar in potency to indomethaein (IDs0- 0.3 mg/ear), although all of them were less effective inhibitors than hydroeortisone (IDs0 = 0.03 mg/ear). In addition, faradiol was more effective than taraxasterol on tumour promotion in two-stage carcinogenesis in mouse skin [79]. The relationship between the hydroxylation of triterpenes and their inhibitory effect was again observed among the triterpenes isolated from the ligulate flowers of Chrysanthemum morifolium, Helianthus annuus (Asteraceae) and Taraxacum platycarpum (Asteraeeae) [80]. The compounds were identified as faradiol, heliantriol B0, heliantriol C and amidiol (taraxastane-type); ealenduladiol and heliantriol B2 (lupane-type), maniladiol and longispinogenin (oleanane-type), brein and uvaol (ursanetype). They were evaluated with respect to their anti-inflammatory activity against TPA-indueed ear edema in mice, and their effects were
Poricoic acid A HOOCt,o.. ~OH
.ooc-
y...
Poricoic acid B
NATURAL TRITERPENOIDS
123
compared with those of indomethacin and hydrocortisone. All the substances inhibited the edema generation, with IDs0 ranging from 0.03 mg/ear to 0.2 mg/ear. The anti-inflammatory activity of these triterpenes was stronger than that of indomethacin, which gave an IDs0 of 0.3 mg/ear on this test. Heliantriol B2, brein and heliantriol C showed a strong inhibitory effect (for the first two, IDs0 = 0.05 mg/ear; 0.03 mg/ear for heliantriol C) comparable to that of hydrocortisone activity. Whether the substance belongs to one or another triterpene type does not seem to be important for the activity, but there is a close relationship between the hydroxylation of triterpenes and the inhibitory effect. Thus, for the A2~ taraxastenes, faradiol showed a marked inhibitory effect (IDs0 = 0.2 mg/ear). Further hydroxylation of faradiol at C-22t~, which yields heliantriol C, substantially enhanced the effect. According to Della Loggia et al. [43], dihydroxy-triterpenes were stronger inhibitors than 313monohydroxy-triterpenes [80]. Eight lanostane-type triterpene acids and four 3,4-secolanostane-type triterpene acids were isolated from the MeOH extract of P o r i a c o c o s . The active principles were identified as pachymic acid, 3-O-acetyl-16o~hydroxytrametenolic acid, dehydropachymic acid, 313-hydroxylanosta7,9(11),24-trien-21-oic acid, dehydroeburiconic acid, and poricoic acids A and B. When topically administered 30 min before application of TPA their IDs0 ranged from 16 ~tg/ear (3-O-acetyl-16t~-hydroxytrametenolic acid) to 44 lag/ear (pachymic acid). In general, their activity against TPAinduced ear edema was similar to that of the reference drug hydrocortisone, and they were much more effective than indomethacin (IDs0 = 300 ~tg/ear) [81]. Nukaya et al. [82] obtained five compounds chemically related to pachymic acid from the MeOH extract of P. c o c o s . Among these compounds, 6ct-hydroxydehydropachymic and 16o~-hydroxytrametenolic acids inhibited the edema formation more potently than pachymic acid. Both 6tx-hydroxydehydropachymic and 16t~-hydroxytrametenolic acids at 5 Ixg/ear showed the same inhibitory effect as pachymic acid at 150 lag/ear. The anti-inflammatory activity of the hydroalcoholic extract of P. c o c o s against some acute and chronic inflammatory processes was established recently. It reduced the TPA-induced edema (80% inhibition at 0.5 mg/ear), but its effect was milder against the AA-induced ear edema (40% inhibition at 0.5 mg/ear). When the extract was assayed in chronic experimental model of inflammation, it caused a 53% reduction in ear thickness together with a 73% decrease in myeloperoxidase (MPO) activity, which shows that leukocyte infiltration into the inflammation site could be prevented by a repeated dose of 1 mg/ear. The main constituents isolated from the extract were identified as pachymic and dehydrotumulosic acids. Topical administration of these lanostanes inhibited the TPA-induced edema with IDs0 of 2.48 and 0.31 ~tg/ear, respectively. Reasons for the greater activity of dehydrotumulosic acid
124
R|OS eta/.
could be found in its structural characteristics: the presence of a free hydroxyl group at position 3, that is blocked by acetylation in pachymic acid, and the presence of a heteroannular 7-8,9-11-dien group, a character eonfering planarity to the molecule [83]. Yasukawa et al. [84] demonstrated the anti-inflammatory activity of some sterols and triterpenes on the TPA-induced ear edema test in mouse. Among them, karounidiol 3-O-benzoate, a D:C-friedooleanane triterpene, showed an interesting inhibitory effect (95%) of the edema formation at a dose of 2 mg/ear; its IDs0 was 0.4 l.tmol/ear. The D:C-friedooleanane triterpene type is the main kind of compound contained in the seeds of Trichosanthes kirilowii (Cucurbitaceae) a species employed in Chinese medicine as an anti-inflammatory agent. Some of the isolated compounds were evaluated for their topical anti-edematous activity against TPA-induced inflammation in mice. 3-Epikarounidiol, 7oxoisomultiflorenol and 3-epibryonolol gave IDs0 ranging from 0.2 to 0.6 mg/ear, and these values approach that of karounidiol 3-O-benzoate. In any case, the anti-inflammatory potency of these triterpenes was lower than that of hydrocortisone [85]. It has been reported that karounidiol and 7-oxo-dihydrokarounidiol have an inhibitory effect on TPA-induced inflammation [86]. Topically applied, they completely inhibited edema generation in a dose-dependent manner. The IDs0 of karounidiol and 7-oxo-dihydrokarounidiol were 0.4 and 0.3 mg/ear, respectively. 12-O-Hexadecanoyl- 16-hydroxyphorbol- 13-acetate (HHPA) was used at a dose of 2 ~tg/ear as an inducer of mouse edema to study the antiinflammatory activity of ursolic acid and some 4,4-dimethylcholestane derivatives. The compounds were administered 30 min before HPPA, and the maximum edema was reached 7 h after application. 200 Bg of ursolic acid reduced the edema by 49%. The functional groups of this triterpene are a hydroxyl group at position 3 and a carboxyl-group at C-28, but they are not essential for the activity. For example, cholesterol has a 31Jhydroxyl group but no carboxyl at C-28, and a structure similar to that of ursolic acid, but it not only was not active at a dose of 200 ~tg/ear, but even promoted inflammation. However, 4,4-dimethylcholesterol showed weak inhibitory activity at the same dose as cholesterol. These results suggested the importance of the 4,4-dimethyl group, common to all triterpene structures, for the anti-inflammatory activity. In this study the authors also observed that the oxidation of the 3-hydroxyl group to a 3oxo group increases the inhibitory activity, while epoxidation of the double bond at C-5 to a 5,6-~-epoxy group diminishes it [87]. The MeOH extract of Rosmarinus officinalis (Lamiaceae) contains 1620% of ursolic acid. Both the extract and the pure triterpene isolated from it were strong inhibitors of TPA-induced inflammation, ornithine decarboxylase (ODC) activity and tumour promotion in mouse skin. Some authors have suggested that the effects could be related to those of
NATURAL TRITERPENOIDS
125
steroidal anti-inflammatory compounds [88]. However, when ursolic acid isolated from Diospyros leucomelas was studied for its mechanism of action in vivo against serotonin-induced paw edema in mice, the triterpene activity appeared to have no relationship with that of glucocorticoids. There was no reduction in the anti-edematous effect in presence of the anti-glucocorticoid receptor progesterone and mRNA or protein synthesis inhibitors such as actynomicin D and cycloheximide [47]. Other mechanisms have already been proposed to explain the anti-inflammatory activity of ursolic acid, and they are discussed below. The inhibitory effect of ten triterpenes was studied on different in vivo models of acute inflammation in order to establish the possible relationship between their chemical structure and anti-inflammatory activity. Compounds belonging to the lupane (lupeol), oleanane (]3-amyrin, erythrodiol, hederagenin, oleanolic acid and oc- and 13-glycyrrhetinic acids) and ursane (o~-amyrin, uvaol and tormentic acid) series were assayed and the results compared with those previously reported for ursolic acid (ursane-type), betulin and betulinic acid (lupane-type). All the topically administered triterpenes were active against TPA-induced ear edema. Erythrodiol, tormentic acid, cz- and 13-glycyrrhetinic acids were the most active (over 80% inhibition). Erythrodiol and uvaol were the most effective compounds against ethylphenylpropiolate (EPP)-induced ear edema, in which the anti-inflammatory action is delayed. In general, the triterpene acids were the most active substances on the TPA test, while their effect was weaker against EPP-inflammation and did not depend on the presence of a carboxyl group. In this last method, the presence of a hydroxymethyl group at position 17 seems to increase the activity in the oleanane series, as occurs with erythrodiol [69]. In connection with the work on the relationship between chemical structure and anti-inflammatory activity, the effect of ursolic acid, betulin, betulinic acid and erythrodiol on a system of chronic dermal edema and cellular proliferation caused by repeated administration of TPA has recently been examined [89]. This experimental model of chronic inflammation has considerable selectivity for corticosteroids and leukotriene synthesis inhibitors. Erythrodiol and ursolic acid were significantly effective and also reduced the neutrophil infiltration detected by MPO activity. The lupane derivatives, betulin and betulinic acid, despite their possible steroid-like mechanism of action [47], were not effective in the chronic model. This result could mean that a six-member E ring of the pentacyclic structure is necessary for the activity against a multiple dose of TPA. The data confirm that a hydroxyl group at the C-28 position is important for the activity, as is also true in the case of erythrodiol, and it may explain the anti-inflammatory effect of this compound in each of the methods. A study was recently undertaken [90] to enlarge our understanding of the mechanism of action of triterpenes as topical anti-inflammatory agents
126
R|OS eta/.
by examining the effect of eleven triterpenes against mezerein-mediated ear edema in mice. Mezerein is an irritant diterpenoid present in some species of Thymelaceae with a certain selectivity for t~, 13 and T-isozymes of protein kinase C (PKC), enzymes involved in the edema formation induced by phorbol-esters. Of the triterpenes tested, erythrodiol was again the most active one (57% at 0.5 mg/ear), in a range similar to the standard drug indomethacin. Topical application of capsaicin on mouse ear causes a neurogenic edema, with a maximum at 30 min after the treatment. The release of 5HT, substance P (SP) and neurokinin-1 (NK-1), which interact with the respective receptors in the ear skin provoke the edema [91,92]. Dexamethasone, histamine H l and/or 5-HT and SP antagonists inhibited the edema, whereas AA metabolism inhibitors were not effective [93]. Glycyrrhetinic acid and three dihemiphthalate derivatives were recently examined for their anti-edematous activity against capsaicin-induced ear edema in mice [94]. Derivative compounds gave IDs0 values ranging from 41 to 53 mg/kg (p. o.); however, glycyrrhetinic acid was ineffective at 200 mg/kg (p.o.). In conclusion, although glycyrrhetinic acid is active in other experimental conditions of inflammation, it has no effect on vasodilation and plasma extravasation induced by the neuropeptides released by the action of capsaicin. In vitro Studies on Inflammatory Mediators
Modulation o f Lipoxygenase Activity
Leukotrienes, for which the 5-LOX is the key synthetic enzyme, are involved in initiating and maintaining a variety of inflammatory diseases such as psoriasis, bronchial asthma, chronic rheumatism and anaphylactic shock. In the family of leukotriene-type mediators, LTB4 is the most potent stimulator of leukocyte responses such as chemotaxis, cell adhesion, superoxide production, and release of hydrolytic enzymes. From the theoretical point of view, products with specific 5-LOX activity inhibition have a high therapeutic value. At the moment there are drugs caffeic acid, phenidone, 3-amino-(1-trifluoromethyl)pyrazoline (BW 755C), eicosatetrainoic acid, nordihydroguaiaretic acid (NDGA) and retinoids--- that are potent, but not specific inhibitors of 5-LOX activity. Since the 5-LOX activity is sensitive to general antioxidants, almost all of the first generation 5-LOX inhibitors belong to the redox type inhibitors. The gum resin of Boswellia serrata (Burseraceae) has been used for the treatment of inflammatory diseases in the traditional Ayurvedic medicine in India. The EtOH extract of the resin significantly decreased LTB4 production in rat peritoneal neutrophils in vitro [95]. The active principles of the gum resin exudate of B. serrata are pentacyclic triterpene acids identified as boswellic acids, whose chemical skeleton belongs to the
NATURAL TRITERPENOIDS
127
ursane type. The prominent form of boswellic acid is the 13-isomer, while the minor components are ot-boswellic acid and 11-keto-13-boswellic acid [49]. These substances have been studied in vitro to demonstrate that the mechanism of inhibition of the 5-LOX activity is not connected with antioxidative properties [96]. These authors therefore employed rat peritoneal PMNL, which when stimulated with Ca2+ ionophore produce mainly LTB4 and 5-HETE from endogenous AA. All the forms of boswellic acid inhibited 5-LOX activity in a concentration-dependent manner, with comparable IC50 values, ranging from 1.5 lxM for acetyl-11keto-13-boswellic acid (AKBA) to 7 IxM for acetyl-13-boswellic acid. At all the tested concentrations the boswellic acids decreased LTB4, its all-transisomers and 5-HETE levels simultaneously. This means that boswellic acids effects on the LTA4 hydrolase and/or glutathione peroxidase, which catalyse the conversion of LTA4 into LTB4 and of 5hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-HETE respectively, should be negligible. The results were compared with glycyrrhetinic acid, which despite its anti-inflammatory activity did not decrease the formation of LTB4 and 5-HETE. The reference drugs were hydrocortisone, which in this system exerted no immediate effects, and the redox type 5LOX inhibitor NDGA, which reduced the LTB4 formation with an IC50 of 0.5 I.tM. In washed human platelets, NDGA inhibited the formation of 12-LOX products as well as the COX product 12-hydroxyheptadecatrienoic acid (12-HHT), with an IC50 of 5 I.tM, while boswellic acids (mixture of o~- and 13-isomers) at concentrations up to 400 BM exerted no effect. In addition, in a cell-free system, the non-enzymatic peroxidation of AA by iron (II)ascorbate was not affected by boswellic acid at concentrations up to 400 BM, whereas NDGA abolished the peroxidation of AA in this test at a concentration of 10 laM. These results suggest that the activity of boswellic acids is selective in the 5-LOX pathway, and the mechanism of inhibition is different from that of the classic antioxidant 5-LOX inhibitors (NDGA, caffeie acid, quercetin) [96]. AKBA inhibited 5-LOX activity without modifying 12-LOX and COX-1 activities. Several in vitro experiments have been performed [59] to identify the mechanism of this novel inhibitor of the LTs synthesis. The activity of AKBA in the cell-free system and purified enzyme suggested that the point at which the inhibition of 5-LOX occurs is different from the AA substrate binding site, where the direct 5-LOX inhibitors (e.g. zileuton and AA-861) act. In contrast with AKBA, other pentacyclic triterpenes such as o~-amyrin or ursolic acid did not inhibit 5LOX activity in the cell-free system at comparable concentrations. In presence of increasing concentrations of the non-inhibitory triterpene amyrin, AKBA's inhibitory effect on 5-LOX activity was antagonised. In contrast with the functional antagonism, the effects of 5-LOX inhibitors from different chemical classes were not modified in presence of increasing
128
RiOS et al.
concentrations of amyrin. To study whether the antagonistic effects of inhibitory and non-inhibitory triterpenes were due to nonselective lipophilic interactions, the inhibitory activity of AKBA was studied in presence of tetracyclic steroids (e.g. cholesterol, cortisone, and testosterone). These four-ring compounds, at comparable concentrations, did not modify the 5-LOX activity or reverse the AKBA-induced inhibition of 5-LOX. In conclusion, AKBA is an enzyme-directed, non-redox based LT biosynthesis inhibitor that interacts with 5-LOX via a pentacyclic triterpene-selective binding site that is different from the AA substratebinding site [59]. The structural requirements of boswellic acid type 5-LOX inhibitors for selective bindings to the effector site and for enzyme inhibitory activity have recently been reported by Sailer et al. [97]. Saponification of AKBA yielded 11-keto-13-boswellic acid with a free 3o~-OH function. This deacetylation slightly diminished the 5-LOX inhibitory potency in intact cells and in a cell free system. A minor decrease in 5-LOX inhibitory activity was found to be caused by a reduction of the carboxyl function of 11-keto-13-boswellic acid to a primary alcohol function. This yielded an 11-keto diol-triterpene, which was still able to inhibit 5-LOX activity. In order to study whether the 11-keto function of pentacyclic triterpenes is enough to inhibit the activity, the compounds without a carboxyl group were assayed and proved to have no activity. 13-Boswellic acid, which lacks the keto function on C-11, only partially inhibited 5LOX activity. The reduction of the carboxyl function of 13-boswellic acid to an alcohol yielding 3~, 24-diol functions caused a total loss of inhibitory activity. However, the existence of an 11-keto group alone in the pentacyclic triterpene is not sufficient to inhibit the 5-LOX activity. It must be combined with an additional hydrophilic group in C-4 of ring A, as demonstrated by the action of AKBA, 11-keto-13-boswellie acid and 11keto diol corresponding to 13-boswellic acid. The data suggest that the binding of pentacyclic triterpenes to the effeetor site is mediated by the pentacyclic ring system, whereas defined functional groups, especially the 11-keto function, are required for inhibiting the 5-LOX activity [97]. Hopanoids are a group of unusual pentacyclic triterpenes that are present in species of bacteria and blue-green algae. Three hopanoids isolated from Zymomonas mobilis were evaluated for their ability to inhibit three AA-metabolising enzymes: 15-LOX from soybean, and human 5-LOX and COX. Only 15-LOX was inhibited by tetrahydroxybacterihopane (THBH) with an IC50 of 10 IxM, but not by the other hopanoids, THBH-glucosamine and THBH-ether. The latter two enzymes were not significantly inhibited at 25 or 50 IxM of THBH. The selective inhibition of 15-LOX by THBH indicates that potentially it has unique pharmacological properties, which are interesting, because most NSAIDs inhibit 5-LOX, 15-LOX and COX [98].
NATURAL TRITERPENOIDS
129
Flowers of Calluna vulgaris (Ericaceae) are used in folk medicine for their anti-inflammatory properties. Using a LOX activity test, extracts from this plant were screened to characterise the principle responsible for this effect. Ursolic acid was isolated from the acetone extract and its antiLOX activity was studied. Its IC50 was identical (about 0.3 mM) for both 5- and 15-LOX. Ursolic acid at a concentration of 1 IxM also inhibited LOX activity in mouse peritoneal macrophages as did NDGA, but ursolic acid was more specific. While NDGA diminished the level of COX metabolites, this enzyme was only slightly affected by the ursolic acid. Inhibition was also studied in human platelets and HL60 leukemic cells, and the results were compared with classic LOX and COX inhibitors. The effect of ursolic acid on macrophages was weaker than that of NDGA or BW755C, while the effect of the triterpene and BW755C on the formation of LOX and COX products was more pronounced than that of NDGA. In conclusion, the LOX inhibitory activity of ursolic acid appears to be dependent upon cell type (macrophage, platelet, granulocyte) [99,100]. In order to obtain more information on the chemical reactions involved in the LOX inhibitory effect of triterpenes, ursolic acid and its analogues (uvaol, oleanolic acid and methyl ursolate) were studied on LOX activity. The best inhibitors of soybean 15-LOX were ursolic and oleanolic acids, with IC50 values of 0.175 and 0.265 mM, respectively. These results showed that the carboxylic group in the position 28 in ursolie acid is implicated in the inhibition of LOX activity because methylation of this functional group abolished it. Other structural features of ursolic acid are also relevant for its inhibitory effect, for oleanolic acid is less active than ursolic acid, and yet they only differ in the position of one methyl group (at C-20 instead of C- 19) [101 ]. To establish the mechanism of action of amyrin acetates as antiinflammatory agents, ct-amyrin acetate, ~-amyrin acetate and 13-amyrin were tested for their effects on the synthesis of 5-LOX products in human neutrophils. None of the triterpenes had any effect on LTB4 synthesis but all reduced 5-HETE synthesis, tx-Amyrin acetate and 13-amyrin acetate inhibited neutrophil synthesis of 5-HETE to the same extent (about 30%). 13-Amyrin inhibited it by 58%, while equivalent concentrations of txamyrin only gave 27%. These data suggest a specific inhibition of glutathione peroxidase, ct-Amyrin, 13-amyrin and their acetates do not appear to affect the 5-LOX enzyme, which catalyses the conversion of AA to 5-HPETE and its subsequent conversion to LTA4, the unstable precursor of the LTs. The results are inconsistent with the greater antiarthritic/anti-inflammatory activity of 13-amyrin acetate in vivo, and for this reason, the authors propose that the selective anti-inflammatory activity of the acetates could be due to the relative effectiveness of the alcohols in inhibiting the 5-HETE synthesis [ 102]. t~-Amyrin and its palmitate and linoleate esters were also tested for COX and LOX activities. The anti-COX effect was assessed using the
130
RiOS et as
carrageenan paw edema in rats, whereas anti-LOX activity was evaluated by measuring the production of 5-HETE and LTB4 by human neutrophils. At a concentration of 50 IxM, t~-amyrin reduced 5-HETE production without affecting LTB4 synthesis, but t~-amyrin palmitate only reduced LTB4 while its linoleate ester had considerable anti-LOX effects. This effect may be due to the competition between the unsaturated fatty acid linoleate and AA in the formation of inflammatory products. The antiLOX effect of o~-amyrin linoleate is similar to that of methotrexate on human arthritic neutrophils; it reduces LTB4 formation by 50%, although o~-amyrin linoleate at 62 ~M caused twice the inhibition of 5-HETE by methotrexate [ 103]. These same authors have studied in vivo and in vitro the effects of the triterpenes lupeol, lupeol-3-palmitate and lupeol 3linoleate on the release of the anti-arthritic joint degradative enzyme collagenase and on the release of LOX inflammatory products by human neutrophils. These compounds hardly inhibited LTB4 formation, and lupeol linoleate was the most active out of the three, with a 30% inhibitory concentration of 27 ~tM. It seems that the mechanism for these effects may be a stabilisation of plasma membranes [71 ].
Human Leukocyte Elastase Activity and Other Hydrolytic Enzymes Human leukocyte elastase (HLE) is a serine protease produced and stored by PMNL and involved in the tissue destruction observed in many inflammatory diseases such as chronic arthritis. In fact, administration of exogenous elastase inhibitors could be a means of protecting tissues from proteolytic attack. Several plant triterpenes (ursolic, oleanolic and 1813-glycyrrhetinic acids) have been evaluated as competitive inhibitors of riLE in vitro [ 104]. Ursolic acid was the most active compound, interacting with the enzyme in a rapid, reversible manner. The authors concluded that the carboxyl group at position C-28 of ursolic acid is involved in binding to HLE, because the presence of a high concentration of NaC1 (1 lttM) in the assay system does not change the mode of inhibition but the Ki value rises from 4 ~tM to 13 IxM. This proves the contribution of electrostatic interactions involving this group. The results were compared with those of uvaol, a pentacyclic triterpene with a similar structure but with an OH group instead of COOH at position 28. Uvaol also proved to be a competitive inhibitor, with a Ki (16 ~tM) similar to that of ursolic acid in high salt solution. These results demonstrate that electrostatic interaction between the 28-carboxyl group and a positively charged group in the enzyme contribute to binding. This conclusion is also supported by the results obtained with other pentacyclic triterpenes belonging to the 13-amyrin series in which oleanolic acid is a stronger inhibitor than erythrodiol. It seems that the binding of triterpene acids to HLE is mediated by the formation of a salt bridge involving the COOH group of inhibitors. The
NATURAL TRITERPENOIDS
131
mechanism of inhibition can be understood if we compare ursolic acid activity with that of oleic acid and cis-unsaturated fatty acid analogues. The data suggest that the substrate-binding domain of HLE is able to bind hydrophobic structures. The boswellic acid derivative, AKBA, decreases the activity of riLE in vitro in a concentration-dependent manner, with an ICs0 value of 15 laM. This result has been compared with those obtained with ursolic acid, amyrin and 18]]-glycyrrhetinic acid, the last of which showed no inhibitory activity on HLE at concentrations up to 20 [tM [105]. Data analyses indicate different mechanisms for the inhibitory actions of the pentacyclic triterpenes AKBA and ursolic acid; inhibition is noncompetitive in the case of AKBA but competitive with ursolic acid. These results are in line with the hypothesis that pentacyclic triterpenes interact with the extended substrate-binding domain in HLE that can bind a variety of hydrophobic ligands [104]. Depending on the substrate length, HLE inhibition can be competitive (ursolic acid) or non-competitive (AKBA). In summary, HLE inhibition has been established for different compounds, but a dual HLE/5-LOX inhibitory property is characteristic of the pentacyclic triterpenes from the boswellic acid series. Although alternative mechanisms of action may contribute to the anti-inflammatory action of pentacyclic triterpenes, these compounds can serve as parent compounds for a new class of HLE inhibitors. Collagenase production and release are partly responsible for the joint destruction that characterises human rheumatoid arthritis. Triterpenes from the lupane and o~-amyrin groups have been studied in vitro to examine their effects on the release of the arthritic joint degradative enzyme collagenase using the rat osteosarcoma. This test and the rat synovial granuloma of adjuvant arthritis are similar; both models are based on connective tissue tumours with bone-invasive properties. The pentacyclic triterpenes assayed have been shown to possess general antiproteolytic effects that can explain the anti-arthritic effects in adjuvant arthritis in rats [71,103]. Effects on Phospholipase A 2A ctivity
PLA2 is an enzyme that is involved in the metabolism of phospholipids and, in consequence, inhibition of this enzyme could be useful for controlling certain inflammatory diseases. Anti-PLA2 activity against three different forms of PLA2 was reported by Jain et al. [50] for two lanostanes isolated from Schinus terebinthifolius (Anacardiaceae) and identified as masticadienoic and masticadienolic acids. According to the authors, these compounds have a novel pharmacophore that interacts with the catalytic site of the enzyme. The two substances differed significantly in their inhibitory potencies in relation to the PLA2 from different sources. Masticadienolic acid, also called schinol, at 0.016
132
RIOS et aL
mole fraction (mF) produced a 50% inhibition of the PLA2 from pig pancreatic enzyme, and a similar value was observed against PLA2 from bee venom. When it was assayed against the recombinant PLA2 from the human synovial fluid enzyme, the ICs0 was 0.05 mF. On the other hand, masticadienoic acid was active against PLA2 from pig pancreas and bee venom, but was ineffective on PLA2 from synovial fluid. These results were compared with those of pentacyclic triterpenes (ursolic and oleanolic acids) and the related lanostane derivatives from Ganoderma lucidum (Polyporaceae), called ganoderic acids [106]. Ursolic acid at 0.1 mF inhibited the activity of the bee venom enzyme by 50% without producing any significant effect on the enzyme from the two other sources, whereas oleanolic acid was totally inactive. Ganoderic acids presented different results depending on the substitution pattern of the rings. Ganoderic acids R and S were active against pig pancreas PLA2 (0.03 and 0.08 mF, respectively), while ganoderic acid T was able to inhibit all the enzymes regardless of the origin. Structure/activity correlation, based on the above mentioned results, indicated that the terpenoid nucleus, the substitutions on the tetracyclic ring, and the aliphatic acidic side chain are important for the activity. In addition, the results of kinetic studies suggest that the mechanism of inhibition was competitive, and that binding to the catalytic site was due to the carboxylic group because when it was methylated, the activity disappeared. The amphipathic character of both triterpenes could explain the minor potency of masticadienoic acid in relation to schinol: the ionised carboxylate group is probably at the surface whereas the tetracyclic nucleus must be in the hydrophobic zone of the bilayer [50]. Two other lanostane-type triterpenes, pachymic and dehydrotumulosic acids, were evaluated as PLA2 inhibitors in a polarographic assay carried out with PLA2 from Naja naja venom [48]. Dehydrotumulosic acid was the most active inhibitor, with an ICs0 of 0.845 mM, around three times lower than that of mepacrine (ICs0 = 2.16 mM), the reference drug used in this study. Pachymic acid exhibited a potency (ICs0 = 2.9 mM) in the same range as mepacrine. The results were in concordance with the structural features indicated above for triterpenes from Schinus but, in addition, it seems that the free carboxylic group present in the side chain can change its position along this chain. lnterleukin-1 Release
Celastrol, from Tripterygium wilfordii (Celastraceae), inhibited mouse IL-1 production in peritoneal macrophages, induced by lipopolysaccharide, and IL-2 production in splenic lymphocytes induced by concanavalin A [ 107]. This plant also furnished three novel tdterpenes named regeol A, B and C, belonging to the D:A-friedooleanane type and nine known triterpenes [42]. In rheumatoid arthritis, the degree of inflammation of the articular synovial membrane has to do with the production of IL-1. For this reason, the
NATURAL TRITERPENOIDS
133
triterpenes were evaluated for their inhibitory activity on IL-1 ct and IL-113 release from lipopolysaccharide-stimulated human peripheral mononuclear cells. Among the compounds, tingenin B, celastrol and derivatives of celastrol showed strong inhibitory activity (100% release inhibition), while regeol A only inhibited by about 50% and the other two new triterpenes were not active.
Lipid Peroxidation and Free Radicals Two of the more widespread triterpenes, oleanolic and ursolic acids, are inhibitors of lipid peroxidation (LP) in vitro, as demonstrated by Balanheru and Nagarajan [108] using rat hepatic microsomes with three different stimulator systems CC14,ADP/NADPH and iron/ascorbate. Pretreatment of the microsomal fraction with the assayed triterpenes always offered a higher degree of protection against peroxidation than did simultaneous and post-induction treatments. When using iron/ascorbate as inducers, both pre- and simultaneous treatment with oleanolic acid reduced malondialdehyde (MDA) production by 90%, a current parameter indicating oxidative rupture of fatty acid residues. The same percentage was obtained for pre-treatment with CC14 or ADP/NADPH. The highest value obtained for ursolic acid was a 61% reduction in the iron/ascorbate system [ 108]. Several well documented studies have shown the antioxidant properties of Glycyrrhiza glabra (Papilionaceae) saponins and sapogenins, and it has recently been reported that L-ascorbic acid-2-(2013-11-oxo-olean- 12-en-29oic acid ethyl ester-3[~-yl hydrogen phosphate) sodium salt (GEPC), a 13glycyrrhetinic acid ester conjugated to vitamin C, has the same properties. This chimaeric compound is a free radical scavenger that is much less active than ascorbic acid on the in vitro 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test, but it is an efficient inhibitor of free radical generation, particularly of hydroxyl generation by the Fenton-type reagent Clgi+/H202 monitored by electronic spin resonance (ESR). In addition, GEPC acts as an iron chelator and has no demonstrated prooxidant activity in the iron~leomycin DNA degradation system. With respect to its effects on LP, 1 mM GEPC strongly inhibited microsomal MDA production induced by Fe3+/ADP/NADPH, in contrast with the very limited effect of glycyrrhetinic acid [ 109]. Quite different are the chemical features of some newer antiperoxidative triterpenoids from Trypterigium wilfordii, because they have highly unsaturated A and B rings with a quinonoid-like structure over a friedooleanane skeleton. The main datum to be noted is that celastrol, the most representative compound of this series, inhibits mitochondrial LP, with an ICs0 = 7 ~tM. Its potency is then 15 times higher than that of t~tocoferol. Furthermore, it has been observed that while celastrol and its acetyl derivative affect the radical chain reaction in a biphasic manner, txtocoferol and pristimerin, a celastrol-methyl ester, do it monotonously.
134
R[OS eta/.
Considering that celastrol and pristimerin act as radical scavengers whereas acetylcelastrol is inactive, and that none of them affect oxygen radical generation, the 2-oxo-3-hydroxy grouping is surely essential for a free radical-related LP inhibition. When operating with mitoplasms (mitochondria deprived of their outer membrane) LP run monotonously in the presence of each triterpenoid, which indicates that acetylcelastrol should affect only the inner membrane. This is in fact confirmed by the increase in the ~ negative potential in neutral egg-yolk phosphatidilcholine liposomes, which brings with it an increase in the stability of the membranes [110]. Metabolism o f Endogenous Corticosteroids
This point probably constitutes one of the triumphs of the rational use, but also misuse, of triterpenoids in therapeutics, because both the antiinflammatory activity and potentially toxic mineralocorticoid side effect of liquorice products are known to depend on an inhibition of glucocorticoid metabolism. More precisely, they depend on the inhibition of 1113hydroxysteroid dehydrogenase (ll-HSD), a widely distributed key enzyme for the inactivation of cortisol---corticosterone in rodents--which catalyses the conversion of the 11-hydroxyl group into an 11-oxo group to give cortisone and 11-dehydrocorticosterone, respectively. This step is thought to be of great importance in the regulation of the access of aldosterone to the mineralocorticoid receptor (MCR), but it is also very important for the alternative access of glucocorticoids to both MCR or GCR and has thus led to the concept of "enzyme-mediated receptor protection" [ 111 ]. As a consequence, glycyrrhetinic acid has been shown to potentiate the slight anti-inflammatory effect of hydrocortisone in the skin; it displaces the dose/response plot to the left and increases the slope. Restraining the activity of 11-HSD is then a valuable goal if we want to increase the efficacy of both hormonal and exogenous corticoids in vivo, and even more so in the case of inflammo-proliferative skin diseases. In these diseases 11HSD is present in larger amounts, as was detected by immunohistochemistry of normal, psoriatic and eczematous human skin [1 12]. In a complete multi-organ screening, the inhibitory effect of glycyrrhetinic acid on 11-HSD in vivo was detectable in many other tissues 3h after i.p. administration, although it disappeared by 24 h. The most sensitive organs were kidney, liver, testis, thymus and spleen [ 113]. Whorwood et al. [1 14] have reported that after continuous administration of 75 mg/kg/day of glycyrrhizin, for 5 days in drinking water to rats, both 1 I-HSD activity and 11-HSD mRNA levels were significantly diminished in selected specific mineralocorticoid (distal colon, kidney) and glucocorticoid (liver, pituitary) tissues. It was further demonstrated that glycyrrhetinic acid had an effect not only on 11-HSD activity and 11-HSD mRNA levels in pituitary GH3 cells, but also on
NATURAL TRITERPENOIDS
135
prolactin (PRL) mRNA levels, which are indicative of hypophysial GCR activation. In this system, glycyrrhetinic acid reduced 11-HSD-related parameters at doses between 10-8-10.5 M, but failed to affect PRL-related parameters. This suggests that there is no direct interaction with GCR. In contrast, the glucocorticoid agonists dexamethasone and RU 28362 did not affect 11-HSD activity although they reduced, as was to be expected, the expression of the PRL gene. Simultaneous presence of corticosterone and glycyrrhetinic acid resulted in a substantial inhibition of PRL mRNA synthesis, and this effect was abolished by the glucocorticoid antagonist RU 38486 but not by the mineralocorticoid antagonist RU 26752. For this reason some authors have concluded that glycyrrhetinic acid protects GCR by means of 11-HSD inhibition, and that its actions do not arise from any direct steroid agonism [ 114]. More evidence of the importance of 11-HSD in the central nervous system feedback regulation of corticosteroid production is the diminished release of corticotrophin-releasing factor-41 (CRF-41) to hypophysial portal blood in the presence of glycyrrhetinic acid and stable levels of circulating corticosteroids. As this effect disappears in adrenalectomised rats, it is evident that glycyrrhetinic acid needs, and therefore acts through, the presence of corticosterone. Moreover, when dexamethasone, which is not extensively metabolised by 11-HSD, is added to the glycyrrhetinic acid treatment in adrenalectomised rats, CRF-41 levels fall, demonstrating the implication of the enzyme [115]. All these assumptions on the physiopathological role of 11-HSD and its modification by certain triterpenoids are well established, but the whole scenario is presently changing due to the recent description of two different isoenzymes: l l-HSD-1, which is bi-directional, NADPdependent, acts with little substrate specificity and has a Km in the mM range, and 11-HSD-2, which at first was considered unidirectional, collocalises with MCR, is NAD-dependent, acts with great substrate specificity and has a Km in the nM range. A further 11-HSD-3 has been characterised from the choriocarcinoma cell line JEG-3 [ 116]. 11-HSD-2 apparently performs the functions associated with the classic mineralocorticoid-like effects, while the widely distributed 11-HSD-1 must be responsible for certain hepatic metabolic processes, including the retro conversion of cortisone into cortisol, and of l l dehydrocorticosterone to corticosterone in rats. The latter conversion accounts for potential enzymatic anti-inflammatory effects based on an increase in these adrenal hormones. This was in fact recently demonstrated in glomerular mesangial cells stimulated with IL-113 and TNF-o~ to release PLA2, an event which is inhibited by 11-hydroxy-glucocorticoids. IL-113 and TNF-ct counterbalance their own pro-inflammatory effects by upregulating the reductase activity of 11-HSD-1, and therefore make 11keto-glucocorticoids appear to be active in this system [ 117].
136
RIOS a aL
Anti-complement A ctivRy
Some of the best known anti-inflammatory triterpenoids have been shown to have inhibitory activity on the complement cascade. A mixture of the aforementioned boswellic acids reduced in a dose-dependent manner the classic pathway activity by as much as 77%, and C3-convertase by 72% [118]. Oleanolic acid showed 85% and 71% inhibition, respectively, in the same tests, at a single dose of 100 l.tg/ml [ 119], and [3-glycyrrhetinic acid inhibited the classic human pathway with an ICs0 of 35 laM. The componem affected was C2. The o~ form of glycyrrhetinic acid was fairly inactive [ 120]. None of these three triterpenoids appreciably inhibited the alternative complement pathway. Histamine Release
In a search for the principles responsible for the histamine release inhibitory activity of Melaleuca leucodendron (Myrtaceae) fruits, some phenolics and triterpenoids were isolated. Ursolic acid was the only triterpenoid that had an effect. At a concentration of 1 mM, it was able to reduce by 95% the amount of liberated histamine from rat mast cells. Other triterpene aldehydes and acids from the same source were inactive [ 121 ]. In a similar biological preparation, the tetracyclic triterpenes penasterone and acetylpenasterol acted with IC50 of 1.5 lttM and 10 I.tM respectively [ 122]. Rather more specific was the work of Lee et al. [ 123], who studied the inhibitory activity of glycyrrhetinic acid on histamine synthesis in mouse mast cells, co-cultured with fibroblasts in order to approach the physiological phenotype of connective tissue mast cells. In these conditions, 50 IttM glycyrrhetinic acid inhibited histidine decarboxylase (HDC) by 80% and PKC-8-mRNA expression, thus suggesting that this isoenzyme regulates HDC activity. Protein Kinase C
One of the key points, possibly the most important one, in the inflammatory and tumour-promoter effects of TPA and related phorbols is the activation of PKC. For that reason many of the compounds biologically opposing TPA are tested for PKC inhibition. Glycyrrhetinic acid showed great homogeneity in the ICs0 for both inhibition of rat brain PKC (IC50 = 450 l.tM) and TPA binding to mouse epidermal membranes. This may mean that there is a causal relationship between these parameters. Glycyrrhizin was a much weaker inhibitor, causing a 23% reduction at 1 mM, in contrast with 90% for glycyrrhetinic acid at this dose [124]. In a later study covering the interaction with rat brain PKC and other protein kinases, the ICs0of glycyrrhetinic acid for PKC
NATURAL TRITERPENOIDS
137
inhibition was reported to be 121 IxM, whereas glycyrrhizin was inactive and betulinic, ot-glycyrrhetinic, oleanolic and ursolic acids had IC50 below 300 l.tM. The potencies were noticeably higher for the inhibition of the rat liver cAMP-dependent protein kinase, which led the authors to discuss whether this fact is useful in explaining previously reported biological effects of triterpenoids [ 125].
NO Synthesis Nitrogen monoxide (NO), widely misnamed "nitric oxide", is considered to have an important role in the regulation of inflammatory processes. When generated by constitutive NO synthase (NOS) it exerts protective and anti-inflammatory properties, in striking contrast with what occurs when inducible NOS activity dominates. For this reason, drugs capable of reducing or abolishing inducible NOS response are thought to be potential anti-inflammatory agents. Pristimerin, a friedooleanane which has been cited above as one of the main active constituents of the plant Trypterigium wilfordii, showed an inhibitory effect on lipopolysaccharideinduced NO production by cultured RAW 264.7 murine macrophages, measured in terms of L-arginine consumption and nitrite accumulation (IC50 = 0.2-0.3 IxM). This inhibition was proven not to depend on a direct enzyme inhibition but on a decrease in NOS induction, which occurred at a transcriptional level by a nuclear factor-kappa B (NF~zB)-mediated pathway. Furthermore, pristimerin 1 gM also reduced the NOS mRNA level, evaluated by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In contrast, no effect was observed on COX-2 mRNA levels, which are known to be also responsive to NF~cB [126]. ABBREVIATIONS AA
=
Arachidonic acid
Ag-Ab
=
Antigen-Antibody
AKBA
=
Acetyl-11-keto-13-boswellic acid
BK
=
Bradykinin
BW 775C =
3-Amino-( 1-trifluoromethy l)pyrazoline
COX
=
Cyclooxygenase
CRF
=
Corticotrophin-releasing factor
DAG
=
Diacylglycerol
DNCB
=
D initrochlorobenzene
138
RiOS a a/.
DPPH
=
1,1-Diphenyl-2-picryl-hydrazyl
EPP
=
Ethylphenylpropiolate
ESR
=
Electronic spin resonance
GCR
=
Glucocorticoid receptor
GEPC acid
=
L-Ascorbic acid-2-(20 13-11-oxo-olean- 12-ene-29-oic ethyl ester-313-yl hydrogen phosphate)sodium salt
HDC
=
Histidine decarboxylase
HETEs
=
Hydroxyeicosatetraenoic acids
HHPA
=
12-O-Hexadecanoyl- 16-hydroxyphorbol- 13-acetate
12-HHT
=
12-Hydroxyheptadecatrienoic acid
HLE
=
Human leukocyte elastase
5-HPETE
=
5-Hydroperoxyeicosatetraenoic acid
11-HSD
=
1113-Hydroxysteroid dehydrogenase
5-HT
=
Serotonin, 5-Hydroxytriptamine
IL-1
=
Interleukin- 1
/.p.
=
Intraperitoneally
IP3
=
Inositol triphosphate
LOX
=
Lipoxygenase
LP
=
Lipid peroxidation
LTs
=
Leukotrienes
MCR
=
Mineralocorticoid receptor
MDA
=
Malondialdehyde
mF
=
Mole fraction
MPO
=
Myeloperoxidase
NDGA
=
Nordihydroguaiaretie acid
NFKB
=
Nuclear factor-kappa B
NK-1
=
Neurokinin- 1
NOS
=
N O synthase
NSAIDs
=
Non-steroidal anti-inflammatory drugs
ODC
=
Omithine decarboxylase
NATURAL TRITERPENOIDS
PAF
=
Platelet activating factor
PGs
=
Prostaglandins
PKC
=
Protein kinase C
PLA2
=
Phospholipase A2
PLC
=
Phospholipase C
PMNL
=
Polymorphonuclear leukocytes
p.o.
per os
139
(Orally)
PRL
=
Prolactin
RT-PCR
=
Reverse transcriptase-polymerase chain reaction Subcutaneously
S.C. SP
=
Substance P
THBH
=
Tetrahydroxybacterihopane
TNF
=
Tumour necrosis factor
TPA
=
12-Tetradecanoylphorbol- 13-acetate
TXs
=
Thromboxanes
REFERENCES [1] [2] [3] [4] [5] [6] [7]
[8] [91 [~0]
[12] [13l [14] [15] [16] [17]
[18]
Mahato, S.B.; Nandy, A.K.; Roy, G. Phytochemistry, 1992, 31, 2199. Mahato, S.B.; Kundu, A.P. Phytochemistry, 1994, 3 7, 1517. Mahato, S.B.; Sen, S. Phytochemistry, 1997, 44, I185. Connolly, J.D.; Hill, R.A.; Boar, R. Nat. Prod. Rep., 1984, 1, 53. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1985, 2, I. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1986, 3, 421. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1989, 6, 475. Connolly, J.D.; Hill, R.A. In Methods in Plant Biochemistry. Terpenoids; Charlwood, B.V.; Banthorpe, D.V., Eds., Academic Press: London, 1991, Vol. 7, pp. 331-359. Connolly, J.D.; Hill, R.A.; Ngadjui, B.T. Nat. Prod. Rep., 1994, ! 1, 91. Connolly, J.D.; Hill, R.A.; Ngadjui, B.T. Nat. Prod. Rep., 1994, 11,467. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1995, 12, 475. Connolly, J.D.; Hill, R.A. Nat. Prod. Rep., 1996, 13, 15 I. Harborne, J.B.; Baxter, H. Phytochemical Dictionary, Taylor and Francis: London, 1993. Harbome, J.B. Nat. Prod. Rep., 1986, 3, 323. Harborne, J.B. Nat. Prod Rep., 1993, 10, 327. Harborne, J.B. Nat. Prod Rep., 1989, 6, 85. Macias, F.A.; Simonet, A.M.; Esteban, M.D. Phytochemistry, 1994, 36, 1369. Bar-Nun, N.; Mayer, A.M. Phytochemistry, 1990, 29, 787.
140
[19]
RiOSeta/.
Shibata, S. In New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutical Activity; Wagner, H.; Wolff, P., Eds.; SpringerVerlag: Berlin, 1977; pp. 177-196. [20] Tang, W.; Eisenbrand, G. Chinese Drugs of Plant Origin. Chemistry, Pharmacology, and Use in Traditional and Modern Medicine, Springer-Verlag: Berlin, 1992. [21] Matsuda, H.; Samukawa, K.; Kubo, M. Planta Med., 1990, 56, 19. [221 Hashimoto, M.; Inada, K.; Ohminami, H.; Kimura, Y.; Okuda, H.; Arichi, S. Planta Med., 1985, 51, 401. [23] Ohuchi, K.; Watanabe, M.; Ozeki, T.; Tsurufuji, S. Planta Med., 1985, 51, 208. [24] Recio, M.C.; Just, M.J.; Giner, R.M.; Mfiflez, S.; Rios, J.L.; Hostettmann, K. d. Nat. Prod., 1995, 58, 140. [25] Hiller, K. In Biologically Active Natural Products; Hostettmann, K; Lea, P.L., Eds.; Clarendon Press: Oxford, 1987; pp. 167-184. [26] Rios, J.L.; Waterman, P.W. Phytother. Res., 1997, 11, 411. [27] Harrison, D.M. Nat. Prod. Rep. 1990, 7, 459. [28] Abe, I.; Rohmer, M.; Prestwich, G.D. Chem. Rev., 1993, 93, 2189. [29] Bevan, C.D.; Marshall, P.S. Nat. Prod. Rep., 1994, 11, 451. [301 Modey, W.K.; Mulholland, D.A.; Raynor, M.W. Phytochem. Anal., 1996, 7, I. [311 Chandel, R.S.; Rastogi, R.P. Phytochemistry, 1980, 19, 1889. [32] Rfos, J.L.; Sime6n, S.; Jim4nez, F.J.; Zafra-Polo, M.C.; Villar, A. Fitoterapia, 1986, 57, 153. [33] Jork, H.; Funk, W.; Fischer, W.; Wimmer, H. Thin-Layer Chromatography. Reagents and Detection Methods, VCH: Weinheim, 1990, Vol. la. [34] Li, T.S.; Li, J.T.; Li, H.Z.J. Chromatogr. A, 1995, 715, 372. [351 Shiao, M.S.; Lee, K.R.; Lin, L.J.; Wang, C.T. ACS Symposium Series, 1994, 547, 342. [36] Gunther, B.; Wagner, H. Phytomedicine, 1996, 3, 59. [37] Gaspar, E.M.S.M.; dasNeves, H.J.C.; Noronha, J.P. HRC-J. High Resol. Chromatogr., 1997, 20, 417. [381 Vitanyi, G.; Bihatsi Karsai, E.; Lefler, J.; Lelik, L. Rapid Commun. Mass Spectrosc., 1997, /1, 691. [39] Wolfender, J.L.; Maillard, M.; Hostettmann, K. Phytochem. Anal., 1994, 5, 153. [40] Heinzen, H.; de Vries, J.X.; Moyra, P.; Remberg, G.; Marttnez, R.; Tietze, L.F. Phytochem. Anal., 1996, 7, 237. [41] Chen, H.R.; Sheu, S.I.J. Chromatogr. A, 1993, 653, 184. [42] Takaishi, Y.; Wariishi, N.; Tateishi, H.; Kawazoe, K.; Nakano, K.; Ono, Y.; Tokuda, H., Nishino H.; Iwashima, A. Phytochemistry, 1997, 45, 969. [431 Della Loggia, R.; Tubaro, A.; Sosa, S.; Becker, H.; Saar, St.; Isaac, O. Planta Med., 1994, 60, 516. [44] Zitterl-Eglseer, K.; Sosa, S.; Jurenitsch, J.; Schubert-Zsilaveez, M.; Della Loggia, R.; Tubaro, A.; Bertoldi, M.; Franz, C. J. Ethnopharmacol., 1997, 57, 139. [45] Agrawal, P.K.; Jain, D.C. Prog. NMR Spectrosc., 1992, 24, 1. [46] Shiojima, K.; Arai, Y.; Masuda, K.; Takase, Y.; Ageta, T.; Ageta, H. Chem. Pharm. Bull., 1992, 40, 1683. [47] Reeio, M.C.; Giner, R.M.; Mfiflez, S.; Gu4ho, J.; Julien, H.R.; Hostettmann, K.; R|os, J.L. Planta Med., 1995, 61, 9. [48] Cu611ar, M.J.; Giner, R.M.; Recio, M.C.; Just, M.J.; M,'tflez, S.; Rios, J.L.d. Nat. Prod., 1996, 59, 977.
NATURALTRITERPENOIDS
[491
[50] [51] [52] [53] [54]
[55] [56] [57]
[58] [59]
[60] [61] [62] [63]
[64] [651 [661 [67] [681 [69]
[70] [71] [72] [73] [74] [75] [76] [77]
141
Ammon, H.P.T.; Safayhi, H.; Mack, T.; Sabieraj, J. J. Ethnopharmacol., 1993, 38, 113. Jain, M.K.; Yu, B.Z.; Rogers, J.M.; Smith, A.E.; Boger, E.T.A.; Ostrander, R.L.; Rheingold, A.L. Phytochemistry, 1995, 39, 537. Wagner, H. Planta Med., 1989, 55, 235. Cavey, D. In Pharmacology and the Skin; Hensby, C.N.; Lowe, N.J., Eds.; Karger: Basel, 1989, Vol. 2, pp.44-88. Bouclier, M.; Cavey, D.; Kail, N.; Hensby, C. Pharmacol. Rev., 1990, 42, 127. Davis, R.H.; Leitner, M.G.; Russo, J.M.; Byrne, M.E.J. Am. Pod. Med. Ass., 1989, 79, 263. Young, J.M.; De Young, L.M. In Pharmacological Methods in the Control of Inflammation; Spector, J.; Back, N., Eds.; Alan R. Liss: New York, 1989; pp. 215-231. Lewis, A.J.; Carlson, R.P.; Chang, J. In Handbook of Inflammation. The Pharmacology of Inflammation; Bonta, J.L.; Bray, M.A.; Parnham, M.J., Eds.; Elsevier Science: New York, 1985; Vol. 5, pp. 371-397. Pay~t, M.; Terencio, M.C.; Ferr~ndiz, M.L.; Alcaraz, M.J. Br. J. Pharmacol., 1996, 117, 1173. Liu, J. J. Ethnopharmacol., 1995, 49, 57. Safayhi, H.; Sailer, E.R.; Ammon, H.P.T. Mol. Pharmacol., 1995, 47, 1212. Heldt, R.M.; Winking, M.; Simmet, Th. Naunyn-Schmiedeberg's Arch. Pharmacol., 1996, 353 (suppl. 4), R142. Singh, S.; Bani, S.; Singh, G.B.; Gupta, B.D.; Banerjee, S.K.; Singh, B. Fitoterapia, 1997, 68, 9, Chawla, A.S.; Kaith, B.S.; Handa, S.S.; Kulshreshtha, O.K.; Srimal, R.C. Fitoterapia, 1991, 62, 441. Kaith, B.S. Int. J. Pharmacog., 1995, 1, 73. Singh, B.; Ram, S.N.; Pandey, V.B.; Joshi, V.K.; Gambhir, S.S. Phytother. Res., 1991, 5, 103. Duwiejua, M.; Zeitlin, I.J.; Waterman, P.G. ; Chapman, J. ; Mhango, G.J. ; Provan, G.J. Planta Med., 1993, 59, 12. Duwiejua, M.; Zeitlin, I.J.; Waterman, P.G.; Gray, A.I. Br. J. Pharmacol., 1993, 108, 236 P. Ansari, S.H.; Ali, M. Fitoterapia, 1996, 57, 103. Ahumada, C.; S~enz, T.; Garc|a, D.; De la Puerta, R.; Fernandez, A.; Marttnez, E.J. Pharm. Pharmacol., 1997, 49, 329. Recio, M.C.; Giner, R.M.; M,~flez, S.; Rtos, J.L. Planta Meal, 1995, 61, 182. Kweifio-Okai, G.; Bird, D.; Field, B.; Ambrose, R.; Carroll, A.R.; Smith, P., Vald~s, R. J. Ethnopharmacol., 1995, 46, 7. Kweifio-Okai, G.; De Munk, F.; Macrides, T.A.; Smith, P.; Rumble, B.A Drug Develop. Res., 1995, 36, 20. Kweifio-Okai, G.; Field, B.; Rumble, B.A.; Macrides, T.A., De Munk, F. Drug. Develop. Res., 1995, 35, 137. Kapil, A.; Sharma, S. J. Pharm. Pharmacol., 1995, 47, 585. Kapil, A.; Inflammopharmacology, 1994, 2, 361. Sugishita, E.; Amagaya, S.; Ogihara, Y. J. Pharm. Dyn., 1983, 6, 287. Rios, J.L.; Giner-Larza, E.M.; M~tflez, S.; Giner, R.M.; Recio, M.C. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. A), 177. Giner-Larza, E.M.; Rios, J.L.; M~fiez, S.; Giner, R.M.; Recio, M.C. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. A), 176.
142
[78] [79]
[80] [81]
[82] [83] [84]
[85] [86] [87]
[88] [89] [901 [91] [92] [93] [94] [95] [96] [97] [98] [99]
[~00] [101] [102] [103]
RiOSa a/. Tabata, M.; Tanaka, S.; Cho, H.J.; Uno, C.; Shimakura, J.; Ito, M.; Kamisako, W.; Honda, C. J'. Nat. Prod., 1993, 56, 165. Yasukawa, K.; Akihisa, T.; Oinuma, H.; Kaminaga, T.; Kanno, H.; Kasahara, Y.; Tamura, T.; Kumaki, K.; Yamanouchi, S.; Takido, M. Oncology, 1996, 53, 341. Yasukawa, K.; Akihisa, T.; Oinuma, H.; Kasahara, Y.; Kimura, Y.; Yamanouchi, S.; Kumaki, K.; Tamura, T.; Takido, M. Biol. Pharm. Bull., 1996, 19, 1329. Kaminaga, T.; Yasukawa, K.; Takido, M.; Tai, %; Nunoura, Y. Phytother. Res., 1996, 10, 581. Nukaya, H.; Yamashiro, H.; Fukazawa, H.; Ishida, H.; Tsuji, K. Chem. Pharm. Bull., 1996, 44, 847. Cu~llar, M.J.; Giner, R.M.; Recio, M.C.; Just, M.J.; M~tflez, S.; Rtos, J.L. Chem. Pharm. Bull., 1997, 45, 492. Yasukawa, K.; Takido, M.; Matsumoto, T.; Takeuchi, M.; Nakagawa, S. Oncology, 1991, 48, 72. Akihisa, T.; Yasukawa, K.; Kimura, Y.; Takido, M.; Kokke, W.C.M.C.; Tamura, T. Chem. Pharm. Bull., 1994, 42, l l01. Yasukawa, K.; Akihisa, T.; Tamura, T.; Takido, M. Biol. Pharm. Bull., 1994, 17, 460. Hirota, M.; Mori, T.; Yoshida, M.; Iriyr R. Agric. Biol. Chem., 1990, 54, 1075. Huang, M.T.; Ho, C.T.; Wang, Z.Y.; Ferraro, T.; Lou, Y.R.; Satauber, K.; Ma, W.; Georgiadis, C.; Laskin, J.D.; Conney, A.H. Cancer Res., 1994, 54, 701. M~tflez, S.; Recio, M.C.; Giner, R.M.; Rios, J.L. Eur. J. Pharmacol., 1997, 334, 103. Mb.flez, S.; Huguet, A.I.; Recio, M.C.; Giner, R.M.; Rios, J.L. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. ,4), 175. Inoue, H.; Nagata, N.; Koshihara, Y. Inflamm. Res., 1995, 44, 470. Inour H.; Nagata, N.; Koshihara, Y. Jpn. J. Pharmacol., 1995, 69, 6 I. Inoue, H.; Nagata, N.; Koshihara, Y. Br. J. Pharmacol., 1993, 110, 1614. Inoue, H.; Nagata, N.; Shibata, S.; Koshihara, Y. Jpn. J. Pharmacol., 1996, 71, 281. Ammon, H.P.T.; Mack, T.; Singh G. B.; Safayhi, H. Planta Med., 1991, 57, 203. Safayhi, H.; Mack, T.; Sabieraj, J.; Anazodo, M.I.; Subramanian, L.R.; Ammon, H.P.T.J. Pharmacol. Exp. Ther., 1992, 261, 1143. Sailer, E.-R.; Subramanian L.R.; Rail, B.; Hoernlein, R.F.; Ammon, H.P.T.; Safayhi, H. Br. J. Pharmacol., 1996, 117, 615. Moreau, R.A.; Agnew, J.; Hicks, K.B.; Powell, M.J.J. Nat. Prod., 1997, 60, 397. Simon, A.; Najid, A.; Chulia, A.J.; Delagr C.; Rigaud, M. Biochim. Biophys. ,4cta, 1992, 1125, 68. Najid, A.; Simon, A.; Cook, J.; Chable-Rabinovitch, H.; Delagr C.; Chulia, A.J.; Rigaud,M. FEBS Left., 1992, 299, 213. Es-Saady, D.; Najid, A.; Simon, A.; Denizot, Y.; Chulia, A.J.; Delage, C. Mediators Inflamm., 1994, 3, 181. Kweifio-Okai, G.; Macrides, T.A. Res. Commun. Chem. Pathol. Pharmacol., 1992, 78, 367. Kweifio-Okai, G.; De Munk, F.; Rumble, B.A.; Macrides, T.A.; Cropley, M. Res. Commun. Chem. Pathol. Pharmacol., 1994, 85, 45.
NATURAL TRITERPENOIDS
143
[104] Ying, Q.L.; Rinehart, A.R.; Simon, S.R.; Cheronis, J.C. Biochem. J., 1991, 277, 52 I. [105] Safayhi, H.; Rail, B.; Sailer, E.R.; Ammon. H.P.T.J. Pharmacol. Exp. Ther., 1997, 281,460. [1061 Hirotani, M.; Asaka, L.; Ino, C.; Furuya, T.; Shiro, M. Phytochemistry, 1987, 26, 2797. [107] L i, X.Y. Int. J. Immunother., 1993, 9, 181. [108] Balanheru, S.; Nagarajan, B. Biochem. Int., 1991, 24, 981. [109] Liu, J.; Mori, A.; Ogata, K. Res. Commun. Chem. Pathol. Pharmacol., 1993, 82, 151. [llO] Sassa, H.; Kogure, K.; Takaishi, Y.; Terada, H. Free Rad. Biol. Med., 1994, 17, 201. [lll] Funder, J.W.; Pearce, P.; Smith, R.; Smith, A.I. Science, 1988, 242, 583. [ll2] Teelucksingh, S.; Mackie, A.D.R.; Burt, D.; Mclntyre, M.A.; Brett, L.; Edwards, C.R.W. Lancet, 1990, 335, 1060. [ll3] Marandici, A.; Monder, C. Steroids, 1993, 58, 153. [ll4] Whorwood, C.B.; Sheppard, M.C.; Stewart, P.M. Endocrinology, 1993, 132, 2287. Ill5] Seckl, J.R.; Dow, R.C.; Low, S.C.; Edwards, C.R.W.; Flink, G. J. Endocrinol., 1993, 136, 47 I. [ll6] G6mez-S~nchez, E.P.; Ganjam, V.; Chen, Y.J.; Cox, D.L.; Zhou, M.Y.; Thanigaraj, S.; G6mez-SAnchez, C.E. Steroids, 1997, 62, 444. Ill7] Escher, G.; Galli, I.; Vishwanath, B.S.; Frey, B.M.; Frey, F.J.J. Exp. Med., 1997, 186, 189. Ill8] Kapil, A.; Moza, N. Int. J. Immunopharmacol., 1992, 14, 1139. [ll9] Kapil, A.; Sharma, S. J. Pharm. Pharmacol., 1994, 46, 922. [1201 Kroes, B.K.; Benkelman, C.J.; Van der Berg, A.J.; Wolbink, G.J.; Van Dijk, H.; Labadie, R.P. Immunology, 1997, 90, I 15. [121] Tsuruga, T.; Chun, Y.T.; Ebizuka, Y.; Sankawa, U. Chem. Pharm. Bull., 1991, 39, 3276. [122] Shoji, N.; Umeyama, A.; Motoki, S.; Arihara, S.; Ishida, T.; Nomoto, K.; Kobayashi, J.; Takei, M. J. Nat. Prod., 1992, 55, 1682. [123] Lee, Y.M.; Hirota, S.; Jippo-Kanemoto, T.; Kim, H.R.; Shin, T.Y.; Yeom, Y.; Lee, K.K.; Kitamura, Y.; Nomura, S.; Kim, H.M. Int. Arch. Allergy Immunol., 1996, ! I 0, 272. [1241 O'Brian, C.A.; Ward, N.E.; Vogel, V.G. Cancer Lett., 1990, 49, 9. [125] Wang, B.H.; Polya, G.M. Phytochemistry, 1996, 41, 55. [126] Dirsch, V.M.; Kiemer, A.K.; Wagner, H.; Vollmar, A.M. Eur. J. Pharmacol., 1997, 336, 211.
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22
145
9 2000 Elsevier Science B.V. All rights reserved
CURRENT STATUS OF THE CHEMISTRY AND SYNTHESIS OF NATURAL ANTIMALARIAL COMPOUNDS AND NATURAL SUBSTANCES USED TO ALLEVIATE SYMPTOMS OF DIABETES (ALDOSE REDUCTASE AND A-GLUCOSIDASE INHIBITORS) K. K A W A N I S H I * and N. R. F A R N S W O R T H
*Kobe Pharmaceutical University, Kobe , Japan Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Illinois, U.S.A. ABSTRACT" Atropine, camptothecin, cocaine, digitoxin, digoxin, morphine, pilocarpinr quinine, taxol, vinblastine and vincristine, among others, are important drugs obtained from higher plants and are used clinically. They have also served as lead compounds for the synthesis and modification of more effective and safer drugs, in many cases. In this chapter, drugs used as antimalarial compounds and for the complications of diabetes (aldosr reductasr and o~-glucosidase inhibitors) will be discussed. Natural product chemists have isolated as little as 1.0 mg of pure compounds from natural sources and have been able to determine their structures using high resolution instrumental techniques. Organic chemists have synthesized thousands of compounds to produce one new drug on the basis of natural product leads, and pharmacologists and biochemists have tested their biological activity. Recently chemists and pharmacologists have worked together to develop techniques for studying structure-activity relationships using computer graphics and have designed new drugs. Biochemists, molecular biologists and pharmacologists have identified many receptors on which drugs act. Thus, mechanisms of drug action at the molecular level are being identified. From the accumulation of these results structure-activity relationships will lead to the preparation of thousands of useful compounds. We must produce drugs in these ways, because we cannot rely on solely on the limited amount of active compounds produced naturally in plants, in many cases, for a number of reasons. However we need to employ plant extracts themselves, because there are millions of people who cannot buy expensive synthetic drugs in the world and these extracts are widely used by them. 1.0 N A T U R A L A N T I M A L A R I A L C O M P O U N D S It has been estimated that about 500,000 million cases o f malaria occur annually and about 1-2 million deaths due to Plasmodium falciparum are involved [1]. Most o f these cases occur in tropical countries and there appears to be little systematic research to discover novel antimalarial drugs. Perhaps one o f the reasons for this lack o f interest is that there is hope that an antimalarial vaccine is the answer, but this is not expected to be available in the near future, if ever.
146
KAWANISHI and FARNSWORTH
There are a few efforts to synthesize analogs of already effective antimalarial drugs, e.g. quinine and artemisinin and these will be discussed in this paper (vide infra). These studies are mainly targeted at finding less toxic entities and/or compounds that overcome the rapid resistance that the malarial parasite seems to acquire. One of the earliest systematic searches for antimalarial drugs from plants was published in 1947 [2], which mainly involved the injection of extracts into infected ducklings. Dichroa febrifuga, used in China for centuries as an antimalarial drug, was found to be active and the alkaloid febrifugine was determined to be the main active principle. However, febrifugine is well known to be too toxic for human use. The marine environment seems to be the area most studied in recent years for novel antimalarial agents [3-9] but a reasonable amount of effort seeking plantderived agents can also be identified [10-26] and activity has centered around the simaroubolides (quassinoids) [27-29], limonoids [30-31], bisbenzylisoquinoline alkaloids [1], as well as other minor classes of compounds.
1.1 Quinine and related compounds Quinine (1) has been known as the principal alkaloid in cinchona bark, which consists of various chemovars and hybrids of Cinchona species (Rubiaceae), such as C. ledgeriana, C. succirubra, C. calisaya, C. officinalis and others, grown and cultivated in tropical countries. A recent review has covered the distribution, cultivation, cytology, tissue culture methods, pharmacology and toxicology of Cinchona and it's alkaloids [32]. Cinchona bark was first mentioned to be used for the treatment of fever in Europe in 1643 and was included in the London Pharmacopoeia as "Cortex Peruanus" in 1677. About 35 quinine type alkaloids have been reported from Cinchona species. The content of quinine is highest in the bark of cultivated trees, while cinchonidine (3), cinchonine (4) and quinidine (2) are more abundant than quinine in C. succirubra and some other varieties of this genus. Quinine is schizontocidal and gametocidal in blood for P. vivax and P. malariae, but not for P. falciparum. Therefore, quinine has been used as a suppressive and therapeutic agent, but not as a prophylactic agent. Quinine, quinidine, cinchonidine and cinchonine contain a quinoline moiety attached through a secondary alcohol linkage to a quinuclidine ring having a vinyl group. Quinine and quinidine each contain a methoxy group, that is absent in cinchonidine and cinchonine. Based on the steric configuration of the secondary alcohol in the molecules, quinine and cinchonidine are (-)-isomers and quinidine and cinchonine are (+)-isomers. Differences in steric configuration do not result in significant changes in biological activity of these alkaloids. Quinine is less potent as an antimalarial but less toxic than quinidine. Cinchonamine
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
147
(5), which contains a 5-vinyl-2-quinuclidyl group attached to an indole ring in addition to an ethanoyl group, has been isolated from H2
H3
H3CO~
~,~
~
8 quinine I
qulnldine 2
H2C~
HO
clnchonldine 3
"I
H2
I~ H
clnchonlne 4
clnchonamine
Remijia purdieana (Rubiaceae), but is devoid of antimalarial activity. Thus, the 5-vinyl-2-quinuclidyl group is not necessary for antimalarial activity. Quinine has been the lead compound for the development of a large number of antimalarial compounds. Quinacrine (6), 6-aminoacridine with 4-diethylamino-l-methylbutyl-, chloro- and methoxy- groups, was used as an antimalarial agent during the World War II. However, quinacrine is not useful for prophylaxis of malaria, is not gametocidal for P. vivax and is toxic. Following the synthesis of thousands of 4-aminoquinolines, chloroquine (7), 4aminoquinoline with attached 4-diethylamino-1-methylbutyl- and chlorogroups, was investigated and found to be an effective agent against the erythrocytic stage of P. vivax, P. ovale, P. malariae and certain strains of P. falciparum. Chloroquine suppresses P. vivax greater than does quinine but does not prevent relapses. Quinine also does not prevent relapses. Chloroquine is metabolized to monodesethylchloroquine (8), which is also highly active as an antimalarial. SN-6911 (9), a 4-aminoquinoline which
148
KAWANISHIand FARNSWORTH
contains 4-diethylamino-1-methylbutyl-, chloro- and methyl- groups, was developed as a more efficient derivative against human malarias.
~H3
( cH3
H N ~ N ~ [
~H3
CH3
: oH3
HN~N~CH3
oc~
el
el
quinacrlne6
chloroqulne 7
H3 H N ~ ~ v l
CI
~H3 ScH3
~
CI" monodesethyl chloroqulne8
v
H
~
: CH3 N
~
CH3
CH3 ~N"
SN 69119
Amodiaquin (10), 4-aminoquinoline which contains 3diethylaminomethyl-4-hydroxyphenylamino and chloro groups, was synthesized and found to be more potent against P. faleiparum than chloroquine. However it is more hepatotoxie than chloroquine and produces granuloeytosis. One of the N-ethyl substituents of chloroquine is 13-hydroxylated to produce hydroxychloroquine (11), which has similar efficient activity against faleiparum malaria, and is less toxic than chloroquine. This compound is preferred over chloroquine in the treatment of rheumatoid arthritis and lupus erythematosus. Many phenanthrenemethanol derivatives were also synthesized during World War II in a search for alternative drugs to quinine. Halofantrine (12) resulted from these studies, which is a 2-(dibutylamino)-ethyl derivative with trifluoromethyl and diehloro groups. Both the (+)- and (-)enantiomers ofhalofantrine are equally active against P. falr in vitro and against P. berghei in mice. The racemate of halofantrine is effective against the asexual erythrocytic stages of several Plasmodium species.
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
149
Halofantrine is metabolized into N-desbutyl halofantrine (13) in humans, and it also has antimalarial activity. H3
~ CH3
CI hydroxychloroquine I I
amodlaquln 10
~
halofantrlne 12
C
H
s
H
N-desbutyl halofantrtne 13
Mefloquine (14), a 4-quinolinemethanol derivative with attached 2piperidyl and 2,8 bis-(trifluoromethyl) groups arose from a synthetic effort on 4-quinolinemethanol. This compound possesses activity against murine malaria and is clinically effective against chloroquine-resistant strains of P. falciparum. A study of the interaction of 4-quinolinemethanol derivatives with DNA produced two analogs of mefloquine, one (15) with a CONH2 in place of CF3 at position 2 of the ring and the other (16) with F in place of CF3 at position 8 of the ring. It was found that mefloquine was more active than either of these two compounds, suggesting that no relationship exists between the reaction with DNA and antimalarial activities [33]. These 4-quinolinemethanol derivatives include four optical isomers but they were tested as their racemates. Four optical isomers of 9phenanthrenemethanol with attached 2-piperidyl and 3,6bis(trifluoromethyl) groups (17) were all prepared and tested for antimalarial activity [34]. They were all active, indicating that stereospecificity was not required in the antimalarial activity.
150
KAWANISHI and FARNSWORTH
HO
EONH 2
CF 3 CF3
CF3
mefloquine 14
F
15
F3(.,
16
CF 3
a-(2-plperldyl)-3,6-bls(trifluoromethyll-9-phenanthrenemethanol 17
Pamaquine (18), the first synthetic 8-aminoquinoline having a methoxy group, was found to have antimalarial activity. Additional analogs were prepared. Pentaquine (19), a 5-isopropylamino-pentylamino-derivative; isopentaquine (20), a 4-isopropylamino-l-methylbutylamino-derivative and primaquine (21), a 4-amino- 1-methylbutylamino-derivative were found to be more active and less toxic than pamaquine. Primaquine is most effective against the late hepatic stages and latent forms ofP. vivax and P. ovale, but is ineffective for suppression of P. vivax malaria. This drug is also active against the hepatic stages of P. falciparum, but not against the erythrocytic stages. Primaquine is used in combination with other drugs for the radical cure of relapses, as well as for prophylaxis. The 4-amino-l-methylbutylamino group in primaquine (21)was changed to a 1,4-pentanediamino group which became quinocide (22) and retained activity. 4-Methylprimaquine (23), which possesses a methyl group at position 4 of the quinoline ring of primaquine was also active and thus derivatives of methyl substitutions at position 4 in the quinoline derivatives were synthesized and tested [35]. Comparing the two derivatives with 3-aminopropylamino group (24) and 5-amino-l-methylpentylamino group (25) to primaquine (21) and 4-methylprimaquine (23),
CHEMISTRYOFANTIMALARIALCOMPOUNDS
H3CO~~NS~ ~
151
H3C _CH3
CH3 pamaquine 18
K~CH3 pentquine 19
H3o
HN~~,~..~
NH2
CH3 Isopentaquine 20
primaqulne
21
the 5-amino-l-methyl-pentylamino derivative (25) showed curative activity (3/5) at 160 and (5/5) at 640 mg/kg against P. berghei in mice, while the 3-aminopropylamino derivative (24) and 4-methylprimaquine (23) caused toxic death (5/5) at 640 mg/kg, and primaquine (21) did toxic death (5/5) at 320 mg/kg.
H 3 C O ~
H3C
H3 ,,~
NH2 (;Ha quinoclde 22
4-methylprimaquine
23
152
KAWANISH!and FARNSWORTH
H3C
~H3
~3
H3C
24
CH3
25
In the series of derivatives of 4-methylprimaquine, having an aryloxy or an alkoxy group at position 5 in the quinoline ring, WR-238,605 (26) with a 3-(trifluoromethyl)-phenoxy group at position 5 and a methoxy group at position 2, WR-242,511 (27) with a n-hexyloxy group, and WR-254,715 (28) with a phenylpentyloxy group at position 5 were promising for most blood schizontocidal activity [36]. However the two former compounds increased the activity at lower dosage range but produced toxicity at higher doses (160-640 mg/kg), although these were more effective than the parent derivative, primaquine (21) in both curative activity and toxicity. Thus, derivative (28) was an excellent candidate in this series.
c~
~H2CH2CH2CH2CH2CHa H3C
H
~
CH3
CH3
WR-238,60526
WR-242,511 27
NH2
CHEMISTRY OF ANTIMALARIALCOMPOUNDS
153
~
H2CH2CH2CH2CH2C6H5
3c
H~,,~~~~
NH2
CH3 WR-254,715 2 8
Quinine led to the development of chloroquine, which consists of an Nalkyl side chain in place of the quinoline ring in quinine. The N-alkyl side chain substituted at position 8 in the quinoline moiety was found to be as effective as the one substituted at position 4 or more. It is interesting that to develop more effective and less toxic drugs, the latest compounds developed possess substituents at positions 4,5,6 and 8 in the ring, such as WR-242,511 and WR-254,715, and in WR-238,605 even more substituents are attached, such as at positions 2,4,5,6 and 8. They were effective against a variety of stages in the life cycle of the parasite including the pre-erythrocytic stage, the drug-sensitive and drug-resistant asexual intra-erythrocytic stage, gametocytes and the intra-hepatic hypnozite stage for P. cynomlgii. In vivo WR-254,715 was more efficacious and less toxic than primaquine 1.2 Artemisinin (qinghaosu) and related compounds
The leaves and flowering tops of the wormwood plant, Artemisia annua (Compositae) have been used as a traditional Chinese medicine for the treatment of fever and malaria. A hot-water extract of this plant was not active in mice infected with P. berghei, but an ethyl ether extract was active. Artemisinin (29) was isolated from the petroleum ether extract of the plant and was found to be a novel sesquiterpene lactone with an endoperoxide group. The history of development of artemisinin and its analogues, as well as a review of the chemistry and pharmacology of this plant and its contained compounds, is available [37-39]. Arteannuin B (35), also isolated from A. annua, contains a ~,-lactone with a cadinane skeleton in the structure [40]. Artemisinin has demonstrated promising antimalarial activity against the erythrocytic stage of P. falciparum and is also active against both chloroquine-resistant and chloroquine-sensitive
154
KAWANISHI and FARNSWORTH
strains of the parasite in vitro and in vivo. Artemisinin is inactive against the liver stages of P. falciparum. It is only poorly soluble in water or oil, which impairs its practical use as antimalarial agent. A search for improved analogs with more potency, water solubility, oral activity and better bioavailablity has been initiated. Hydrogenation of artemisinin gave two different compounds. One of the peroxide oxygen atoms of artemisinin was deoxylated to give the epoxide, deoxyartemisinin (30) by Pd/CaCO3. The lactone group of artemisinin was hydrogenated to the lactol, dihydroartemisinin (31 1, R = H 9major, 32 1, R = H 9minor) by sodium borohydride [41 ]. I]-Dihydroartemisinin (31 1, R = H) has been isolated from A. annua. The activity was greater for dihydroartemisinin (31 1, R = H) than for artemisinin, but not for deoxyartemisinin. Relative activities of artemisinin and deoxyartemisinin analogs were compared and the latter showed very little activity, while the former was active [42]. Artemisinin is metabolized to deoxyartemisinin (30), dihydrodeoxyartemisinin (33 1, R = H) and Crystal-7 (36) [43]. They were not active against P. berghei in mice. Dihydroartemisinin was dehydrated to obtain anhydrodihydro-artemisinin (37), with the double bond between positions 9 and 10, and an ether (38) [44]. The former was inactive but the latter was active. Ethers, esters and carbonates of dihydroartemisinin have been prepared to obtain novel and more active derivatives [45]. Atter examination of the ethers, including the epimers at position 10, artemether (31 2, R = C H 3 ) and arteether (31 3, R = CH2CH3), they were found to be about twice as active as artemisinin, but less active than dihydroartemisinin. Artemether has been isolated as a natural constituent ofA. annua. Arteether was found to be 34 times more active than chloroquine against the W-2 (Indochina) clone of P. falciparum (normally resistant to chloroquine) and three times less active against the D-6 (Sierra Leone) clone (normally resistant to mefloquine). Artemether was two times more active and eight times more active than mefloquine against the W-2 and D-6 clones, respectively. Both artemether and arteether are more oil soluble than artemisinin and are currently in clinical trials. Esters (32 4, R = C(=O)-alkyl or-aryl), of which ct-epimers were mainly obtained, were more active than the ethers. Sodium artesunate (32 5, R = C(=O)-CH2CH2COONa), the half succinic acid half-ester of dihydroartemisinin is water-soluble and shows potent antimalarial activity. Therefore, this can be administered intravenously. However it is uncertain whether this derivative is pharmacologically effective because of its sensitivity to hydrolysis. In considering this result new ether derivatives of dihydroartemisinin, which are stable and water soluble derivatives have been prepared [46]. Dihydroartemisinin was condensed with esters of aliphatic or aromatic carboxylic acids with hydroxy groups to produce mainly ethers with the t-configuration. Ethyl 2-(10dihydroartemisininoxy) acetate (31 4, R = CH2COOCH2CH3) and methyl
CHEMISTRYOF ANTIMALARIALCOMPOUNDS
155
p-[(10-dihydroartemisininoxy)methyl] benzoate (31 5, R = C H 2 - C 6 H 4 COOCH3) were active against the D-6 and W-2 strains, in vitro and they were as active as the parent compound, dihydroartemisinin. These two ethers, ethyl acetate and methyl benzoate were changed to potassium acetate (31 6, R = CH2COOK) and potassium p-methyl benzoate (31 7, R = CH2-C6H4-COOK) derivatives, respectively. The latter retained activity but the former did not. The potassium carboxylates were converted to the corresponding acids (31 8, R = CH2COOH and artelinic acid, 31 9, _CH3 5 .H ."~ H "." '
34 H
3
C
.CH3 H ~ H ~ '~
~
H3C
9. "'H H J
IH"~ , ~ ~ . , , ~ H
0
0
-d'.:
artemlsinin
29
.CH3 H ~ H H3C~ '
I"'H
Hl,.~ OR
~"C H
H :0, ~...,H 3
RO"=~
arteether
artelinir acid
R-
H 1 CH3 2 CH2CH3 3 4 CH2COOCH2CH3 CH2C6H4COOCH3-p 5 6 CH2COOK CH2C6H4COOKp CH2COOH CH2C6H4COOH-p
"ella
H 32
31
dihydroartemisinin
_CH3 :. H
H
~ ~
-/.:
artemether
30
deoxyyartemlslnln
R=
H CH3 CH2CH3 C(=O)-alkyl or aryl
sodium anesunate
C(=O)-CH2CH2COONa C(=O)-alkyl or aryl
156
KAWANISH! and FARNSWORTH
R = CH2-C6H4-COOH). The acetic acid derivative was unstable but the pmethyl benzoic acid (artelinic acid) was not. However, this was less active than its methyl benzoate or potassium benzoate derivative. Ethers of dihydroartemisinin, which contain a sugar moiety, were prepared for enhanced water solubility and high antimalarial efficacy [47]. Acetylated dihydroartemisinin sugar derivatives (39, 1,2 and 4) were obtained, and then they were hydrolyzed to be deacetylated ones (39, 5,6 and 8). The acetylated derivatives were substantially more active than the deacetylated ones in two P. falciparum malaria parasite clones designated as Sierra Leone clone (D-6) and Indochina clone (W-2). 3-Hydroxy-5,6isopropylidene-D-glucosides both protected and unprotected at 1,2dihydroxy groups with isopropylidene (39 3 and 7) were active in two P. falciparum malaria parasite clones. The acetylated forms were active against P. berghei in mice, while the deacetylated or unprotected forms were inactive.
H :
N
.CH3 ~ H
I'"',"
H
i
I"","
OR
H
33
34
I R= 2
.CH3 '.. H
H CH2CH 3
Carbonates (32 6, R = (CO)O-alkyl or-aryl), of which the o~-epimers predominate, are oil-soluble and are the most active in the series. The reduction products of deoxyartemisinin, deoxydihydroartemisinin (33 1, R = H and 34) and deoxydihydroarteether (33 2, R = CH2CH3) did not show noteworthy activity against D-6 in vitro [41]. Artemisinin (29), with an endoperoxide, is active but not deoxyartemisinin (30), which shows the parallel necessity for the endoperoxide moiety for antimalarial potency. DRing contracted analogs of artemisinin (40 and 41) were prepared, and antimalarial activity against W-2 and D-6 clones was found to be greater in (40) than in (41) [48].
CHEMISTRY OF ANTIMALARIALCOMPOUNDS
.CH3 H - .:. H
157
.CHaH H ". H3 C O ~ - ~ K
6" ~ H
H3
., CH3
0 arteannuin B
O cryatal-7
35
36 .CH3
H.-:H H
.CH3 ,-" H
H
a
C
~
~
.3c
I'"'H T
Hk,
~,CH3
CH3 ! '" H anhydrodlhydroartemlsln
37
H3~., r ~I .~ H CH3 38
Tricyclic 1,2,4-trioxanes (42 1-8) were prepared, and (42 7) and (42 8) were stable at room temperature and active against P. berghei in mice [49]. Trioxanes related to 6,7,10-trioxaspiro[4,5]decane (43 1-7)and 1,2,5troxaspiro[5,5]undecane (44 1-7) which contained 1,2,4-trioxane in their structures, were synthesized and screened against P. berghei in mice [50]. The former derivatives were more active than the latter. Trioxanes (43 1, R l = R2 = H) and (43 3, RI = C1, R2 = H) at a 30 mg/kg dose showed more than a 90% suppression of parasitemia.
158
KAWANISHI and FARNSWORTH
~-~3
H -
./-
H
I"',"
H~...I
~'CH3
OR
~H2OX
39
~ X~ O X
f H20X
/".,~ ox I OX
H2OX OX ~.ss] H
9H~ ~
1 H3C
Xl /",~ I
"0
X
ox
OX
xkc.3
I OX
OX
I X = COCH 3 5 H
2 X = COCH3 6 H
X = 7
H
R
4 X = COCH3 8
H
CH 3
H
_CH3 H
.CH3
H 3 C ~ "0"* C. H'~
"'~ "*ssCH3 4O
41
"CH3
Dispiro-l,2,4,5-tetraoxanes (45 1-3) were prepared with the intact peroxide functional group and tested for antimalarial activity [51]. Dispiro- 1,2,4,5-tetraoxanes, 1(S), 10(R)-dimethyl-7,8,15,16tetraoxodispiro[5,2,5,2]hexadecane (45 3) was as active as artemisinin, both in vitro and in vivo. Based on the resistance index (ICs0(W-2)/ICs0(D6)), 2,4,11,13-tetramethyl-7,8,15,16 tetraoxodispiro[5,2,5,2]hexadecane
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
159
(45 1) was effective against W-2, and 3,12-dimethyl-7,8,15,16tetraoxodispiro[5,2,5,2]hexadecane (45 3) was effective against D-6.
0---
42 I 2 3 4 5
42
R=
6
H CH 3 OCH 3 F Cl
O--
_8 7 8
Ri
43 1 2 3 4 5 6 7
3\2..~...o ~ 45
Rl= H F CI OCH 3 CH 3 H CI
R2= H
R=H Cl
44
H H H H CH3 CH3
~~2 48
I
O--O
45
3
2
160
KAWANISHIand FARNSWORTH
CH
H ~ ~I
H ~H3I. :. I ), OCH3/
48
47
1 R = CH3 2
Y=
H2(~'~
I
CH2CH=CH2
2 ~ C O O C H
H Hs
3
!
Q
C, Z =
Z
48
y -
O
3
~~'~COOH
p
l
R Q
2
O=-CH2CH3 x 2
0
3
O=-CH2CH3 x 2
S
x2Z=O
4 ~ C O I ~ cH2CH3 \~ ~'l " " CHzCH3 ~CH3 5 ~COOCH2CH2N~ CH3 6
~CH3 CHzNCO0~- CH3 --CH 3 /CH3
7
I~CH3
8
Y = S.Z=0x2 1
/ CH2CH3
I~CH2CH3 9
49
2 3
Trioxane ethers (46 1-4) were as effective as the lower alkyl ethers of dihydroartemisinin except (46 1, R = CH3) [52]. From the size of the substituents of the trioxane ethers the benzyl ether was the most active. Trioxane carboxylate esters (47 1-6) and carbamate esters (47 7-9) were tested against W-2 and D-2 elones. The trioxane monomethyl
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
161
terephthalate ester (47 2, R = C6H4--C O O C H 3 ) and trioxane monodiethylamino terephthalate ester (47 4, R = C6H4-CON(CH2CH3)2) were one to three times more active than artemisinin against both clones. The trioxane N,N-diethylcarbamate ester (47 8, R = N(CH2CH3)2) and trioxane N,N-diphenylcarbamate esters (47 9, R = N(C6H5)2) were two to seven times more active than artemisinin. The trioxane diphenyl phosphate esters (48 1, R = O - C 6 H 5 and Z = O), trioxane diethyl phosphate ester (48 2, R = O-CH2CH3 and Z = O) and trioxane diethyl phosphorothioate (48 3, R = O-CH2CH3 and Z = S) were more active than artemisinin. The trioxane diphenyl phosphate ester (48 1, R = C 6 H 5 and Z=O) was the most active of the three compounds against the W-2 clone, and the trioxane diethyl phosphate ester (WR279,137) (48 2, R = OCH2CH3 and Z=O) was the most against the D-6 clone. Trioxane ptoluenesulfate ester (49 1, R = C 6 H 4 - C H 3 - p ) , trioxane 2(methoxycarbonyl)-benzenesulfate ester (49 2, R = C 6 H 4 - C O O C H 3 - 0 ) and trioxane 5-(dimethylamino)-l-naphthalenesulfate ester (49 3, R = C I o H 6 N(CH3)2 (C-5)) were more or less as active as artemisinin. From a prediction of the comparative molecular field analysis (CoMFA) trioxanes, four 8a,9-seco artemisinin analogs (50 1-4) which have no lactone were prepared and tested [42]. Their relative activity between predicted and actual activity were very similar and they were three to ten times more active than artemisinin.
H
Ra
H I 2 3 4
H3C
Rl
= H
H H OCH a
R2 =
H OCH 3 OCH2CH 3 H
R3 = CH 3
H H H
SO
Antimalarial activity of 4,5-secoartemisinins (51 1-4) which consisted of 1,2,4-trioxane with a lactone has been reported [53]. (-)-5-Nor-4,5secoartemisinin (51 2, RI = CH3, R2 = H, R3 = CH3) was as effective as artemisinin against the D-6 clone. The same analogs which were prepared by using the prediction in CoMFA analysis showed that the differences of activity were small between the predicted and actual values [41 ]. Artemisitene (52 I, Rl = R2 = H) has been isolated from ,4. a n n u a [54]. Artemisitene, its hydroperoxide (53) and its alcohol (54) were the starting compounds for preparation of derivatives [55]. Thirteen derivatives were
162
KAWANISH!and FARNSWORTH
~,CH3~2
RI
R3 H9
7
8
I 2 3 4
RI = CH 3 CH 3 CH 3 H
R2 = H H CH 3 H
R3 = H CH 3 CH 3 CH 3
4 H'5~~1~
prepared and screened. 9-Desmethyl-9-ethylideneartemisinin (52 2, Ri = H, R2 CH3) was as effective as artemisinin against the D-6 clone and three times weaker than artemisinin against the W-2 clone. 9,9aDihydroxyartemisinin (55) and one of the isomers of epoxyartemisitene (56) were ineffective against both clones, while both isomers of epoxydihydroperoxyartemisitene (57) were active but 0.5-1 times less active and one fourth less active than artemisinin against the D-6 and W-2 clones, respectively. The lactol, epoxydihydroartemisitene (58)was as active as artemisinin against the D-6 clone. =
CH . 3H
H
oO~2
H - CH 3H
1
CH
H -3H
O ~ OOH
52
artemisitene
OH
53
1 R] = H 2 H
1 Rl = H 2 CH 3
_CH3
CH 3
..H" H
.
_CH3
.H::
H~H
"6;.,,.
H~' .. OH
:. 0
o /~H
55
HO H
H
II
IH H
58
0
-H
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
_CH3 LI
.=2
I-.I
H
_CH3 " H
OOH
OH
58
,,,
,,,, ,,
2
CH 3 " H
H
Rl O "or H O H O
H
..:
R2
CH3 - H 9
R1 H
H- 6 , , ~ 60
R2
R! -
13 !4 15
CH2CH2CH2CH2C6H5 CH2CH2COOCH2CH 3 H
16 17 i8 19 20
CH2CH3 CH2CH2CH 3 CH2CH2CH2CH3 CH2CH2CH2CH2CH 3 CH2CH2CH2C6H5
'R! =
,
R2--
CH2CH2CH 3 CH2CH2CH2CH3 CH2CH(CH3)2 CH2CH2COOCH2CH 3 CH2CH2C6H5 CH2CH2CH2C6H4Cl.p CH2CH2CH2CH2C6H5 CH2CH3 CH2CH2CH2CH3 CH2CH2C6H5 CH2CH2CH2CH6H4CI.p
1
, ,,,,,
CH2CH 3
3 4 5 6 7 8 9 !0 I! !2
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
_CH3 ~. H
H
57
1
163
CH3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH2CH3 CH2CH2CH3 CH2CH2CH2CH 3 CH2CH(CH3)2 CH2CH2CH2CH2C6H5 CH2CH2C6H5 CH2CH2CH2C6H4CI-p CH2CH2COOCH2CH3 ,, CH2CH2COOH
R2=
H H H H H H H H (CH2)3CH3 (CH2)3CH3 (CH2)BCH3 (CH2)3CH3 (CH2)3CH3 (CH2)BCH3 CH3 CH 3 CH3 CH 3 CH 3 CH3
CH 3 H CH2CH3 CH2CH2CH3 CH2CH2CH2CH 3 CH2CH2CH2CH2CH 3 CH2CH2CH2C6H5 CH2C H2CH2C6H4Cl-p H H H H H H H H H
164
KAWANISH! and FARNSWORTH
3-Substituted artemisinin derivatives (59 1-8, R2 = H and 59 9-14, R2 = CHECHECHECH3) were synthesized in an attempt to find more active derivatives than artemisinin and to design further analogs by structureactivity relationships [56]. The 3-propyl analog in the former (59 2, R ! C H 2 C H E C H 3 , R2 = H) was the most active of the eight against the D-6 clone and twenty times more than artemisinin. The 3-(3'-carbethoxyethyl) analogs (59 5, R l - CHECH2COOCHECH3, RE = H and 3-(4-phenylbutyl) (59 8, Rl = CHECHECHECHEC6H5, R2 = H) were similarly effective and two times more than artemisinin. However the 3-(2-phenylethyl) analog (59 6, Rl = CHECHEC6H5, R2 = H) possessed no activity. In the latter group the analog substituted with 3'-carbethoxyethyl at position 3 and butyl at position 9 (59 14, Ri -- CHEC H 2C O O C H 2C H 3, RE = CH2CH2CHECH3) increased activity six times more than the parent compound (59 5, RI = CH2CH2COOCHECH3, R2 = H). The 3-(2phenylethyl analog in the latter group (59 1l, RI = CH2CH2C6H5, R2 = CHECHECH2CH3) had no activity. lO-Deoxoartemisinin (60 1, R1- RE = CH3) possessed 6.5 times more activity than artemisinin against the D-6 clone. Two groups of 10deoxoartemisinin derivatives, one group substituted at positions 3 and 9 (60 3-8) and the other group substituted at position 3 (60 9-17) were prepared [57]. In the former group the 9-(p-chlorophenyl)propyl analog (60 8, Rl = CH3, R2 = CH2CH2CH2C6H4-CI-p) was 70 times more active than artemisinin, followed by the 9-butyl (60 5, Rl = CH3, R 2 CH2CH2CH2CH3) and 9-phenylpropyl analogs (60 7, RI = CH3,, R2 = CH2CH2CH2C6H5). The compounds, in which ethyl, butyl and p-chlorophenylpropyl groups were substituted at position 3 (60 9, R l - CHECH3, R2 = H, 60 11, Rl = C H E C H 2 C H 2 C H 3 , RE = H, and 60 15, R l CH2CH2CH2C6H4-CI-p), RE = H, respectively) had lower activity compared with the compounds substituted at position 9 (60 3, Rl = CH3, R2 = CHECH3), (60 5, Rl = CH3, R2 = CHECHECHECH3) and (60 8, R 1 = CH3, R E - CH2CHECHEC6H4-CI-p).
In the 3-substituted analogs with 9-methyl in the artemisinins (10-oxo derivatives) (59 16-20), ethyl and propyl analogs substituted at 3 (59 16, R i - C H E C H 3 , RE = CH3 and 17, Ri - CHEC H2CH3, RE = CH3, respectively) were the most active and about 12 times more active than artemisinin, while the ethyl and propy! analogs substituted at position 9 in 10-deoxoartemisinin (60 3, Rl = CH3, RE- CHECH3 and 4, R ! - CH3, RE= CHECHECH3 were nine and five times more than artemisinin, respectively. 4-(p-Substituted phenyl)-4'-(R or S)-[ 10(ct- or 13-)-dihydroartemisi ninoxy] butyric acids (61 1-16) were designed to improve efficacy and increase solubility either in water such as sodium artelinate which possessed substantially less central nervous system toxicity in rats and dogs, or in oil such as artemether and arteether [58]. The methyl esters (61 1-9) and three of the free acids (61 10,13 and 14) had IC50 values less than 1 mg/ml, and were as active as the lipophilic arteether in vitro, while
CHEMISTRYOF ANTIMALARIALCOMPOUNDS
165
artelinic acid had an IC50 of more than 1 ng/ml. R-diastereomers were mostly more potent than the corresponding S-diastereomers. pChlorophenyl (61 11, RI = CI, R2 = H, R([3)) and p - b r o m o p h e n y l derivatives (61 14, RI = Br, R2 = H, R([3)) showed four and ten times more activity than artelinic acid against both the W-2 and D-6 strains in vitro respectively, p-Fluorophenyl (61 12, Rl = F, R2 = H, R([3)) and pmethoxyphenyl derivatives (61 16, Rl = OCH3, R2 = H, R([3)), which possessed strong electron-withdrawing and strong electron-donating functions, respectively, were as active as artelinie acid or less. The in vivo antimalarial activity against P. berghei in mice correlated well with the in vitro data.
CHH
H H
a
C
l 2 3 4
5
~
H-OCH o
H
RI
I
CH2CH2COOR 2
61
6 7 8 9 1o I1 12 13 14 15 16
Rl =
C! CI CI F
R2 =
CH3 CH 3 CH 3 CH 3
*Si~) R([3) R(cx) R(fl)
F
r
S(fl)
Br Br OCH 3 OCH 3 Ci CI F F Br Br OCH3
CH 3 CH 3 CH 3 CH 3 H H H H H H H
R(~) S(~) R~) R(tz) S([~) R(~) R(fl) S(fl) R(~) S(~) R(~)
In studies of derivatives of artemisinin for antimalarials, compounds with different substituents on artemisinin, 10-deoxoartemisinin, and tricyclic trioxane derivatives which lack the lactone ring present in tetracyclic artemisinin, and the compounds with side chains at position 3 and 9, and stereoselectivities of the side chain at position l0 in the structure of artemisinin have been synthesized and tested for antimalarial activity to obtain more potent compounds. On the other hand, a trioxane moiety is absolutely necessary to be effective for antimalarial activity. Thus tricyclic 1,2,4-trioxanes, 6,7,10-trioxanespiro[4,5]decanes, 1,2,5trioxaspiro[5,5]undecanes, and so on, which do not consist of the ring junctions as in artemisinin have been synthesized and tested. Some of them have been shown to be more active than artemisinin and dihydroartemisinin. Recently and crucial to the typical physiological pathway involving heme-promoted activation of trioxanes into metabolites cytotoxic to the malarial parasites, a reaction pathway proceeding via a carbon-centered radical has been suggested to be important for antimalarial activity [59,60]. These results could make progress to produce better nonalkaloidal antimalarial agents.
166
KAWANISHI and FARNSWORTH
Antimalarials which are related to derivatives based on the natural products, quinine and artemisinin have been discussed here. Antimalarials from other categories have been synthesized and screened in vitro and in vivo. However, newly developed drugs have not been successful, because strains of P. falciparum which are relatively or absolutely resistant to their action have emerged from this approach. The mechanisms of antimalarial actions are uncertain. In addition, drugs having less side effects do not seem to be possible with analogs of these types. Studies on the biology of P. falciparum, a knowledge of mechanisms involved in drug actions, toxicity and side effects, and the chemistry of drug preparations must be combined to obtain effective drugs. 2.0
NATURAL SUBSTANCES SYMPTOMS OF DIABETES
USED
TO
ALLEVIATE
2.1 Aldose reductase inhibitors
Insulin therapy is effective in normalizing glucose levels and has improved the elimination of keto-acidosis coma as a cause of death in diabetics. Chronic diabetes is accompanied by complications such as cataracts, neuropathy, retinopathy and nephropathy. However insulin therapy has not been effective in eliminating them. In diabetes mellitus, the increased availability of glucose results in the formation of sorbitol through the polyol pathway in insulin-insensitive tissues such as lens, nerve and retina. Aldose reductase converts glucose to sorbitol only at high glucose levels in plasma and tissue in diabetes, while it does little in normal cases. Sorbitol can be converted in turn to fructose by polyol dehydrogenase. Polyols will be accumulated to high levels in cells and will result in a loss of osmotic integrity and cellular damage because they are not readily metabolized and they do not penetrate cell membranes easily. These reactions lead to the development of complications in chronic diabetes. Diabetic rats treated with aldose reductase inhibitors decrease the accumulation of polyols [61-65]. Some of the aldose reductase inhibitors have been used for prevention of some pathologies of chronic diabetes as well as in the treatment of diabetes complications. Tetramethyleneglutaric acid (TMG) (62) and alrestatin (AY-22,284) (63)) are known aldose reductase inhibitors. From natural products, a number of flavonoids have been reported to have aldose reductase inhibitory activity. Quercetin (64 1, R = H), quercitrin (64 2, R = Lrhamnose), rutin (64 3, R = rutinose (6-O-ct -L-rhamnosyl-D-glucose)) and myricitrin (65 2, R = L-rhamnose) were much more effective inhibitors of aldose reductase from rat lens than TMG (62) and alrestatin (63) [66]. Quercetin with 3',4'-dihydroxy substitution in ring C was more potent
CHEMISTRY OF ANTIMALARIALCOMPOUNDS
167
than morin with 2',4'-dihydroxy substitution in ring C (66). Derivatives of 3',4' dihydroxyflavone were designed and tested for inhibitory activity on aldose reductase from both rat and bovine lens [67]. In this series methylation of the 7-hydroxy or methylation of the 8-hydroxy enhanced the inhibitory activity of the derivatives with 5,7,8-trisubstitutions and 5,6,7,8-tetrasubstitutions. 3',4'-Dihydroxy-5,6,7,8-tetramethoxy flavone was the most potent inhibitor in this derivatives. Forty-four flavonoid derivatives, as well as chlorogenic acid, catechins, anthocyanins and coumarins, which were mostly isolated from plants [68] were evaluated according to the degree of hydroxylation and the state of oxidation in ring B. In the simplest flavonoids, trihydroxyflavones, apigenin (4',5,7trihydroxyflavone) and 4'-methoxyapigenin (5,7-dihydroxy-4'methoxyflavone) were active, and the former was slightly more active than the latter. In tetrahydroxyflavones, 3',4'-dihydroxy derivatives with two COOH
TMG 6 2
63 alrestatln, AY-22, 284
~ ~ H
2'
7
3O ' Hm4, OH
H
OH
~
0 OH
OH
OR
OH
O 65
64 querceUn quecltrin rutln
OH
I R=H L-rhamnose 2 6-O-a-L-rhamnosyl- I-D-glucose 3 4 D-glucose HO H
myrlcetln myricltrin
, OH
T OH
-o. 0 morin 6 6
I 2
R= H L-rhamnose
168
KAWANISHI and FARNSWORTH
hydroxyls in ring A were more active than the 3'-hydroxy series with three hydroxyls in ring A. Luteolin (3',4',5,7-tetrahydroxyflavone) and orientin (3',4',5,7-tetrahydroxy and 8-C-glucoside) showed similar activity. Fisetin (3',4',3,7-tetrahydoxyflavone) and eriodictyol (2,3-dihydroluteolin, 3',4',5,7-tetrahydoxyflavone) were also potent. In pentahydroxyflavones, quercetin (64 1, R = H), quercitrin (64 2, R = L-rhamnose) and quercitryl2"-acetate still possessed inhibitory activity at 10-7 mol. In hexahydroxyflavones, myricitrin (65 2, R = L-rhamnose) was potent at 107 mol. In the series of derivatives with hydroxy groups at the same position after closing and opening of the oxide ring B, hesperetin (3',5,7trihydroxy-4'-methoxyflavone), hesperidin (7-rutinoside), hesperidin chalcone and chlorogenic acid, chlorogenic acid was only potent at 10-7mol (67) [68]. In the series of catechins and anthocyanins derived from 4',3,5,7-tetrahydroxyflavone, in which the substituents were analogous to those of kaempferol (4',3,5,7-tetrahydroxyflavone), pelargonin (3,5-bis (glucosyloxy)-4',7-dihydroxy flavylium chloride) (70) were inactive at 10-5 mol. (-)-Catechin ((2R,3R) 4',5,7-trihydroxy flavan-3-ol) (69) was more potent than (+)-catechin ((2R,3S) 4',5,7-trihydroxy flavan-3-ol) (68), but was not as active as quercetin. No derivative in this series had potent activity. In the coumarins, esculetin (71 1, R = H) was active at 10-7 mol, while esculin (6,7-dihydroxycoumarin 6-glucoside (71 2, R = D-glucose) was not. 5,6,7-Trisubstituted flavone derivatives, which were isolated from plants or synthesized, were screened against aldose reductases from both rat and bovine lenses [69]. Axillarin (3',4',5,7-tetrahydroxy-3,6dimethoxyflavone) was the most potent inhibitor, which was 19 times more active than quercitrin on the enzymes from both sources, followed by cirsiliol (4',5-dihydroxy-6,7-dimethoxyflavone) and 7-methoxyaxillarin (3',4',5-trihydroxy-3,6,7-trimethoxyflavone). In the next series, 8methoxy-5,6,7-trisubstituted flavone derivatives were weak comparing with the 5,6,7-trisubstituted flavone derivatives. No differences in inhibitory activity was shown between 5,6-dihydroxy-7,8dimethoxyflavone derivatives and 5,7-dihydroxy-6,8-dimethoxyflavone derivatives. In the derivatives of 6-hydroxy-5,7,8-trimethoxyflavone, 5hydroxy-6,7,8-trimethoxyflavone and 5,6,7,8-tetramethoxyflavone, 3',4',6trihydroxy-5,7,8-trimethoxyflavone were the most active, followed by 4'hydroxy-5,6,7,8-tetramethoxyflavone and 4', 6-dihydroxy-5,7,8trimethoxyflavone, which were similar or more potent compared with quercitrin (62 2, R = L-rhamnose) on rat lens aldose reductase. Isoflavone derivatives had no inhibitory activity. Esculetin, coumarin with 6,7dihydroxy substitutions (71 1, R = H) possessed inhibitory activity, while coumarins with 3-hydroxy, 6-methoxy, 7-methoxy and 6,7-methylendioxy substitutions did not [68,69]. Monoglycosides had a higher activity than the corresponding aglycones and oligosaccharides [70]. 4',5,7-Trihydroxy3,6-dimethoxyflavone (72) was isolated from ,4canthospermum australe
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
OH " ~
169
OH OOH
HOOC"'?HO~
~)Oy 0 67
-
i,i
o.
.=m
OH
ee
, OH
OH 69
CI" H
OH [
_ O-D-glucose -D-glucose 70
.
71 esculetln I esculln 2
72
R = H D-glucose
(Compositae) as an aldose reductase inhibitor, which was 18 times more effective than quercitrin [71]. Matteuorienate A (73 1, R! = H, R2 = OCH3) and B (73 2, Rl = R2 = H) were found in Matteuccia orientalis (Polypodiaceae) [72]. They were 6.6 times more active than quercetin for rat lens aldose reductase. Matteucinol (74 1, Ri = R2 = H, R3 = OCH3), 7O-13,-glucopyranosyl matteucinol (74 2, Rl=D-glucose, R2 = H, R3 = OCH3), demethoxymatteucinol (74 3, Rl = R2 = R3 = H) and 7-O-13 -glucopyranosyl demethoxymatteucinol (74 4, RI = D-glucose, R2 = R3 = H) were less active. Dicrotalic acid (75), which is a moiety of
170
KAWANISH! and FARNSWORTH
mattcuorienate A and B did not show inhibitory activity. Methyl esters of matteuorienate A and B (73 3, Rl = CH3, R2- OCH3 and 73 4, R l - CH3, R2 = H, respectively) were inactive. Studies on structure activity relationship of flavonoids suggest that hydroxy groups substituted at positions 7 and 4' enhance inhibitory activity.
R,O0~C'~
o
OH
~H3
]
"'"'~ 73
OH
P~
0
A B matteuorlenate A methyl ester B matteuorienate
CH 3
R3
HOO~~HH~
-,Ill
74 matteuctnol
I Rz =H R2=H R,3=OCH3 2 R l = D-glucose R2=H R3 = OCH 3 3 RI=H I~ = H R 3 = H
4
COOH
dlcrotallcacid
75
R l = D-glucose R2=HR 3 =H
COOH
HO~
COOH HO0
7e
O
OC H~r--CH--CH2--0
5H cromolyn 7 7
b
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
171
For development of potent aldose reductase inhibitors based on flavonoids, the aromatic 2-phenyl substituents of flavonoids were replaced with a non-aromatic carboxyl group. The 4-oxo-4H-chromene ring system of flavonoids was necessary for inhibitory activity [73]. From structure similarities of 2-chromonecarboxylic acid (76) with the antiallergy agent, cromolyn (77), a variety of antiallergy agents were examined for a search of aldose reductase inhibitors, such as quinolone, coumarin, oxanilic ester, xanthone, 11-oxo-11H-pyrido[2,1-b]quinazoline, 1,4,6,9-tetrahydro-4,6dioxo-pyrido[3,2-g]quinoline, 1,6-dihydro-6-oxo-2-phenyl-pyrimidine, 3,4-dihydro-4-oxothieno[2,3-d]pyrimidine and others [74]. The chroman system was combined with a hydantoin ring to result in the formation of spirohydantoin with a potent aldose reductase inhibition activity, sorbinil (CP 45,634) ( 78 1, R = H). A methyl group was added at position 2 in the chroman ring to develop M79,175 (78 2, R = CH3). The chroman ring was replaced with a planer fluorene ring to produce the spirohydantoin, alconil (AL 1567) (79), which had increased inhibitory activity. Alrestatin (63) and tolrestatin (AY-27,773) (80), which were previously known aldose reductase inhibitors and containing the N-benzoylglycine fragment, contributed significantly to the inhibitor-enzyme interaction. They were developed as more potent organic acids such as [3-4(-bromo-2fluorobenzyl)-4-oxo-3H-phthalazin- 1-yl]acetic acid, statil (ICI 128,436) (81)) and (E)-5-[(E)-2-methyl-3-phenyl-propenylidene]rhodanine-3-aeetie acid, ONO-2235 (82). From 54 hydantion and thiohydantion derivatives 1(4-bromophenylsulfonyl) hydnation (83) was deeveloped to be as potent sorbinil (78 1, R = H) [75] and its inhibition was due to its non-ionized form and was a non-comparative type [75]. This was expanded to preparing N- benzenesulfonylglycine derivatives (84 1-3) [76]. In this series, 13 -naphthylene derivative (84 1, R = 3,4-CH=CH-CH=CH-) was three times more active than the o~ - naphthylene group (84 2, R = 2,3CH=CH-CH=CH-), which both were more active than alrestatin (63). A 4-benzoylamino analog (84 3) was as effective as 13 -naphthylene (84 1) [77]. 5-Arylthiazolidine-2,4-diones (85) were derived from l(4bromophenyl-sulfonyl)hydantoin (83) [78]. 5-(3,4-Dialkoxyphenyl)thiazolidine-2,4-dione derivatives (85) showed potent inhibitory activities in both assays; aldose reductase and lens swelling, and were more potent than 5-(4-alkoxyphenyl)thiazolidine-2,4-dione derivatives. To the contrary, the 5-(2,4-dialkoxyphenyl) and 5-(trialkoxyphenyl) derivatives had inferior activity. Alrestatin (63), tolrestatin (80) and ONO-2235 (82) were altered to produce the 2-oxoquinoline derivative, ICI 105,552 (86). This led to the simple 2-oxoquinoline-1-acetic acid derivative (87 1), which contained a N-acylglycine moiety [79]. The derivatives of 2-oxoquinoline1-(2-propanoic acid) (87 2) and -1-(3-propanoie acid) (87 3) were not as potent as the derivative of 2-oxoquinoline-1-acetic acid (87 1).
172
KAWANISHI and FARNSWORTH
O
O•'-NH
H
o
,R
78
79
sorbinll (CP45, 634) I R = MP 79, 175 2
~
O
H CH3
alconil AL1567
I R = H 2 F
Hs
CH2(X)OH
H3
*~
~F3
Br COOH
tolrestaUn, AY-27, 773
statll (ICI-128, 436) 81
80
~H2COOH o
Dr
~
~ SO2--N ~ _
83
ONO-2235 82
R•• 1 2 3
SO2"-- NHCH2COOH
84 R = 3,4-CH=CH-CH=CH2,3-CH=CH-CH=CH4-NHCOC6H5
O
CHEMISTRY OF ANTIMALARIALCOMPOUNDS
/ R2'
\
Sy 85
RI = 3-OCH3 3-OCH3 3-OCH3 3-OCH2CH3 3-OCH2CH3 3-OCH2CH3 3-OCH2CH3
R2=
~H2COOH
NH O 4-OC4H9 4-OC5H11 4-OC6H13 4-OC4H9 4-OC5H 1! 4-OC5H1l(iso) 4-OC5Hl3(iso)
~H3
~
~ ~ N ~ C o H3
Cl
CH3
R
Cl
ICI 105, 552
173
87
I R = CH2COOH 2 CH(CH3}COOH 3 CH2CH2COOH
86
The 4(3H)-quinazolinone ring was derived from the 4H-1-benzopyran4-one ring in the flavones to obtain 2-(arylamino)-4(3H)-quinazolinones (88 1-9) [80]. The 4'-COOH analog (88 9, R1 = OCH3, R2-- COOH) was the most effective but was much less active compared to sorbinil. I
i
~ 1
~ ]
OH 1 2
3 4 5 6 7 8 9
RI=
2'
H
H H H H H OCH3 OCH3 OCH3
3'
R2
NH(CH2)n~ _ _' . ~6f 4'5'
H4 O
88 R2 -
H H 4'-OH 4'-COOH 4-SO3Na T-OH, 4'-COOH H H 4'-COOH 6
n-I
0 0 0 0 0 I
0 0
K A W A N I S H I and F A R N S W O R T H
174
In the series of benzopyran-2-ones (coumarins), 5,6-dihydroxybenzopyran-2-one with CH2COOH at position 4 (89 15, R~ = 5,6-(OH)2, R2 - C H 2 C O O H ) was as active as sorbinil, while benzopyran-2-one with CH2COOH at position 4 (89 3, R! = H, R 2 - CH2COOH) was 30 times less active [81 ]. 1
~
O ~
R1
89 ! 2 3 4 5 6 7 8 9 10 1i 12 13 14 15
Rl =
5,7-(OH) 2 5,8-(OH) 2 H 7-OH 7-OH 7-OH 7-OAe 7-OCH 3 7-OCH 3 7-OCH3 7-OCH3 7-CI 7-CH2CH3 5,7-(OH) 2 5,6-(OH) 2
O 3
R2
R2 =
CH3 CH3 CH2COOH CH2COOH CH2COOCH3 CH2CONH2 CH2COOH CH2COOH COOH CH=CHCOOH CH2CH2COOH CH2COOH CH2COOH CH2COOH CH2COOH
O
~
/ ORl HOH2C~O 91
\0--~ 0
R3O /
90
1
2 3 4
R1=
L
CHO
1-4
H
CH3 CH3 SO3K
R2 =
H
CH3 CH3 SO3K
R3 =
H
CH3 CH3 SO3K
R4 =
H
H SO3K SO3K
Ellagic acid (90 1, Rl = R 2 = R 3 = R 4 = H) and its derivatives are widely distributed in the plant kingdom. Ellagic acid is reported as an aldose reductase inhibitor [82] and its two derivatives (90 2, Rl = R2 = R3 = CH3, R4 = H and 90 3, Rl = R2 = R3 = CH3, R4 = SO3 K) were also found to be
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
175
aldose reductase inhibitors [83]. The latter was more active than sorbinil, while the former was not. Other derivatives were synthesized and had potent inhibitory activity. Sulfonylated compounds had increased activity. The sulfonylated compound (90 4, R1 = R2 = R 3 = R 4 = SO3K) was the most potent, which was ten times more active than sorbinil. Ellagic acid was also isolated as an aldose reductase inhibitor from the Kampo Medicine, Hachimi-jio-gan, besides 5-hydroxymethyl-2-furfuraldehyde (91), which had 100 times weaker activity than ellagic acid [84].
22
IH
,H
4 19
18 danshenol A 92
1
danshenol B 92 2
~H
dihydrotanshinone t a n s h i n o n e I A 15
18
1 92
O.
-sl'l H
3
cryptotanshinone 92
4
5
t a n s h t n o n e IIA A15
6
The novel abietane-type diterpenes, danshenol A (92 1) and B (92 2), along with four known ones, dihydrotanshinone I (92 3), cryptotanshinone (92 4), tanshinone I(92 5) and tanshinone IIA (92 6) were isolated from the roots of Salvia miltiorrhiza (Labiatae) [85]. Inhibitory activity was found in these six abietane-type diterpenes with activity more or less equal to quercetin. Danshenol A was the most potent, which was 56 times more than quercetin. Flavonoids and related phenols, such as catechins and coumarins have been found to be aldose reductase inhibitors. Compounds containing a
176
KAWANISH! and FARNSWORTH
glycine moiety in the molecules including hydantoin analogues, have been developed as synthetic aldose reductase inhibitors. Flavonoids and coumarins have been modified further by combining these with Ncontaining compounds to produce more effective analogs. Abietane-type diterpenes, which do not contain phenolic groups in their structures have recently been found. Further studies to identify different types of basic structures will help to develop more potent inhibitors. 2.2 a-Glucosidase inhibitors
Diabetes mellitus is characterized by fasting hyperglycemia and postprandial increases in plasma glucose levels. It is important to diagnose the type of diabetes in patients. The level of postprandial blood glucose in hyperglycemia, hyperinsulinemia and hypertriglyceridemia is usually high. The dietary carbohydrate components, starch and sucrose, are broken down enzymatically to monosacchaddes by ~-glucosidases before they are absorbed. Inhibition of intestinal a-glucosidases causes a delay in the digestion of starch and sucrose. Inhibitors of these enzymes can act to delay carbohydrate absorption, improve the metabolic state and possibly prevent the development of diabetic complications. Several potent aglucosidase inhibitors have been isolated from natural sources. Compounds that are structurally similar to aldoses or natural products that are ~glucosidase inhibitors have been synthesized. Some of these have been
H
~
O~[N~~
~
(OH
H3q
acarbose 93 1 (BAY G542 I)
- " ' ~
~OH
OH
oI-hl
OH HO
OH AL 5562 9 3
2
"~2
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
177
used clinically. A study of the synthesis of compounds with potent inhibitory activity has allowed for putative mechanisms for the enzymatic hydrolysis of disaccharides to be proposed and prototype transition-state analogues with potent inhibitory activity of o~-glucosidase have been synthesized [86]. In the culture broths of Actinoplanes strains SE 50, SE 82 and SB 18 carbohydrates were produced. One of them was acarbose (BAY G5421) (93 1), which consists of a pseudo-tetrasaccharide, and including a valienamine (95) or acarviosin (96 1) group. It potently inhibited brushborder glucoamylase, dextrinase, maltase and sucrase and pancreatic o~ -amylase in vitro and in vivo [87]. Acarbose is structurally similar to oligosaccharides which are derived from starch digestion and attaches to the carbohydrate binding site of the tx-glucosidase enzyme. A15662 (93 2) which consists of a pseudo-heptasaccharide in including two valienamine (95) or acarviosin (96 1) groups, was also an ct-glucosidase inhibitor [87].
r:2o. voglibose 94
CH20H valienamine 95
The pseudo-amino sugar, voglibose (94), which consists of a cyclohexanetetrol with 2-amino 2-deoxy glycerol or a valiolamine with a glycerol group was obtained from a culture broth of Streptomyces hygroscopicus subsp, limoneus. Voglibose inhibits intestinal ct -glucosidases which are responsible for digestion of disaccharides such as maltose and sucrose [87]. HOH2q
Rl
acarviosin oligobiosaminide
methylacarviosin methyloligobiosaminide
I 2 3 4
Rl=
H H CH3 CH3
R2 =
H OH H OH
178
KAWANISHI and FARNSWORTH
OAt: 96 96
5 a-{ISl 6 a-{IR)
8
7 13
Acarviosin analogues (96 1-8) were synthesized and the structureinhibitory activities were elucidated [88]. Oligobiosaminide (96 2) is a core structure of acarbose. Methyl acarviosin (96 3) is an cc-amylase inhibitor. Methyl acarviosin (96 3), methyl oligobiosaminide (96 4), the 1,6 anhydro derivative of acarviosin with 1S configulation (96 5) and 3,6-anhydro derivative of methyl acarviosin (96 8) were highly active against yeast cc -glucosidase, and free pseudo-disaccharide (96 7) was only potent against Jack bean ct-mannosidase. 1 2
HOH2C HOH -O ' ~. ~ ~, , , ~ ~ 'N
_ . . ~ H2C ~/~ __~ .Ro " !, O OH ~ ~Ox[ 97
R2 ~Rl
3 4 5 6 7
R!= H OH H H H H n
R2=
OH H N3 NH2 NHAe F S-C6H4CH3-P
8
H
H
9
H
H
R3= OH OH OH OH OH OH OH OH H
Syntheses and measurement of inhibitory activity for 1,6-anhydro analogs of methyl acarviosin (97 1-9) have been reported [89]. Two analogs (97 8 and 9), which are both deoxy derivatives at position 2 in anhydro glucose, were the most active against yeast a-glucosidase, which were 10 and 5 times more active than methyl acarviosin (96 3) and 1,6anhydroacarviosin (96 5), respectively. The results suggest that the absence of a polar function or introduction of a hydrophobic portion around the imino linkage was likely to increase their inhibitory activity. Polyhydroxylated piperidines from natural sources, which have structures and shapes resembling monosaccharides have been found as o~ -glycosidase inhibitors. They competitively inhibit glycosidases whose substrates they most closely resemble. 1-Deoxynojirimycin (moranoline) (98 1), was isolated from Mori Cortex (root bark of the mulberry tree, Morus bombycis (Moraceae)), leaves of Jacobinia suberecta (Acanthaceae)
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
179
and a culture filtrate of Streptomyces species and Bacillus species [90]. 1Deoxynojirimycin was a deoxy derivative of nojirimycin (98 11) which was found in Streptomyces spp. and synthesized [91]. The enzyme inhibitory activity of 1-deoxynojirimycin was related to its similarity in chemical structure to glucose. Derivatives with an alkyl group substituted at the N of 1-deoxynojirimycin were prepared and tested for hydrolysis by sucrase from rabbit intestine. 1-Deoxynojirimycin and N-methyl 1deoxynojirimycin (98 2) showed the most potent inhibitory activity. NEthyl 1-deoxynojirimycin (98 3) was 20 times less active than the former derivatives, and the isobutyl derivative (98 4) was least active in the series. From the leaves of M. bombycis, were isolated 1-N-methyl-1deoxynojirimycin (98 2), 2-O-ct,-D-galactopyranosyl-D-arabinitolyl 1deoxynojirimycin (98 5), fagomine (1,2-dideoxynojirimycin (98 6)), 1,4dideoxy- 1,4-imino-D- arabinitol (99 1), 1,4-dideoxy- 1,4-imino-(2-O-[3-Dglucopyranosyl)-D-arabinitol (99 2), lct,2[3,3cx,4[3-tetrahydroxynortropane (calystegine B2) (105 6) and 1-deoxynojirimycin (98 1) [92]. Their inhibitory activities on t~-glucosidase have not been reported. NButyl l-deoxynojirimycin (98 10) was found to be an inhibitor of the glucosyltransferase-catalyzed biosynthesis of glucosylceramide besides an inhibitor of the r and human immunodeficiency virus replication in vitro [93]. Two derivatives of 1-deoxynojirimycin were developed as an ct-glucosidase inhibitor. One of them was miglitol (BAY M1099) (98 7), of which the 2-hydroxyethyl group was substituted at the N in the ring, and the other was emiglitate (BAY O 1248) (98 8), an ether of miglitol, such as a p-ethoxybenzoate group substituted at N in the ring [87,94]. Both inhibited sucrase and maltase in vivo. Emiglitate reduced postprandial blood glucose and insulin levels after a carbohydrate load administered even up to 17 h after giving the substance to rats. OH
98
l-deoxynojirimycin N-methyl- 1-deoxynoj irimycin
fagomine migilitol (BAY M ! 099) emiglitate (BAY O 1248) N-butyl- 1-deoxynoj irimycin
! 2 3 4 5 6 7 8 9 10
R2
RI = OH OH OH OH O-tx-D-galactose H H H H H
R2 -
H CI{3 CH2N3 CH2CH(CH3)2 H H CH2CH2OH CH2CH2-O-C6H4COOC2H5 CH2CH2-~-C6H4 COOC2 H5 CH2CH2CH2CH3
180
KAWANISH! and FARNSWORTH
OH
OH
HO-HO._~,~ OH noJirimycin
98 11
1-deoxymannoJlrimycln 98 12
99
1 2
R--- H D-glueo~
HO
Fagomine (1,2-dideoxynojirimycin) (98 6) was first found in the seeds of buckwheat, Fagopyrum esculentum (Polygonaceae) and Castanospermum australe (Leguminosae) and the glucoside of fagomine (98 9) which was later found in the seed of Xanthocercis zambesiaca (Leguminosae), both failed to show inhibitory hydrolysis by a-and [3glucosidases, r a- and 13-galactosidases and 13-glucuronidase [95, 96]. However, fagomine reduced blood glucose levels in streptozotocin-diabetic mice significantly, while deoxynojirimycin was only weakly active [97]. Protonated 1-deoxy nojirimycin (98 1)and 1deoxymannojirimycin (98 12) might stimulate the charge of the corresponding glycosyl cation, but not change their configuration to the flattened form (100). This was proposed on the base of X-ray crystallographic studies of the lysozyme-inhibitor complex [98]. Amidines (101 1,5 and 8), amidrazones (101 3,6 and 9) and amidoximes (101 4,7 and 10) of monosaccharides were synthesized to study the desired flattened configuration of the transition state [99]. Glucoamidine (101 1), glucoamidrazone (101 3) and glucoamidoxime (101 4) were found to be inhibitors of 13-glucosidase, and glucosamidine and glucoamidrazone to also be inhibitors of a-mannosidase. Mannoamidrazone (101 6) was found to be the specific and most potent inhibitor against r and galactoamidoxime (101 10) inhibited both r and 13-galactosidases. It has been proposed that mechanism of enzymatic glycosidase hydrolysis involves a transient oxocarbonium with a flattened chair conformation stabilized by an active catalytic residue with a complementary charge (101 12), and the structure, shape and charge of the amidine derivatives (101 1, 5 and 8) closely resembles the transient glycosyl cation as potent glycosidase inhibitors [100-102]. A study of the aglycon moiety of the glycosides was undertaken by using benzylamidine (101 11) [ 103]. Benzylamidine inhibited r [3 -mannosidases, reduced the binding constant for 13-glucosidase and did not inhibit l~-galactosidase. Benzylamidine is an inhibitor with rather narrow specificity in comparison
C H E M I S T R Y OF A N T I M A L A R I A L COMPOUNDS
181
with glucoamidine (101 1) and mannoamidine (101 5). These results showed that the enzyme-aglycone interaction might contribute to the stereoselectivity of this binding.
~ H
Glu35
.,l O - - - R
ASP52 transition state
100
OH N HOHo
I01
I 2 3 4
R = NH 2 N(CH3) 2 NH-NH 2 NH-OH
101
5 6 7
R = NH 2 NH-NH 2 NH-OH
R
OH
H
R
H
OH N
I01
HO
R
8 R = 9 10
H~x,/H"
.......
benzylamidine I 0 1
e~
11
I
transition state 1 0 1
12
NH 2 NH-NH 2 NH-OH
182
KAWANISH! and FARNSWORTH
Phenyl a-D- and [3-L- glucopyranosides and their acetates (102 1-7) were prepared and hydrolyzed by yeast tx-glucosidase [104]. 2Chloromethyl-4-nitrophenyl a-D-glucopyranoside (102 1) showed the most potent inhibitory activity in this series. The 2-chloromethyl-phenyl derivative (102 6) which had no nitro group in the benzene ring, and the 2chloromethyl-4-nitrophenyl 13-L-glucopyranoside (102 7), in which the OH
102 O RI
~OR3
!O , ~ /~~ .
R! =
CH2CI CH3 CH3
R2=
NO2 H NO2
CH2Br CH2CI CH2CI
R3=
NO2 NO2 H
H Ae Ae Ae Ae H
~" R2 CH2CI
OH
HO~~~O
I 2 3 4 5 6
(OO NO2
102 7
phenyl group was oriented opposite to (102 1) were less active. 2-Deoxy2,2-difluoroglycosides attached to a 2,3,6-trinitophenyl group (103 3 and 6) were investigated for use in structural and mechanistic studies of ct -glucosidase inhibition [105]. 2,4,6-Trinitrophenyl 2-deoxy-2,2-difluoro103
OH 9
I
X
~R 2
.
I 2 3
Rl = H F F
~
N
O
R2 =
H H X
2
O2N"
r /
103 4 R~ = H 5 F ~I 6 F
OH
\R2
R2=
H H X
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
183
a-glucoside (103 3) and 2,4,6-trinitrophenyl 2-deoxy-2,2-difluoro-ct -maltoside (103 6) inactivated yeast a-glucosidase and human pancreatic or-amylase, respectively. The indolizidine alkaloid, swainsonine (104 1) was isolated from Swainsonia canescens (Leguminosae) and an Ipomoea spp. Q6 (aff. calobra) (Convolvulaceae) and found to be a potent inhibitor of t~mannosidase [ 106, 107]. Castanospermine (104 7) which consists of a tetrahydroxylated indolizidine with different orientations of the hydroxy groups from swainsonine was isolated as the major alkaloid from the seeds of Castanospermum australe (Leguminosae) and its 6-epimer, 6-epimercastanospermine (104 8) and fagomine (98 6) were also obtained [96]. Castanospermine (104 7) was a potent inhibitor of t~- and 13 -glucosidases and its 6-epimer (104 8) was an inhibitor of only a -glucosidase. Australine, a pyrrolizidine alkaloid (104 9) was also isolated from the same plant and found to be potent and specific inhibitor of amyloglucosidase. Rosmarinecine, trihydroxypyrrolizidine (104 10)
H
H
swainsonlne
104
H
8-epi-swainsonine
1
104 2
1,8-dlepl-swainsonine
104 3
OH HO
H
OH 8a-epl-swainsonine
OH 104 4
2,8a-diepi-swainsonine
104 5
8,8a-dlepi-swainsonine 104 6 8
HO H
H
~ HO" 7
castanospermine 104 7
6-epi-castanospermine 104 8
rosmarinecine 104
I0
australine 104 9
184
KAWANISHI and FARNSWORTH
and fagomine (98 6) had no inhibitory activity, both of which possess three hydroxy groups in their rings. From a study of a structure-activity relationships of swainsonine, swainsonine (104 1) and the 8,8a diepimer (104 6) completely inhibited acidic a-mannosidase, while its 8a-epimer (104 4) was only weakly inhibitory (108). 8-Epimer (104 2), 1,8-diepimer (104 3) and 2,8a diepimer (104 5) had no appreciable effect on -mannosidases. OH HO
V7
6
OH HOW
OH
===-
HO
OH
H
calysteglne A 3
105 1
H ~ HO
calysteglne
A5 105 2
H HO
105 3
calystegine A 6
,OH HO ~ H
HO"V calysteglne
H HO
A7 105 4 H
OH
calysteglne B 1
105 5 H
OH
H ~ HO
132 105 6
calystegine
H
H
OH
H calystegine B a
105 7
calystegine B 4
105 8
calysteglne B s
105 9
Trihydroxy or tetrahydroxy nortropanes, calystegine A3 (105 1) [ 109], Bl (105 5) and B2 (105 6) have been isolated from Calystegia sepium and Convolvulus arvensis (Convolvulaceae) and were reported to be potent inhibitors of 13-glucosidase and a-galactosidase [110]. They were also isolated from Physalis alkekengi var.francheti (Solanaceae) which showed glycosidase inhibitory activity [111]. Calystegines were also obtained from Solanum species, including potatoes, eggplant and the leaves and fruits of S. dimidiatum (potato weed) (Solanacease). Calystegines A3,As,A6,A7,BI,B2,B3,B4,Bs,CI,C2,NI, N-methyl calystegine B2 and Ci, l~-amino-31B,41B,5o~-trihydroxycycloheptane, fagomine and 6deoxyfagomine (105 16) were isolated from Lycium chinense (Solanaceae) [ 109]. N-Methyl calystegine BE(105 13) was a more potent inhibitor of a -galactosidase than the parent compound but not inhibitory for other
CHEMISTRYOF ANTIMALARIALCOMPOUNDS
185
glycosidases. N-methylation products of calystegines A3 and B4 markedly enhanCed inhibition of a-galactosidase but not 13-glucosidase. . H 2 N ~ H
HO ~ H HO" V
HO-V
calysteglne C2 105 11
calystegine Cl 105 10
H
a ~OH O ~ V
o".
calysteglne Nl 105 12
3 ..,,,~-- OH OH
H
-OH
O
HO" v
N-methyl-calystegine B2 1015 13
~
OH -OH
N-methyl-calystegtne Cl 105 14
N"2
H O ~
3 I ~-amino-3~.4~,5a-trihydroxycycloheptane 105 15
H3C-~ ~,HN
6-deoxyfagomine 105 16
Synthetic compounds derived from the alkaloids described above were also tested for inhibitory activity against (x-glucosidases. 1,4,6-Trideoxy1,4-imino-D-mannitol (106 2) was ten times more active than swainsonine (104 1) against jackbean ct -mannosidase (112). 1,4-Dideoxy- 1,4-imino-Dmannitol (106 1) was as active as 1,4,6-trideoxy-l,4-imino-D-mannitol (106 2). 2R-Hydroxymethyl-3R,4S-dihydroxypyrrolidine (1,4-dideoxy1,4-imino-D-arabinitol (D-AB-1) (107 2), and its enantiomer (L-AB-1) (107 3) were more specific in inhibitory activity against tx-glucosidase than 13-glucosidase, while 2 R , 5 R - d i h y d r o x y m e t h y l - 3 R , 4 R dihydroxypyrrolidine (DMDP) (107 1) inhibited both enzymes with less potency [ 113]. Other type of amino sugars, 1,4-dideoxy- 1,4-imino-Ltalitol (108 1) and 1,4-dideoxy-l,4-imino-D-libitol (108 2) were synthesized from D-mannose, and the former was a specific inhibitor of human liver lysosomal (x-mannosidase in vitro [ 114]. 1,4-Dideoxy-l,4imino-D-mannitol (108 3) was a powerful and specific inhibitor of several tx -mannosidases [114]. Other amino sugars and their derivatives, 1,4dideoxy-l,4-imino-L-allitol (DIA (109 1), its N-methyl derivative (Nmethyl-DIA ) (109 2)) and its N-benzyl derivative (N-benzyl DIA) (109 3)) were synthesized and their enzymatic activities were measured [ 115].
186
KAWANISHI and FARNSWORTH
HO~,~H 5|'"I R
106
H.HN
I R= 2
CH2OH CH3
I
HOH2~
HO.27
.0.2 I HOH2C
CH2OH DMDP
107 l
D-AB-I 107 2
OH CYB-3 107 4
L-AB-1 107 3 HOH2C~
~OH ""
HOH2C
..,, OH
HOH2C 1,4-dloxy- 1,4-
1,4-dloxy-l,4imino-D-talitol 108 1
1,4-dloxy-l,4imino-D-mannltol 108 3
imino-L-ribltol 108 2
H+/R
1,4-dloxy- 1,4imino-L-allitol 109 cr
HOH2C
H
I R= 2 3
OH CH 3 CH2C6H 5
"OH
Oil
2 110
DIA was a specific inhibitor against ~-D-mannosidase and N-benzyl-DIA was a specific inhibitor against ~-D-L-fucosidase, while N-methyl-DIA was not an inhibitor against any glycosidase. Synthetic 1S,2R,3S,4R,5Rmethyl[2,3,4-trihydrox-5-(hydroxymethyl)cyclopentyl]amine (110), of which the amino group is not in the ring, was an inhibitor of a mannosidase [116]. It was proposed that the a-amino function of the compound was appropriately positioned to be protonated in the enzyme
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
187
active site by an a -oriented carboxylic acid and four hydroxy groups of the compounds are oriented as in the mannoyl cation with the geometry of the five-membered ring.
R22
3 4 5
6 7
8 9 10 I 1 12 13 I 4
13 16 17
II IV 20
21 21
OH
OCH,
H
OH OH OCH) OCH,
OCH] OCW] OH OCH] OH
OH OH
OH OH OH OCHl OH OH OCIf) OCH, OH OH OH OH 011
H 011
WHI OCH3
H OH OCH] OH H H OH OH
H OH OCH,
11
OH OH OH OH 0I1 0H OH OH OCH) OCOCH,
32
OCII]
OCH3
33 14
OCH] OH OH
OCIOH2 I OCH3
OH
OCJHll
23 24
25 26 27 18 19 30
3s 36 37
la 39 40
H
R]'
OCH) OH OH
OCII] OCH, OCH]
OH OH OCHl OH OCH] OH OCH) OCH) 0CHJ
OH OH
OH OCH) OH OH 011 OH OH
R4= OCHl
111
OH OCH,
H
n7- n
H H
H H H I1
ti
H
II
XH3
H H
H H
H H
OCH) OCH3 OCH3 OH
n
OCH]
n
OCH3 H OH OCH3
H H H H OCH]
H H
Rs-
OCH] OCH, OH 0I( OH
Rs-H
H OCHj OCH] OH
on
n
H
OH
OH
on
H H
OH OH
II
OCHl OCIl) OH OH 0H H H OH OH
OCHj H OCH]
H OCH] OCHl
OH OH
OH
H
0H
H H H
H OCHf OCH] OH OCll,
H OCH,
H OCHl H
H H
n
OCHl
OH
H H H
OH
H
OH 0 I1
H H
OH OH
H
OH OH
H H
H
H
H
OH OCR)
H
H
0 I1 OH OH
OH
OH OH
H
OCH3
0 II
OH
H
OH
0H OH
H OH
H
OH
I1
OH Ii
OCH)
OCH] OCOCH] OCH3 OCHj
OH OCOC3H7 ococHl
OH
ococn3
OH OH OH OCH3 OCOCH,
OH
H OH
OH
H H
ocn,
OCH3
OH OH
OCIZHZJ OCI(LH17
OCIZH2S OCl2H2$ OCH3 OCH, OCH, OCH3
OH
H
0 II
I1
OH
H
OCH3 H
H H
OH
H H II
Oll OH
H
on
0H
OCIZHZ5
H
on
H
H
H
OH OH
H
0c4'~1104
on
OH OH
OCOCiH7 OCOCll, OPO]NmZ OH OH OH 0H OH OH OH OH
H
n H
H H
H H H H
H H
Forty flavones (111 1-40) were tested for activity as inhibitors of rat small intestine a -glucosidase [ 1 171, of these 3',4',5,6,7-pentahydroxyflavone (111 24) showed the most potent inhibitory activity against sucrase, followed by 3',4',5,6,7-pentahydroxy-3-methoxyflavone(111 27). An active extract of tea polyphenols was analyzed against rat small intestinal sucrase and a -glucosidase [118]. Catechins and theaflavins (112 1-8) were isolated from the tea and tested for inhibitory activity on small intestinal sucrase and a -glycosidase. The four catechins (1 12 1-4) inhibited sucrase more than a -glucosidase, while the four theaflavins (112
KAWANISHI and FARNSWORTH
188
5-8) inhibited a -glucosidase to a greater degree than sucrase. (-)Epigallocatechin gallate (112 4) and theaflavin digallate (112 8) were the most potent inhibitors in each group, which might drive from affinities of the esterified polyphenols for the enzyme proteins. Gallic acid itself did not inhibit either sucrase or ct -glucosidase. OH 112
I 2 3 4
oH
~
'%tOR I
R I --- H OH H OH
R2 =
H H X X
~R 2 OH "--"
112
O
5 6 7 8
R1 -
OH
H H X X
R2 --
H X H X
OH
I
OH
Acarbose, voglibose, miglitol and emilitate have been used clinically for diabetes mellitus. The two former compounds have been found in microbial broth and the latter two were developed synthetically from 1deoxynojirimicyn, which is obtained from plants and in culture filtrates of organisms. As described above many a-glucosidase inhibitors have been found in natural sources and derivatives have been synthesized. Some of them are rather broad inhibitors. Chemical structures of the inhibitors against a-glucosidases are similar to carbohydrates which can be hydrolyzed to monosaccharides or compounds similar to monosaccharides. More effective drugs will undoubtedly be developed, when it is known how the enzymes react with the substrate and the inhibitors by measurements of keat, KM and KI values and kH/kD for different glucoside substrates and inhibitors [ 119]. a-Glucosidase inhibitors have been shown to have anti-tumor and antiHIV activities, in addition to decreasing the levels of postprandial blood glucose in hyperglycemia.
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
189
REFERENCES [1] [21 [3] [4]
[5] [6] [7]
[8] [9] [10] [ll] [121 [13] [14]
[15] [161 [17]
[18] [19]
Marshall, S.J.; Russell, P.F.; Wright, C.W.; Anderson, M.M.; Phillipson, J.D.; Kirby, G.C.; Warhurst, D.C.; Schiff, P.L.Jr. ,4ntimicrob. Agents Chemother., 1994, 38, 96. Spencer, C.F.; Kioniussy, F.R.; Rogers, E.F.; Shavel, J.R.Jr.; Easton, N.R.; Kuehl, F.A.Jr.; Phillips, R.F.; Walti, A.; Folkers, K.; Malanga, C.; Seeler, A.O. Lloydia, 1947, 10, 145. Angerhofer, C.K.; Pessuto, J.M.; Konig, G.M.; Wright, A.D.; Sticher, O. J. Nat. Prod., 1992, 55, 1787. Bhakuni, D.S.; Dhawan, B.N.; Garg, H.S.; Goel, A.K.; Mehrotra, B.N.; Scimal, R.C.; Srivastava, M.N. Indian J. Exp. Biol., 1992, 30, 512. Sayed, K.A.; Dunbar, D.C.; Goins, C.R.; Cordova, T.L.; Perry, T.L.; Wesson, K.J.; Sanders, S.C.; Janus, S.A.; Hamann, M.T.J. Nat. Toxins, 1996, 5, 261. Falch, B.S.; Konig, G.M.; Wright, A.D.; Sticher, C.K.; Angerhofer, C.K.; Pezzuto, J.M.: Bachmann, H. Planta Med., 1995, 61, 321. Konig, G.M.; Wright, A.D.; Sticher, C.K.; Angerhofer, C.K.; Pezzuto, J.M. Planta Med., 1994, 60, 532. Okami, Y. Pure ,4ppl. Chem., 1982, 54, 195 I. Wright, A.D.; Konig, M.; Angerhofer, C.K.; Greenidge, P.; Linden, A.; Desqueyroux-Faundez, R. J. Nat. Prod., 1996, 59, 710. Amorim, C.Z.; Marques, A.D.; Balao, R.S. Mere. Inst. Oswaldo Cruz, Rio de Janeiro, 1991, 86, 177. Antoun, M.D.; Gerena, L.; Milhous, W.K. Int. J. Pharmacognosy, 1993, 31, 255. Ayudhaya, T.D.; Nutakul, W.; Khunanek, U.; Bruhsith, J.; Chawaritthumrong, P.; Jewaechdurongkul, Y.; Pawanunth, K.; Yongwaichjit, K.; Webster, H.K. Bull. Dep. Med. Sci. (Thailand), 1987, 29, 22. Benoit, F.; Valentin, A.; Pelissier, Y.; Diafouka, F.; Marion, C.; Kone-Bamba, D.; Kone, M.; Mallie, M.; Yapo, A.; Bastide, J.M. Am. J. Trop. Med. Hyg., 1996, 54, 67. Carvalho, L.H.; Brandao, M.G.L.; Santos-Filho, D.; Lopes, J.L.C.; Krettli, A.U. J. Med. Biol. Res., 1991, 24, 1113. Connelly, M.P.E.; Fabiano, E.; Patel, I.H.; Kinyanjui, S.M.; Mberu, E.K.; Watkins, W.M. Ann. Trop. Med. Parasitol., 1996, 90, 602. Cordell, G.A.; Angerhofer, C.K.; Pezzuto, J.M. Pure ,4ppl. Chem., 1994, 66, 2283. Gessler, M.C.; Nkunyak, M.H.H.; Mwasumbi, L.B.; Heinrich, M.; Tanner, M. Acta Tropica, 1994, 56, 65. Gessler, M.C.; Tanner, M.; Chollet, J.; Nkunya, N.H.H.; Heinrich, M. Phytother. Res., 1995, 9, 504. Khalid, S.A.; Farouk, A.; Geary, T.G.; Jensen, J.B.J. Ethnopharmacology, 1986, 15, 201.
[20] [21] [22]
Misra, P.; Pal, N.L.; Guru, J.C.; Katiyar, J.C.; Tandon, J.S. Int. J. Pharmacognosy, 1991, 29, 19. Nkunyga, M.H.H.; Weenan, N.; Bray, D.H.; Magani, Q.A.; Mwasumbi, L.B.Planta Med., 1991, 5 7, 341. O'Neill, M.J.; Bray, D.H.; Boardan, P.; Phillipson, J.D.; Warhurst, D.C. Planta Med., 1985, 5, 394.
190
[23] [24] [25] [26] [27] [28] [29] [301 [311 [32] [331 [341 [351 [36] [37] [38] [39] [40] [41]
[42] [431 [44] [45] [46] [47]
[48] [49]
[50] [51] [52] [53] [54]
[551
KAWANISHI and FARNSWORTH
Omulokoli, E.; Khan, B.; Chhabra, S.C.J.Ethnopharmacology, 1997, 56, 133. Phillipson, J.D.; O'Neill, M.J. Fitoterapia, 1987, 5, 394. Phillipson, J.D.; Wright, C.W. Planta Med. Suppl., 1991, 57, 53. Tantchou, J.; Sherman, I. Rev. Sci. Technol., 1985, 2, 127. Bray, D.H.; O'Neill, M.J.; Phillipson, J.D.; Warhurst, D.C.J. Pharmacol. Suppl., 1987, 39, 85. O'Neill, M.J.; Bray, D.H.; Boardman , P.; Phillipson, J.D.; Warhurst, D.C.; Peters, W.; Suffness, M. Antimicrob. Agents Chemother., 1986, 30, 101. Trager, W.; Polonsky, J. Am. J. Trop. Med. Hyg., 1981, 30, 531. Bray, D.H.; Warhurst, D.C.; Connolly, J.D.; O'Neill, J.M.; Phillipson, J.D. Phytother. Res., 1990, 4, 29. MacKinnon, S.; Durst, T.; Amason, J.T.; Angerhofer, C.J.; Pezzuto, J.M.; Sanchez-Vindas, P.E.; Poveda, L.J.; Gbeassor, M. J. Nat. Prod., 1997, 60, 336. Sharma, A.; Tewari, R.; Gaunuya, A.K.; Virmani, O. Curr. Res. Med. Aromat. Plants, 1987, 9, 34. Davidson, M.W; Griggs, B.G.; Boykin, D.W.; Wilson, W.D.J. Med. Chem., 1997, 20, 1117. Carroll, F.I.; Blackwell, T. J. Med. Chem., 1974, 17, 210. LaMaontagne, M.P.; Markovac, A.; Menke, J.R.J. Med. Chem., 1977, 20, 1122. Nodiff, E.A.; Chatterjee, S.; Musallam, A. Prog. Med. Chem., 1991, 28, 1. Klayman, D.L. Science, 1985, 228, 1049. Hien, T.T.; White, N.J. Lancet, 1993, 341, 603. Woerdenbag, H.J.; Pras, N.; W. van Uden; Wallaart, T.E.; Beekman, A.C.; Lugt, C.B. Pharmacy Worm & Science, 1994, 16, 169. Jeremic, D.; Jokie, A.; Behbud, A.; Stefanovic, M.Tetrahedron Lett., 1973, 3039. Brossi, A.; Venugopalan, B.; Gerpe, L.D.; Yeh, H.J.C.; Flippen-Anderson, J.L.; Buchs, P.; Luo, X.D.; Milhous, W.; Peters, W. J. Med. Chem., 1988, 31,645. Avery, M.A.; Gao, F.; Chong, W.K.M.; Mehrotra, S, ; Milhous, W.K.J.J. Med. Chem., 1993, 36, 4264. Zhu, D.; Huang, B.; Chen, Z.; Yin, M. Chem. Abstr., 1981, 94, 167365a. Li, Y.; Yu, P.; Chen, Y.; Li, L.; Gai, Y.; Wang, D.; Zheng, Y. Chem. Abstr., 1982, 97, 92245n. Cao, M.; Hu, S, ; Li, M.; Zhang, S. Chem. Abstr., 1984, 100, 34720h. Lin, A.J.; Klayman, D.L.; Milhous, W.K.J. Med. Chem., 1987, 30, 2147. Lin, A.J.; Li, L." Andersen, S.L.; Klayman, D.L.J. Med. Chem., 1992, 35, 1639. Haynes, R.K.: Vonwiller, S.c.; Wang, H.Tetrahedron Lett., 1995, 36, 4641. Singh, C.; Misra, D.; Saxena, G.; Chandra, S. Bioorg. Med. Chem. Left., 1992, 2, 497. Singh, C.; Misra, D.; Saxena, G; Chandra, S. Bioorg. Med. Chem. Lett., 1995, 5, 1913. Vennerstrom, J.L.; Fu, H.; Ellis, W.Y.; Ager, A.L.; Wood, J.K.; Andersen, S.L.; Gerena, L.: Milhous, W.K.J. Med. Chem., 1992, 35, 3023. Posner, G.H.; Oh, C.H.; Gerena, L.; Milhous, W.K.J. Med. Chem., 1992, 35, 2459. Avery, M.A.; Gao, F.; Chong, W.K.M.; Hendrikson, T.F." Inman, W.D.; Crews, P. Tetrahedron, 1994, 50, 957. Acton, N.: Klayman, D.L.; Planta Med., 1985, 51,441. Acton, A, ; Karle, J.M.; Miller, R.E.J. Med. Chem., 1993, 36, 2552.
CHEMISTRY OF ANTIMALARIAL COMPOUNDS
[56] [57]
[58] [59] [60] [61] [62] [63] [64] [651 [66]
[67] [68]
[69] [70] [71] [72] [73] [74] [75] [76]
[77] [78] [79]
[801 [81]
[82]
[83] [84]
191
Avery, M.A.; Mehrotra, S.; Bonk, J.D.; Vroman, J.A.; Goins, D.K.; Miller, R. J. Med. Chem., 1996, 39, 2900. Avery, M.A.; Mehrotra, S.; Johnson, T.L.; Bonk, J.D.; Vroman, J.A.; Miller, R.J. Med. Chem., 1996, 39, 4149. Lin, A.J.; Zikry, A.B.; Kyle, D.E.J. Med. Chem., 1997, 40, 1396. Posner, G.H.; Ho Oh, C.; Wang, D.; Gerena, L.; Milhous, W.K.; Meshnik, S.R.; Asawamahasadka, W. Jr. Med. Chem., 1994, 3 7, 1256. Avery, M.A.; Fan, P., Karle, J.M.; Bonk, J.D.; Miller, R.; Goins, K. J. Med. Chem., 1997, 40, 1885. Hayman, S.; Kinoshita, J.H.J. Biol. Chem., 1965, 240, 877. Robison, W.G. Jr.; Kador, P.F.; Kinoshita, J.H. Science, 1983, 196, 1177. Terashima, H.; Hama, K.; Yamamoto, R.; Tsuboshima, M.; Kikkawa, R.; Hatanaka, I.; Shigeta, Y. J. Pharmacol. Exp. Ther., 1984, 229, 226. Showalter, H.D.H., Johnson, J.L.; Werbel, L.M., Leopold, W.R.; Jackson, R.C.; Elslager E.F.J. Med. Chem., 1984, 27, 255. Sarges, R.; Schnur, R.C.; Belletire, J.L.; Peterson, M.J.J. Med. Chem., 1988, 31, 230. Varma, S.D.; Mikuni, I.; Kinoshita, J.H. Science, 1975, 188, 1215. Okuda, J.; Miwa, I.; Inagaki, K.; Horie, T.; Nakayama, M. Chem. Pharm. Bull., 1984, 32, 767. Shambhu, D.V.; Kinoshita, J.H. Biochem. Pharmacol., 1976, 25, 2505. Okuda, J.; Miwa, I.; Inagaki, K.; Horie, T.; Nakayama, M. Biochem. Pharmacol., 1982, 31, 3807. Shimizu, M.; Ito, T.; Terashima, S.; Hayashi, T.; Arisawa, M.; Morita, N.; Kurokawa, S.; Ito, K.; Hahismoto, Y. Phytochemistry, 1984, 23, 1885. Shimizu, M.; Horie, S.; Arisawa, M.; Hayashi, T.; Suzuki, S.; Yoshizaki, M.; Kawasaki, M.; Terashima, S.; Tsuji, H.; Wada, S. Ueno, H.; Morita, N.; Berganza, L.H.; Ferro, E.; Basualdo, I. Chem. Pharm. BulL, 1987, 35, 1234. Kadota, S.; Basnet, P.; Hase, K.; Namba, T. Chem. Pharm. Bull., 1994, 42, 1712. Kador, P.F.; Sharpless, N.E. Biophys. Chem., 1978, 8, 81. Kador, P.F.; Kinoshita, J.H.; Sharpless, N.E.J. Med. Chem., 1985, 28, 841. Inagaki, K.; Miwa, I.; Yashiro, T.; Okuda, J. Chem. Pharm.Bull., 1982, 30, 3244. DeRuiter, J.; Brubaker, A.N." Garner, M.A., Barksdale, J.M.; Mayfield, C.A.J. Pharmacol. Sci., 1987, 76, 149. Mayfield, C.A.; DeRuiter, J. J. Med. Chem.. 1987, 30, 1595. Shoda, T.; Mizuno, K.; Imamiya, E.; Tawada, H.; Meguro, K.; Kawamatsu, Y.; Yamamoto, Y. Chem. Pharm. Bull., 1982, 30, 3601. DeRuiter, J.; Brubaker, A.N.; Whitmer, W.L.; Stein, L.Jr. J. Med. Chem., 1986, 29, 2024. DeRuiter, J.; Brubaker, A.N.; Millen, J.; Riley, T.N.J. Med. Chem., 1986, 29, 627. Brubaker, A.N.; DeRuiter, J.; Whitmer, W.L.J. Med. Chem., 1986, 29, 1094. Shimizu, M.; Horie, S.; Terashima, S.; Ueno, H., Hayashi, T.; Arisawa, M.; Suzuki, S.; Yoshizaki, M.; Morita, N. Chem. Pharm. Bull., 1989, 37, 2531. Terashima, S.; Shimizu, M.; Nakayam, H.; Ishikura, M.; Ueda, Y.; Imai, K.; Suzuki, A.; Morita, N. Chem. Pharm. Bull., 1990, 38, 2733. Shimizu, M.; Zenko, Y.; Tanaka, R.; Matsuzawa, T.; Morita, N. Chem. Pharm. BulL, 1993, 41, 1469.
192
[85] [86] [87]
[88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99]
[loo] [lO1] [102] [103] [104]
[105] [106] [~07]
[108] [109] [llO] [lll] [ll2] [1131 [114]
[1151
KAWANISHI and FARNSWORTH
Kasimu, R.; Basnet, P.; Tezuka, Y.; Kadota, S.; Namba, T, Chem. Pharm. Bull., 1997, 45, 564. Liu, P.S.J. Org. Chem., 1987, 52, 4717. Bischoff, H. Eur. J. Clin. Invest., 1994, 24, Suppl. 3. Ogawa, S, ; Shibata, Y, ; Kosuge, Y.; Yasuda, K.; Mizukoshi, T.; Uchida, C. J. Chem. Soc. Chem. Commun., 1990, 1387. Ogawa, S.; Aso, D. Carbohydr. Res., 1993, 250, 177. Yoshikuni, Y. Agr. Biol. Chem., 1988, 52, 121. Inouye, S.; Tsuruoka, T.; Niida, T. Tetrahedron, 1968, 23, 2125. Asano, N.; Tomioka, E.; Kizu, H.; Matsui, K. Carbohydr. Res., 1994, 253. 235. Platt, F.M.; Neises, G.R.; Dwek, R.A.; Butters, T.D.J. Biol. Chem., 1994, 269, 8362. Venter, H.L.; Joubert, P.H.; Loweings, A. Eur. J. Pharmacol., 1990, 183, 1014. Evans, S.V.; Hayman, A.R.; Fellows, L.E.; Shing, T.K.M.; Derome, A.E.; Fleet, G.W.J. Tetrahedron Lett., 1985, 26, 1465. Molyneux, R.J.; Benson, M.; Wong, R.Y.J. Nat. Prod., 1988, 51, 1198. Kimura, M.; Chen, F.; Nakashima, N.; Kimura, I.; Asano, N.; Koya, S. d. Traditional Medicines, 1995, 12, 214(Japanese). Perkins, S.J.; Johnson, N.; Phillips, D.C.; Dwek, R.A. Biochem. J., 1981, 193, 553. Papandreou, G., Tong, M.K., Ganem, B. J. Am. Chem. Soc., 1993, 115, 11682. Perkins, S.J.; Johnson, L.N., Phillips, D. C.; Dwek, R.A. Biochem. J., 1981, 193. 553. Tong, M.K.; Papandreou, G.; Ganeum, B. J. Am. Chem. Soc., 1990, 112, 6137. Pan, Y.T.; Kaushal, G.P.; Papandreou, G.; Ganem, B.; Elbein, A.D.J. Biol. Chem., 1992, 267, 8313. Bleriot, Y.J.; Genre-Grandpierre, A., Tellier, C. Tetrahedron Lett., 1994, 35, 1867. Briggs, J.; Haines, A.H.; Taylor, R.J.K.J. Chem. Soc. Chem. Commun., 1992, 1039. Braun, C.; Brayer, G.D.; Withers, S.G.J. Biol. Chem., 1995, 270, 26778. Colegate, S.M.; Dorling, P.R.; Huxtable, C.R. Aust. J. Chem., 1979, 32, 2257. Molyneux, R.J.; McKenzie, R.A., O'Sullivan, B.M.; Elbein, A.D.J. Nat. Prod, 1995, 58, 878. Cenei di Bello, I.; Fleet, G.; Namgoong, K.; Tadano, K.; Winchester, B. Biochem. J., 1989, 259, 855. Asano, N.; Kato, A.; Oseki, K.; Kizu, H.; Matsui, K. Fur. J. Biochem., 1995, 229, 369. Molyneux, R.J.; Pan, Y.T.; Goldmann, A.; Tepfer, D.A.; Elbein, A.D. Arch. Biochem. Biophys., 1993, 304, 81. Asano, N.; Kato, A.; Miyauehi, M.; Kizu, H.; Tomimori. T.; Matsui, K.; Nash, R.J.; Molyneux, R.J. Eur. J. Biochem., 1997, 248, 296. Eis, M.J.; Rule, E.C.; Wurzburg, B.A.; Ganem, B. Tetrahedron Lett., 1985, 26, 5397. Soofield, A.M.; Fellows, L.E.; Nash, R.J.; Fleet, G.W.J. Life Sci., 1986, 39, 645. Fleet, G.W.J.; Son, J.C.; Green, D.St.C.; Cenei di Bello, I.; Winchester, B. Tetrahedron Lett., 1988, 44, 2649. AI Daher, S.: Fleet, G.; Namgoong, S.K.; Winchester, B. Biochem. J., 1989, 258, 613.
CHEMISTRYOF ANTIMALARIALCOMPOUNDS
193
[116] Farr, R.A.; Peet, N.P.; Kang, M.S. Tetrahedron Lett., 1990, 31, 7109. [117] Miwa, I.; Okuda, J.; Horie, T.; Nakayama, M. Chem. Pharm. Bull., 1986, 34, 838. [118] Honda, M.; Hara, Y. Biosci. Biotech. Biochem., 1993, 57, 123. [ 119] Kempton, J.B.; Withers, S.G.Biochemistry, 1992, 31, 9961.
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22 9 2000 Elsevier Science B.V. All rights reserved
195
A DIRADICAL ROUTE TO BIOACTIVE NATURAL PRODUCTS AND THEIR ANALOGS R. DANIEL LITTLE*
and M I C H A E L M. OTT
Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA In this chapter we describe the development and application of the intramolecular diyl trapping reaction (cycloaddition) to the total synthesis of hirsutene, capnellene, coriolin, and hypnoph~lin, as well as its application toward the synthesis of aphidicolin and an analog of taxol ~v. ABSTRACT:
LINEARLY FUSED TRICYCLOPENTANOIDS. DIYL TRAPPING REACTIONS. The linearly fused tricyclopentanoids have attracted widespread interest. Their biological activity and interesting molecular architecture have made them the focus of many synthetic endeavours [ 1]. Those discussed in the first portion of this chapter include hirsutene (1), A(9.12)-capnellene (2), coriolin (3), and hypnophilin (4). H
..
OH
..,,,
hirsutene (I)
"
A(9,12)-capnellene (2)
--,,/...,,, o.
n
corioltn (3)
_o
.H
hypnophllin (41 Fig. (I). Tricyclopentanoid target structures.
196
LITTLE and OTT
Our approach to the synthesis of these materials was coupled with the development of the diyl trapping reaction [2]. While the process formally corresponds to a [3+2] cycloaddition, we have refrained from using this description because it is so closely associated with 1,3-dipolar cycloadditions [3]. Both the intermolecular and intramolecular versions (Fig. 2) of the trapping reaction have been explored. In each instance, a diylophile intercepts a short-lived trimethylenemethane-like (TMM) diyl (highlighted in bold) in a transformation that leads to the formation of two new sigma bonds and one (intermolecular) or two (intramolecular) new rings. This powerful process has proven of interest mechanistically and is of synthetic utility [2]. When generalized in the manner portrayed in Fig. (2), one can see that the intramolecular cycloaddition pathway can provide access to the carbon framework of a host of natural products. It is a remarkable testament to the beauty of chemistry that these short-lived intermediates are capable of engaging in exceptionally selective transformations.
6
Fig. (2). lntramolecular diyl cycloaddition modes.
The interrnolecular diyl trapping reaction provides a direct and simple route to the linearly fused tricyclopentanoid ring system. As illustrated in the first example of this pathway, heating the dimethyl diazene 9 to 70-75 ~ in the presence of an excess of cyclopentenone afforded a 90-98% yield of cycloadducts 10-12 [4].Unfortunately, the process displayed ~ o
.."" H
N (90-98% 1.3:1:3)
H
9
H
H
I0 II
i
I
,,"
0
"
H
H
II el
H
12 i
ii
i
0
BIOACTIVE NATURAL PRODUCTS
197
essentially no stereo- or regioselectivity, the ratio of cis-syn to cis-anti adducts being a modest 3"1. What is interesting, however, is the fact that the cis-syn isomers 11 and 12 dominate. Later, we will see that the cis-anti adduct invariably corresponds to the major product of an intramolecular diyl trapping reaction. The opportunity to conduct a diyl trapping reaction depends upon the availability of bicyclic azo compounds to serve as the diyl precursor. A basic approach to these systems consists of fulvene formation [5], DielsAlder cycloaddition, selective saturation of the A-5,6 pi bond, and conversion of the biscarbamate to the diazene linkage,J6] as depicted in Fig. (3). A.,~ B
B
A
B
A
B
A
A
Y 0
13
14
15
16
17
Fig. (3). Generalized route to diyl precursors.
Hirsutene (1) At one time hirsutene (1) was considered a reasonably challenging target structure. It has evolved into a virtual playground to showcase new methodology [1]. This relatively simple sesquiterpene hydrocarbon is believed to be a biosynthetic precursor to the more complex bioactive substances like coriolin (3) and hypnophilin (4). The stereochemical challenge is similar to that of the more complex structures in that methodology must lead to the cis-anti-cis ring junction stereochemistry. Given that the intermolecular diyl trapping reaction is most efficient with electron deficient diylophiles [2], and that this effort marked our first exploration of the intramolecular process, we elected to synthesize and examine the chemistry of diazene 19, even though we were aware to access hirsutene the ester would have to be removed at a later stage [7]. CO2CH3 C02CH 3
.% 1
H
18
Fig. (4). Analysis of the hirsutene (1) problem.
N N
19
198
LITTLE and OTF
Diazene 19 was synthesized in the manner portrayed below. Thus, treatment of anhydride 20 with sodium borohydride selectively reduces one carbonyl to a methylene unit. Reduction of the resulting lactone with DIBAL followed by a Wittig reaction and oxidation with PCC afforded aldehyde 22. When treated with cyclopentadiene in the presence of diethylamine in methanol, 22 undergoes a smooth and efficient conversion to fulvene 23. Diels-Alder cycloaddition to the azodicarboxylate 24 proceeded rapidly, a characteristic of reactions with this electron deficient chlorinated dienophile [8]. Selective reduction of the endocyclic n bond using diimide generated in situ, followed by the electrochemical reductive cleavage of the biscarbamate led to diazene 19 [6]. OH I. Ph3P=CHCO2Me ~ CH3CN(80-96%}
I. NaBH4174%)=
O(OyO
2. DIBAL,Et20 (';'60+)
/%
2. PCC, CH2CI2 (83-92%}
2O
21
~ , = C O 2 C H
3
CO2CH3
'o' 22
CpH, Et2NH, (91%)
MeOH: AcOH
I. ClaCH202CN=NCO2CH2CCI3 (24} ~_____~-'~/,, N N
2. KO2CN=NCO2K,AeOH(87%,2steps) ~ 3. -1.75 V, DMF,LICIO4;KaFe(CN)6(55~
CO2CH3
' ~ ~ ~.
%//
19
23 Scheme 1. Synthesis of first diazene used en route to hirsutene (1).
With diazene 19 in hand, we were in a position to explore the first intramolecular diyl trapping reaction. These transformations are exceptionally easy to conduct, simply requiring heating or photolysis, removal of the solvent and isolation of the product(s). As with many diyl trapping reactions, the course of the process was monitored by TLC, reflux being discontinued when the diazene was consumed. In this
, , ~ . ~ s~
CO2Me
,CO2CH3 "
,~H
H ":
H ~CO2CH3 .," H &
/J,,. H ~N ~
19 I
18 III
II
H 25
BIOACTIVE NATURAL PRODUCTS
199
instance, cycloadducts 18 and 25 were obtained in an 87:13 ratio and a combined isolated yield of>85%. The cis-anti adduct 18 predominated, as is customary for intramolecular cycloaddition. This is in contrast to the outcome of the intermolecular diyl trapping reaction of 9 with cyclopentenone discussed previously; there, the cis-syn adducts formed preferentially [4]. In an effort to understand the origin of the selectivity, we developed a model that views the cycloaddition as a kinetically controlled process, where the lowest energy transition state corresponds to the extended pseudochair formulation, 26, illustrated below [7].
J H 18
2 6 . E = CO2CH 3 I
I
I III
III
We initially postulated the existence of bonding secondary orbital overlap interactions between the carbonyl carbon of the ester, E, and the odd electron centers located on the diyl ring of 26. To test this notion, we synthesized an analog of diazene 19 bearing a Z-substituted diylophile. As illustrated, if secondary interactions dominate, then the cis-syn adduct 28, resulting from the folded pseudochair transition state conformation 27, should correspond to the major product. In practice, the cis-anti adduct 29 still predominated, a 75"25 ratio of 29 to 28 being obtained. g; H
H
H
~
H
tt
27
~1
predicted ... if s e c o n d a r y Interactions control ring J u n c t i o n s t e r e o c h e m l s t r y II
I
IllIllll
29
IIIII
It is noteworthy that the Z-diylophile stereochemistry was maintained in this instance. This piece of information, combined with the fact that it
200
LITTLE and 0 1 T
was also maintained using the diazene 19, indicates that these intramolecular diyl trapping reactions are stereospecific in-so-far as the diylophile stereochemistry is concerned. Based upon this information, we conclude that cycloaddition to electron deficient diylophiles is either a concerted process, or that formation of the second sigma bond occurs at a rate which exceeds that of bond rotation and loss of stereochemistry [9]. With an efficient and selective means of tricyclopentanoid synthesis in hand, our objective turned toward the conversion of 18 to hirsutene (1). It is obvious that the task requires the removal of the ester located at C7 and elaboration of the A-ring x bond. To do so, 18 was first converted to aldehyde 30 via reduction with LAH followed by oxidation using PCC/Celite.
_CHO
CO2CH3
H
~
]. LAH,Et20{>95o~)
H
~
H 18
n 30
Attempts to decarbonylate 30 using Wilkinson's catalyst in refluxing 1,2-dichloroethane met with mixed success. A particularly annoying feature of the transformation was the fact than an olefin, isomeric with the desired product was also formed. For some unknown reason, the relative amounts of these materials varied from run-to-run. It was reasoned that the C-C ~ bond was participating and that it would be best to partially elaborate the A-ring before removing the functionality at C7 (Scheme 2). To this end, 18 was reduced with LAH, the resulting alcohol protected as the dimethyl tert-butyl silyl ether 31 and the n bond was then elaborated using borane in THF followed by oxidation with PCC. While it is usually assumed that reactions occurring at the bridgehead of a linearly fused tricyclopentanoid afford the more stable cis-, rather than trans-ring fusion, the presence of the bulky silyl ether assured the hydroboration would occur on the side away from it and guaranteed a cis-ring fusion. Cleavage of the silyl ether under standard conditions, followed by oxidation afforded ketoaldehyde 32 and set the stage for another attempt to remove the aldehyde. This time, treatment of the ketoaldehyde 32 with 1.2 equiv of Wilkinson's catalyst in refluxing 1,2-dichloroethane proceeded satisfactorily to afford ketone 33 in yields ranging from 76 to 91%. Unlike the decarbonylation of 25, no side reactions were detected.
BIOACTIVE NATURAL PRODUCTS
201
CO2CH 3 H
9
H
/
OTBDMS
"
H
u
I. LAH, Et20 (>95%) .
.
.
.
.
H
.
2. TBDMSCI, imidazole DMF (95%) H H 18
31
I. BH 3, THF; PCC (54-57%) 2. TBAF (89-93%) H
H
CHO (Ph3P)3RhCI. CICH2CH2CI reflux, 54 h {76-91%)
il H H
"r~ IIIH
O 33
0
H "~-V 32
S c h e m e 2. Tricyclopentanoid interconversions; hirsutene precursor.
The synthesis was completed by introducing the angular methyl group by using a standard blocking-alkylation-deblocking sequence illustrated below. Spectral data for ketone 35 matched those of an authentic sample [10]. Since 35 had been converted to hirsutene (1), the synthesis was complete.
.,"'~
H 1. NaOMe. EtOCHO 1 9 6 % 1 9n-BuSH, p-TsOH 182%} 33
/
'3. KOBu-t, t-BuOH. Mel (62%? ~ ~ ~ ~ ~ _
KOH. ethylene 9 glycol(51%1 ~
34 I
I
H
~
O I
I
35
I
Before closing this section it is worthwhile to point out that our initial plan calling for the use of an electron deficient diylophile in order to ensure cycloaddition and avoid diyl dimerization was overly conservative. Thus, as illustrated, the unactivated diazene 36 served admirably as a diyl precursor, leading to a respectable yield of the cis-anti tricyclopentanoid 37. The latter was easily converted to ketone 35, thereby culminating a much shortened synthesis of hirsutene (1) [ 11 ].
202
LITTLE and O T T
/~
_O.IMInTHF'
.~
36
~.~t +
37
38
Coriolin (3) and Hypnophilin (4) Coriolin (3) was first isolated from the mycelial cake of C o r i o l u s c o n s o r s by Umezawa and coworkers in 1971 [12], and has attracted widespread interest due primarily to its interesting molecular architecture and antitumor properties. Ten years later, Anke, Steglich and coworkers isolated and characterized the linearly fused tricyclopentanoid hypnophilin (4) [13]. It too is biologically active, displaying activity toward grampositive and gram-negative bacteria, fungi, and yeasts, as well as antitumor activity. We elected to showcase the diyl trapping reaction with the synthesis of both coriolin (3) and hypnophilin (4) [14]. H
3.,
,i
>
, 39
OH
4O
HO
I
~_~
BsIS
42 Scheme
H
3. Strategy toward coriolin (3) and hypnophilin (4).
41
OH
BIOACTIVE NATURALPRODUCTS
203
Commercially available dihydro-5-(hydroxymethyl)-4,4-dimethyl2(3H)-furanone (42) was chosen as the starting material since it incorporated the essential structural features that are present in the acyclic chain of diazene 41. To synthesize both coriolin (3) and hypnophilin (4), enone 39 was selected as a common intermediate. While 39 had previously been converted to coriolin (3), its conversion to hypnophilin (4) had not been accomplished. The furanone 42, although commercially available, could also b e obtained in large amounts by epoxidation of 3,3-dimethyl-4-pentenoic acid with 3-chloroperoxybenzoic acid (MCPBA) in chloroform at room temperature (84%). After protection of the primary alcohol 42 as a benzyl ether, the carbonyl unit was reduced with diisobutylaluminum hydride in ether at-78 ~ to afford the diastereomeric pair of lactols 43 in 97% yield and a ratio of approximately 2"1. The lactols were methylated with ptoluene sulfonic acid in methanol to provide the functionalized tetrahydrofurans in nearly quantitative yield. The benzyl group was removed by hydrogenolysis over palladium hydroxide on carbon to afford the alcohols 44 in 94% isolated yield; use of other catalysts, such as palladium on carbon, gave less reproducible results. OH ~
I. PhCH2Br, Ag20, DMF(76%}
~.
2: D~B~.. r~o (9Wo)
l'""t
HO...,ff'
1.
~.,
Bn 0 - - - ~
1%
42
MeOH,/>TsOH (>95~
2. H2, ed(OH}2 (94%)
I
I
I
I
0 "~ \ / )----/..,,
.o--/
|
"
44
43
III
~Me
I
I
II
The conversion of 44 to the corresponding aldehydes was carried out by using a Swem oxidation. Thus, treatment of 44 with oxalyl chloride in dimethylsulfoxide at-60 ~ followed by triethylamine, afforded aldehydes .
.
.
.
.
.
.
.
.
OMe I. (COCl)2, DMSO; Et3N O
HO
I. EN=NE
. . . . . . .
2. CpH, pyrrolidlne/AcOH, - (, % MeOH(55%, two s t e p s ) ~
$... 2. diimide (91%, 2 steps [l ~ "t E = CO2Me) ~
\\// 45
I
I Ill
OMe
I
l",,. ~ ""
/i ~ N/ E 4 6 , E = CO 2CH2CCl3 47, E = CO2Me
204
LITTLE and OTF
which were sufficiently pure to allow them to be used directly. Treatment with methanol, cyclopentadiene and pyrrolidine led to the formation of fulvenes 45 [5]. While after 24 minutes no starting material could be detected, the reaction was still incomplete. Instead, an intermediate formed and was transformed very slowly into the required fulvenes 45. Prolonged reaction times led to decomposition of the fulvene which had formed, so that it proved advantageous to work-up the reaction after 24 hours. Under the slightly acidic work-up conditions the intermediate was converted to the starting material, which was recycled. After several recyclings, the yield of fulvenes ranged from 40-45%. While attempting to improve the yield, it was discovered that the rate of fulvene formation increased dramatically when acetic acid was added to the reaction mixture. Thus, treatment of the aldehydes with 1 equiv of acetic acid, 2 equiv of pyrrolidine and 2.5 equiv of cyclopentadiene in methanol at room temperature gave, after 12 hours, the fulvenes 45 in 55% isolated yield over two steps (viz., Swern plus fulvene formation); no starting material could be recovered. With the fulvene unit destined to become the carbon framework of the diyl in hand, the next task called for preparation of the bicyclic skeleton of diazene 41. This was accomplished by carrying out a Diels-Alder reaction between the fulvenes 45 and either bis(2,2,2-trichloroethyl) azodicarboxylate in ether at 0 ~ for 1 hour, or dimethyl azodicarboxylate in ether at 0 ~ for 3 days. The endocyclic ~ bond of the resulting adduct was selectively hydrogenated using diimide to afford 46 and 47 in 82% and 91% isolated yield, respectively (two steps). Both 46 and 47 consisted of a mixture of diastereomers. However, this was of no importance with respect to the remainder of the sequence since the methyl ether bearing carbon was destined to be converted to an sp 2 hybridized center. In addition, from previous studies, there was evidence to suggest that the diyl would exist as a time-averaged planar intermediate and that the existence of diastereoisomers about the exoeyclic ~ bond (tether oriented toward the front or back) would not have any bearing upon the stereochemical outcome.[15] Thus, we were confident that each of the diastereomers would lead to the same products. Having assembled the bicyclic framework, attention was directed toward introduction of the diylophile. Deprotection of the masked aldehyde in 46 and 47 was most efficiently accomplished using 70% aqueous acetic acid at 50-60 ~ for 5 days (85% and 95% yield, respectively). The dimethyl dicarbamate was subjected to saponification with potassium hydroxide in refluxing ethanol for 1.5 hours, whereafter the in situ oxidation with potassium ferricyanide at 0 ~ gave rise to the dizaene 48 in yields ranging from 76-86%. We were gratified to observe that treatment of 48 with triphenylphosphonium methylide in THF at room temperature led to the desired diyl precursor 41 in 56-83% yield.
BIOACTIVE NATURAL PRODUCTS
205
OH
I. AcOH 47
Ph3P=CH2
% 9
Ss
.,..~
,,
]]
THF (56-83
2. KOH, EtOH; K3Fe(CN) 6 (76-86%)
//N N 48
41
Ill
II
I I
II
The intramolecular diyl trapping reaction was studied in a variety of solvents (THF, MeOH, acetonitrile), and the diyl was generated both thermally and photochemically [ 14]. The photo-induced deazetation of 41 in methanol at -6 ~ afforded the desired tricyclopentanoid 40 in an excellent 84% yield. The transition state model portrayed by 49 nicely rationalizes the stereochemical outcome. The solvent study revealed that its choice had essentially no effect upon the product ratio at any given temperature. However, we did discover that methanol, a solvent which had not been utilized previously in intramolecular 1,3-diyl trapping reactions, was very useful for low temperature studies.
]\
MeOH
l OH
41
I
49
Ill
40
I
I Ill
III
Illl
To determine whether enthalpic or entropic factors were responsible for controlling the product distribution, the cycloaddition was conducted at several different temperatures. In analogy with previous results, there was reason to believe that both thermally and photochemically initiated extrusion of nitrogen would lead to the same 1,3-diyl, thereby allowing one to examine the chemistry over a reasonably large temperature range [15]. In practice, the product ratio (40: sum of minor products = EMnr)
206
LITTLE and OTF
varied from 4.7:1 in refluxing methanol, to 9.1:1 when the reaction was initiated photochemically at 6 ~ to 30" 1 when initiated photochemically at -60 ~ A plot of ln(40/EMnr) vs 1/T afforded a straight line from which we concluded that the variation in product ratio had its origins in enthalpic (AAH~t -2.19 kcal/mol), rather than entropic factors (AAS~-3.5 eu[ 14]). All attempts to convert 40 or its hydroxyl protected derivatives to the enone needed to complete the functionalization of the A-ring were unsuccessful. Most often, treatment of benzoate 50 with a variety of oxidizing agents (e.g., with Collins reagent) led to complex reaction mixtures wherein, in addition to small amounts of the desired enone 51, enone 52 was the major product. H
H
H
H
R :
h
OR
40, R = H 50, R = COPh
H
9
~)COPh
51
0
OCOPh
52
Fig (5). Isomeric enones formed during allylie oxidation.
Consequently, alternative modes of enone formation were examined. To set the stage for the use of a Rickborn-Crandall sequence to accomplish this objective [16], we treated alcohol 40 with MCPBA in methylene chloride, expecting to form a single epoxide. We were surprised to discover the formation of an 80/20 mixture of two adducts, 54a and 55a, in >90% yield. The ratio proved to be dependent upon the size of the -OR unit appended to C l l. When a bulky silyl ether was used (53), a single product formed, 55e. Desilylation afforded the minor epoxy alcohol 55a which was produced in the initial epoxidation of 40. X-ray analysis of the corresponding benzoate 55b, the major adduct derived from the epoxidation of 50, demonstrated conclusively the presence of a trans-fused A,B-ring system. Thus, even though the parent trans-fused bicyclo[3.3.0]octane is -~8 kcal/mol less stable than its cis-fused counterpart, such systems can be assembled, and in high yield. Furthermore, it is interesting to note that despite the fact that the C~l OR unit is formally on the o~-side of the tricyclic framework, the BC-ring fusion positions the substituent so that it is above the ~ bond of the A ring. Large R groups would, therefore, be expected to direct epoxidation to the opposite side, as observed. Heating the epoxides in THF with either (n-Bu)2NLi or LDA led to the expected diol 56 in 45% yield (two steps). All attempts to oxidize selectively the allylic alcohol met with failure; selective protection of the secondary hydroxyl group in 56 as a benzoate ester and oxidation using PCC afforded the desired enone 51 in 76% yield after crystallization.
BIOACTIVE NATURAL PRODUCTS
T a b l e 1.
207
Epoxidation Ratios
H
H
H
H
H
H
MCPBA '#1 I
H
"
Na2CO3'
OR
40, 50, 53
0 ~
>90%
,
H
54a-c
Starting aikine ,,
O
" _ OR
"'"~)
H 55a-c
" " OR
R
Products
ratio
40
H
54a, 55a
80/20
50
COPh
54b,55b
SiMe2Bu-t
54c, 55c
,, ,,,,,
L
,
53 ,,,,
...... i' 27/73
0/100
,,
~ / ~ . . , , t t /
1. MC PBA. CHCI3. 0 ~ .,
_ H 40
. _ OH
'
2. (n-Bu)2NLi. THE (45%, two steps)
HO H 56
I
: OH
"'%
I. PhCOCI, pyr (89%) 2. P C C / C e l l t e (85%)
H
H
--.
o
H
51 ~llillli
li
II
i
I
i
O120 Ph i
We next addressed the problems posed by the addition of a methyl group to the hindered fl-carbon of enone 51, a carbon which is encumbered by being doubly substituted, and in a less obvious fashion, is hindered by virtue of the fact that the C-ring C ll methine hydrogen is pointed directly toward it. Thus, we were not surprised to discover that a simple Gilman reagent failed to produce the desired 1,4-addition product 57. In contrast, treatment of 51 with the higher order euprate, Me2CuCNLi2, in the presence of boron trifluoride etherate, converted it to the desired product 57 in a stellar 93% isolated yield [ 17].
208
LITTLE and OTF
H
H
Me2CuCNL|2, BF3, THF, -50 *C 193%1
H _
9 ~ -
OCOPh
~)COPh
51
57 II
IIII
To introduce the 7r bond and generate enone 58, the triquinane 57 was first converted into a 3:2 mixture of two regioisomeric trimethylsilyl enol ethers using LDA in THF at -78 ~ followed by the addition of TMSCI; the major isomer resulted from enolate formation toward Cr (note 57, below). Treatment with palladium acetate in acetonitrile converted it to the required enone 58, while the minor isomeric silyl ether was reconverted to the starting ketone 57. Enone 58, which has previously been synthesized and converted into the target molecule 59 by Koreeda and coworkers, was isolated in 40% yield, along with 50% recovered starting material; intermediate 58 proved to be identical in all respects to the material synthesized by the Koreeda group [ 18].
H
H
r
o11
: ~
I
lq "
ocom 57
H I. LDA.THF:::TMSCI = 2. Pd{OAc)2, CHsCN (40%, two steps)
" ~ O
bR 58, R = COPh 59, R=H
With a formal total synthesis of coriolin (3) accomplished, we turned our attention toward hypnophilin (4). In practice, it was found that treatment of enone 59 with LDA in THF at-78 ~ followed by trapping of the resulting enolate with formaldehyde at-30 ~ led to a mixture of the diastereomerie diols 60 in 85% yield (Scheme 4). The mixture was treated with tosyl chloride and pyridine in dichloromethane at room temperature; TLC analysis revealed that some dienone 39 was formed even under these reaction conditions. After 4 days, tosylation of the primary hydroxyl group was complete and the elimination reaction was
BIOACTIVE NATURAL PRODUCTS
209
H LDA, THF.-78 ~
.CH20, . . .-30. .~
185~
"~~"
--
o
-\ 9-
.
-"
m
OH
OH
59
60
1. TsCI, pyr 2. DBU (80%) H
"
H H202. T H F / H 2 0
I1"
R
2c03
_._
:"
\ OH
4, hypnophlltn
39
Scheme 4. Completion of the hypnophilin (4) synthesis.
accomplished upon addition of 1,5-diazabicyclo[5.4.0]undec-5-ene (DBU). The dienone 39 was obtained in 80% yield and proved identical to the material synthesized previously by Schuda and Heimann [19]. Monoepoxidation of the endo-cyclic ~ bond in dienones similar to 39 has already been described. Thus, under reaction conditions applied by Danishefsky and coworkers [20], dieneone 39 was converted to hypnophilin (4) in 50% yield; some starting material (30%) was recovered. Bisepoxidation could also be detected, but that material decomposed during the column chromatographic purification on silica gel. Synthetic hypnophilin (4) displayed spectral data which were in full accord with those of natural material.
A9(12)-Capnellene (2) [21]. Bridged vs Fused Regioselectivity In 1974, a manuscript appeared describing the characterization of a sesquiterpene that was isolated from the methylene chloride extracts of the soft coral Capnella imbricata (Coelenterata, Octocorallia)collected off Sewaru, Leti Island, Indonesia [22]. The skeletal type was given the name capnellane, and the structure and absolute configuration of the compound, named A9~12)-capnellene-3fl,8fl,10a-triol (61), was secured by singlecrystal X-ray analysis. Over a period of several years, other capnellols were isolated from C. imbricata. In addition to these alcohols, two hydrocarbons, A9Cl2)-capnellene (2), the presumed biogenetic precursor of
210
LITTLE and OTF
the alcohols, as well as precapnelladiene (62) [23], the putative immediate precursor to the tricyclopentanoid skeleton found in 2, were also isolated from C. imbricata. H
H
HOll'"
61, R=OH
2
62
Fig. (6). Capnellenr (2) and other marine natural products.
It appeared as though the trapping reaction would be ideally suited for a total synthesis of A9(IE)-capnellene (2) [21]. The plan, illustrated in Scheme I, required construction of the bicyclic diazene 63, which was to serve as the direct precursor to the required diyl. From the outset we were aware of the potentially more stringent requirements that were to be imposed upon the present trapping reaction. Our immediate attention was directed to the fact that an unactivated diylophile was to be used and therefore that the diyl trapping reaction might be slowed sufficiently to allow dimerization of the diyl to compete with cycloaddition. Given the positive results obtained with the unactivated diazene 36, however, the prospects for success in the present instance also seemed high. H
.-
2, 691121-capnellene
63
Fig. (7). Diazene precursor to capnellene (2).
Diazene 63 was prepared in short order from readily available starting materials. Thus, acid 64, prepared in a standard manner from the isobutyric acid dianion and 3-methyl-3-butenyl p-toluenesulfonate, was first reduced with lithium aluminum hydride and then oxidized by using PCC/Celite to afford the expected aldehyde in 88% yield. In preparation for the Diels-Alder reaction that was to be used to assemble the bicyclic framework found in 63, the aldehyde was converted to fulvene 65. Initially, we attempted to accomplish this objective using methodology
BIOACTIVE NATURAL PRODUCTS
211
developed by Freisleben [24]. Our past experience with the procedure provided very good yields of fulvenes that were otherwise often difficult to obtain without significant competing processes. In the present instance, treatment with cyclopentadiene and diethylamine in methanol at room temperature for 6 h led only to the recovery of unreacted aldehyde. Presumably, the neopentyl nature of the carbonyl carbon provides sufficient steric encumbrance so that the initial iminium ion forming step of the sequence does not occur at an appreciable rate under these conditions. Fortunately, since the aldehyde is devoid of ct-hydrogens, the problems were overcome by treating it with lithium cyclopentadienide in THF at room temperature [25]. In this way, fulvene 65 was obtained in yields ranging from 67-80% after chromatography on neutral alumina. A Diels-Alder reaction between 65 and dimethyl azodicarboxylate followed by selective reduction of the endocyclic ~ bond of the resulting adduct using diimide in dichloromethane provided 73-91% yields of the dimethyl dicarbamate precursor to diazene 63, which in turn, was obtained in 78% yield after saponification and oxidation using aqueous potassium ferricyanide.
I. LAH, Et20 2. PCC/Cellte, CH2C12 (88%, 2 steps) 3. CpLl, THF (67-80%)
64
65 I. MeO2CN=NCO2Me 2. KO2CN=NCO2K, AcOH (73-91%1 3. KOH, EtOH; K3Fe(CN)6 (78-86%) 63 I
I
I
The diyl trapping reaction did lead to the expected linearly fused product. However, cycloaddition was slowed to the point where it proved necessary to slowly add the diazene to a refluxing solution of THF to assure a low concentration of the diyl. Otherwise, it dimerized in a process that is characteristic of the triplet spin state [26]. Furthermore, the linear adduct was accompanied by the formation of nearly an equal amount of the previously unobserved bridged regioisomer. These materials proved difficult to isolate as hydrocarbons and were converted as a mixture to
212
LITTLE and O'IT
ketones 66 and 67 v i a hydroboration-oxidation (56% from 63). The structure of the bridged ketone 67 was confirmed by x-ray crystallographic analysis. Once separated and characterized, it proved a simple matter to convert the linearly fused ketone 66 to the natural product using a Wittig reaction in DMSO.
{a}THF, reflux, syringe p u m p (b) BH 3, THF; PCC 0
'N
:-
63 II
66
I
I
I
II
67 I
The most obvious difference between the present case and those discussed earlier is the existence of the gem methyl unit appended to the carbon adjacent to the diyl, and the placement of a methyl group on the internal carbon of the diylophile. As illustrated in 68, a reasonable transition state representation for a concerted cycloaddition leading to the linearly fused product, the positioning of these substituents sets up several energy raising nonbonded interactions. That illustrated between the gem methyl unit and the diyl ring hydrogen is alleviated if the alternative transition state formulation (69) leading to the bridged adduct is adopted. But what if these cycloadditions are not concerted, but instead occur in a stepwise manner? Then, the first step leading to the linearly fused material corresponds to a 5-exo-trig cyclization onto an alkene that is substituted with a methyl group (70 to 71a), while the bridged adduct results from a 6-endo-trig cyclization onto the opposite end of the alkene (70 to 71b). In analogy with monoradical chemistry [27], one notes that in cases such as these, the rate of the 6-endo-trig cyclization exceeds that of the alternative, though the rate differences are generally small.
68
H
Fig. (8). Transition state formulations.
BIOACTIVE NATURAL PRODUCTS
(
9
213
" ca 2
70
"~
7 1 a (5-exo path} r
71b {6-endo path)
"~
Fig. (9). Monoradical-like cyclization modes.
In an attempt to sort out the relative importance of geminal substitution the nature of the substituent appended to the diylophile, we synthesized and examined the chemistry of the diyls derived from diazenes 72a-e, the first two having geminal substituents but no alkyl group on the diylophile, the third devoid of the geminal substituents but possessing a hydroxymethylene unit on the diylophile. To our surprise, geminal substitution played an imperceptible role. The only one of the three systems to provide a significant quantity of the bridged cycloadduct was 72e. vs
Table 2.
Effect of Substitution on Linear/Bridged Ratios a
R'
N
72a-c
R,
major product/comments
H
linear
|
a, CH 3 b, OCH3
c,H
,t,,
linear CH2OH
essentially !'1 bridge/linear
To address the possibility that both the singlet and triplet diradicals could be participants, we examined the chemistry of 72e in the presence of molecular oxygen. The reaction of TMM diyls with oxygen serves as a diagnostic for the intervention of triplet chemistry [28]. While in principle both the singlet and triplet diyl can react, a combination of the low oxygen concentration available under the reaction conditions, combined with the
214
LITTLE and O T [
short lifetime and rapid rate of intersystem crossing of the singlet to the triplet, allows only the triplet diyl to react. In the present case, bridged adduct formation was completely quenched, and the amount of linearly fused adduct 73 was reduced from 44 to 17%. These results indicate that all of the bridged and a portion of the linearly fused adducts are triplet derived. Table 3.
~
/~CH~OH
_~i I
CH2OH
CH3CN, reflux N
N 72c
73
no oxygen ,
,,,,
,,,
,,
|,
,,,
74
1"1.2 linear/bridge; 90% combined yield ,
with oxygen
,
no bridge & yield of linear decreases from 44 to 17%
I
The chemistry of diazenes 75a-e illustrates that linearly fused adducts are formed very efficiently when an electron withdrawing group is appended to either carbon of the diylophile. Product formation could not be quenched with molecular oxygen, indicating that these materials are singlet derived. The differing reactivity patterns can be understood using frontier molecular orbital theory, coupled with the Berson cascade mechanism [29]. According to the latter, when a diazene is heated or irradiated, the first formed interceptable intermediate is the singlet diyl. We were originally of the opinion that intramolecular cycloaddition would occur fast enough to render intersystem crossing (ISC) to the triplet manifold non-competitive. This does indeed seem to be the case when an electron withdrawing group is appended to the diylophile. But as evidenced by the findings described above, this is not always true; triplet chemistry can and does intervene. We suggest that the reason cycloaddition to the singlet diyl is rapid is related to the existence of a comparatively small diyl HOMO/diylophile LUMO energy gap, AE, the situation most likely to occur when an electron withdrawing group is appended to the diylophile. If AE is made larger, then that rate ought to decrease, thereby providing time for ISC to become competitive. Conceptually, this is an easy objective to achieve merely replace the electron withdrawing group with an alkyl substituent.
BIOACTIVE NATURAL PRODUCTS
Table 4.
215
Influence of Electron Withdrawing Substituents on Product Ratio
EWG
EWG
N 75a-c
76a-c
77a-c
yield (%)
% linear
a, C O 2 C H 3
88
>95
b, C H O
93
88
98
91
EWG
c, C O C H 3 ,,,
Since the triplet diyl affords the bridged cycloadducts, we wondered whether it would be possible to optimize production of the triplet and simultaneously optimize the amount of bridged product formed. One motivation for so doing was the recognition that there are many bioactive natural products that possess the [3.2.1 ] subunit. If we were able to obtain these materials selectively, then the possibility of efficiently accessing the natural products would obviously exist. Once the triplet forms, it can choose either a stepwise 5-exo-trig or a 6endo-trig cyclization pathway for the initial carbon-carbon bond forming event. The former converts 80 to 81, an intermediate which will suffer progressively larger nonbonded interactions as the size of the diylophile substituent, Rf, increases. The alternative cyclization, 80 to 82, alleviates these, particularly if the radical site bearing Rf assumes either a planar or time-average planar geometry, thereby allowing it to maximize its separation from the remainder of the molecule. The preferences for 5- vs 6-membered ring formation are expected to be similar to those expressed in monoradical cyclizations, and this appears to be the case from the capnellene results described above. We reasoned therefore, that to optimize formation of the bridged cyeloadduct one should append a large alkyl group, Rf, to the internal carbon of the diylophile in 78. This will assure a large AE and ought to significantly tip the balance toward the selective formation of bridged cycloadducts. To add versatility and the possibility of functional group elaboration, a funetionalized alkyl group can be selected.
216
LITTLE and OTT Rf
D
N
,,-,.-,-,-,,.-.]m,-
Rf 79
78
5-exo,trlg
Rf
~ e
I' I I
--~
I
II i
Rf 6-endo,trig
80
Scheme 5. Cyclization modes - stepwise pathways.
The equation illustrated below demonstrates the dramatic influence the size of the alkyl group has on determining the bridged/linear regioisomer distribution. Thus, for the comparatively small CH2OH unit of 72e, a 1.2:1 ratio was obtained, while for the large dimethyl ketal found in 83, it increased to 16:1 (84/85) [30].
/"1 ~ L---~N~N
THF. reflux. ~ I raM. 3-4h
/ CtOMe)2CH3
83 _
"
.
.
.
.
.
CIOMel2CTIa
84 .
.
.
JI
85 I
I
I
_
/I .
.
.
.
.
.
I
The influence of both steric and electronic factors is illustrated in the chemistry of diazenes 86a and 86b[31 ]. The dimethyl ketal 86a affords the bridged adduct 87a to the near exclusion of its fused counterpart 88a. In stark contrast, the corresponding enone 86b provides the linearly fused adduct 88b nearly exclusively. From a detailed examination of these substrates, the following points have emerged: 1) The presence of geminal alkyl substituents adjacent to the first carbon exocyclic to the diyl ring has no apparent influence on the regiochemical outcome of intramoleeular diyl trapping reactions; 2) The regiochemical controlling factor turns out to be directly correlated with the
BIOACTIVE NATURAL PRODUCTS
Table 5. H
217
Bridged Regioselection OCH 3 C O
3
~
~, OCH 3
.........
J
THF, reflux
----<.//"
R
>
R
N 86a,b |
ii,
87a,b
, i
bridge/linear
R i
.
.
.
.
88a,b
i
ii
.
a, C(OCH3)2CH3
16"1
b, COCH3
1:>18
.,,
yield (%) ..
i
|
ii
.
,i
nature of the substituent appended to the diylophile. Electron withdrawing groups (EWG) attached to either the terminal or internal carbon results in the selective formation of linearly fused systems. In contrast, the presence of an alkyl group on the internal carbon of the diylophile leads to variable amounts of both linear and bridged adducts, depending upon the size of the substituent. The larger the group, the greater the preference for formation of the bridged material. This point is discussed in detail in the next section of this chapter. 3) Both the singlet and the triplet diyl play an important role, the former providing a linear adduct when an electron withdrawing group is appended to the diylophile, the latter being the precursor of the bridged isomer. In summary, the simple marine natural product A9(12)-capnellene (2) presented an unexpected challenge, one which ultimately forced us to revise our transition state model for the intramolecular diyl trapping reaction. While we were surprised to obtain the bridged adduct, its formation and discovery coupled with the spinnoff that has resulted from efforts designed to understand why it was produced, constitute what we believe to be the most important feature of our capnellene effort. This is one of those not-so-rare times when the unexpected proved of greater interest/importance than original objective(s). As a result of these studies we are now able to selectively form either the bridged or the linearly fused regioisomer by design. As the remainder of this chapter delineates, we are using this ability to gain access to aphidicolin (89) and taxol | analogs.
218
LITTLE and O'IT
Taxol (90), Baccatin III (91), and Target Analog 92 It is fair to say that taxol | (90) is one of the most important materials of this, or any century. Its successful use in the treatment of several forms of cancer is clear and well-established [32].
RO '""
6.
o
-: . . . ~ o
OBz
Ph
j
6A~
O
t.
BzHN
= R, taxol |
90
OH H
-
R, baccatln III, 9 1
Fig. (10). Taxoi | (90), baccatin III (91), and target structure 92.
While many ingenious approaches to the skeleton have been developed, only four total syntheses have been recorded.[33] As indicated recently by Danishefsky and coworkers, the availability of a renewable source of baccatin III (91) reduces the impact new total synthesis might have upon the supply problem [33h]. The development of general strategies that will allow the construction of a wide range of analogs whose bioactivity might equal or exceed that of the naturally occurring materials, clearly represents a laudable and noteworthy objective. Our target for application of the intramolecular diyl trapping reaction was tricycle 92 [34], a system possessing much of the functionality found in the B-ring of the natural product; the gem methyl unit is absent, though its role in defining bioactivity is unknown. Key to the successful implementation of our plan was the construction of aldehyde 97 and its subsequent conversion to the diazene 96 on a multigram scale; see Scheme 6. Scale, and the ability to scale up, are clearly important issues as they relate to the ability to utilize adaptations of this chemistry to construct useful amounts of bioactive materials. As indicated earlier in this chapter, the dioxolane subunit appended to the diylophile is critical to guarantee formation of the bridged cycloadduct, and is designed to serve as a synthon for the C1 hydroxyl group of the natural products [35]. Conversion of 95 to the target structure 92 was predicated upon the successful isomerization of the C-C ~-bond to the
BIOACTIVENATURALPRODUCTS
.... ~ ~ . , o
13~ .....~
[
219
~
~
r
F[~
r
9:$
92
94
F = functionality
H r
97
Scheme
96
95a, 9 5 b
6. Diylcycloadditionrouteto analogtaxol| 92.
tetrasubstituted position shown in 94. The remaining steps include alkylation of the enone 94, oxidative cleavage of the olefin followed by an intramolecular alkylation to afford the ABC ring system 92 common to taxoids. We believe that the route offers many opportunities for the construction of more elaborate materials. For example, an OR-unit positioned at the central carbon, Cr, of the tether that links the diylophile to the diazene in 96 becomes the important oxygen functionality found at C13 of the natural products. Furthermore, the use of a functionalized alkylating agent could incorporate the oxetane or provisional functionality to allow its construction at a later stasge. These are but two of many options. THF served as an inexpensive, readily available starting material. Ring opening using sodium iodide and benzoyl chloride in acetone [36], followed by alkylation of acetyl acetone, the addition of paraformaldehyde and deacylative methylenation in DMSO, afforded vinyl ketone 99 [37]. This material was most conveniently used without purification m a sequence consisting of ketalization, removal of the benzoate, and Doering oxidation to provide aldehyde 97 in a 60% yield overall [38].
220
LITTLE and O'IT Nal. BzCI THF
I~
Me2CO
OBz 98
O
O
{a}
DMSO. K2CO3 (b} {CH20)n
O
O
(a} HOCH2CH2OH, H*,CHaCH(OMe}2
O B
z
O
~
(b} KOH (c} SO3.pyr, DMSO, EtaN 99
97 Scheme 7. Assembly of tether and diyiophile.
Treatment of 97 with cyclopentadiene and diethylamine in methanol afforded fulvene 100 in a 90% yield. A Diels-Alder reaction with diethyl azodicarboxylate, followed by reduction of the A-5,6 g bond of the adduct using diimide, led efficiently (>95%) to the biscarbamate 101. The diazene linkage was unveiled in a customary fashion, to provide 20 grams of diazene 96 in an overall yield of 35%fi'om THF.
CpH. Et2NH. MeOH (90%}
100 (a} DEAD I {>95%} (b} N2H2
{a) KOH. EtOH (b} K3Fe(CN}e (83%} 98 1o~. E - COCt
Scheme 8. Entry to diyl procursor 96.
BIOACTIVE NATURAL PRODUCTS
221
With substantial quantities of diazene 96 in hand, we were able to perform the diyl trapping reaction on a 20 gram scale, by far the largest ever used. The transformation proceeded superbly, affording 16 grams (80% isolated yield) of a 1:1 mixture of stereoisomeric bridged cycloadducts 95a and 95b [39]. In dramatic contrast to the first diyl trapping reactions where one typically used small quantities, sealed tubes, or found occasion to use a syringe pump, a simple dropping funnel was used to achieve a slow addition of diazene 96 to refluxing solvent. Many times intramolecular diyl trapping reactions are even easier to perform, simply calling for heating a solution of diazene in a given solvent until the starting material disappears [2]. Slow addition, as in the case of 96, is used when cycloaddition involves the triplet diradical to minimize or avoid entirely, dimerization of the diyl, a known triplet state process.
CH3CN, reflux __
{8o%} I0: I (bridged:linear}
@
\N~,~ N
102
96
~
ll,..
1
95a {a-H}, 95b ~-H}
The results are a clear and welcome validation of the hypothesis put forth earlier in this chapter. That is, given a choice between the 5-exo-trig or a six-endo-trig cyclization pathways portrayed below, the triplet diyl 103 prefers the latter to avoid energy-raising nonbonded interactions between vicinal substituents (see 104), and to allow the dioxolane unit to position itself as far from sterically demanding locations as possible, as is the case in structural representation 105. Here, the radical site to which R is bound can assume a planar or time-average planar geometry to minimize steric interactions. Isomerization of the double bond, in preparation for the oxidative cleavage designed to deliver the eight-membered ring, proved more challenging than anticipated. For example, attempts to do so using
222
LITTLEand O T r
5-exo,trlg @
! I !
R
I !
R= R 6-endo,trig 103 105 Scheme 9. Comparison of cyclization modes.
rhodium (III) chloride in refluxing ethanol afforded the di-substituted alkene 106 [40]. Since this reagent isomerizes double bonds to the thermodynamically preferred site, the results suggest that the tetrasubstituted olefin is not the most stable in this instance. Molecular mechanics calculations support this conclusion, consistently placing the di-substituted isomer 106 at lower energies than either the tri- and tetrasubstituted materials. Iio.-
I1,,-
RhCl 3
(
,
EtOH, reflux O 95a,b
106
Allylic oxidations were also explored using PCC, Collins reagent, and selenium dioxide. While the desired enone 108 formed, the less strained, less substituted olefin 107 was invariably the dominant product. Use of the Collins reagent afforded the best yields (40%), but produced the same inseparable mixture of regioisomers (3" 1, 1t)7:108). O
o_""i
107 III
I
I
CO ~~ 108
BIOACTIVE NATURAL PRODUCTS
223
A clean reaction of 95a,b occurs with singlet oxygen, but affords an 85% yield of allylic alcohol 109. In this and each other instance, the less strained isomers were produced, prompting the exploration of a new strategy.
a. O2/TPP
('
b. N2H 2
185%1
95a,b iiiin
i
109 I lnnlllllllllll
I
II
I
Ill
Ideally, we imagined a regiospecific electrophilic oxyphenylselenation, placing the phenylselenyl unit at the bridgehead carbon of 95 (95 to 110). Subsequent oxidation of selenium, followed by a regiospecific syn elimination of the resulting selenoxide, could insert the double bond in the desired position, 111. However the literature suggested that the first step was likely to occur with the opposite regiochemistry [41 ].
llo*.
X PhSe-X -PhSeOH
I
110
III
Yet, as the following analysis shows, there was reason to believe that the process might occur in the desired manner. The first point to consider is the rt facial preference for selenonium ion formation. In this case, addition to 95 should occur syn to the ring junction hydrogen in order to assure formation of the significantly more stable cis-fused [3.3.0] subunit 112;[14] addition to the opposite face would afford the trans-fused counterpart 113.
224
LITTLE and OTI"
-
ph Set(~
.-" .. y
.,,,..
-
112
,,,..
% 9. . .
%
m
95 PhSe(~
%
'"'T-
-
-"
113 Scheme
I0. Selenonium ion formation- facial selectivity.
Assuming 112 is preferred, the question then becomes one of whether the nucleophile will attack at the bridgehead carbon Cx, or at Cy. The former would require the development of substantial carbocation character since a direct backside attack at Cx cannot occur (112 to 114, Scheme 11). Nucleophilic attack from the topside of the cation 114 would lead to an energetically unfavorable trans-fused [3.3.0] ring system and is unlikely to occur. Attack from the bottom ought to be retarded by sterie interactions with the phenylselenyl unit, and is also considered unlikely. What about a direct backside nucleophilic attack at the secondary center, Cy? This appears reasonable as it ought to encounter minimal steric inhibition and lead to a cis-fused [3.3.0] adduct, 115. Thus, despite literature precedent, we were hopeful that the reaction would occur in the desired manner.
BIOACTIVE NATURAL PRODUCTS
225
SePh
c~
~
,,...
..--"-.
Coo" 112
y
I I I I I I I I I I
~
,,,.. |
~.o
114
I I I I I I I I I I I I I I
%
115 Scheme 11. Regioselective ring opening of selenonium ion.
While the addition did not occur using PhSeSePh, PhSeC1, or PhSeBr, the operation could be carried out conveniently and reproducibly via the addition of 95a to phenylselenyl trifluoroacetate, generated in situ from PhSeBr and AgOCOCF3 [42,43]. The trifluoroacetate unit hydrolyzed during workup to consistently afford a 93% yield of product 116. We were exceptionally pleased - the addition proved both regiospecific and efficient [44]. With the phenylselenyl unit in place, the oxidative syn elimination was examined. Compound 116 was oxidized with MCPBA and the reaction mixture added to refluxing carbon tetrachloride, but without success; the desired product could not be detected. To facilitate elimination, alcohol 1 1 6 was oxidized to the corresponding ketone 118, the thought being that the conjugative stabilization associated with formation of an enone might also lower the activation barrier. In practice, a Swem oxidation, followed by treatment with MCPBA at -78 ~ and addition of the reaction mixture to refluxing carbon tetrachloride, did lead to the formation of enone 94 as a single product in near quantitative yields. The overall transformation from the
226
LITTLE and OTF
?"
|lle-
a. "PhSeOzCCF3" b. K O H / E t O H (93%)
116
95a
a. MCPBA b. CCI 4, reflux OH
?N 1% o 117
bridged system 95a to the enone 94 could now be accomplished consistently in 68-78% overall yield, and on scales greater than 10 grams. PhS_e / ~ )
O
b. CCI 4, reflux
~.~0
..
118 I
III
I
The viability of the proposed route to the eight membered ring was confirmed by the ozonolytie cleavage of silyl ether l19b. This material was readily available from enone 94 via a selective 1,2-reduction using DIBAL-H, and protection of the resulting allylie alcohol l19a as a silyl ether. Verification of the structure was obtained by NMR using HMQC TOCSY [45]. A particularly noteworthy observation was that the chemical shift difference between the methylene protons, H~ and H,, was 1.9 ppm. This remarkably large separation suggests that the material exists in a conformation wherein the earbonyl units are oriented as shown, with syn proton, Hs, resonating at 3.76, and the anti, Ha, at 1.89 ppm. A similar
BIOACTIVE NATURAL PRODUCTS
227
difference was observed for the methylene protons located at C3 (taxol | numbering), one appearing at 1.89, the other deshielded considerably, appearing at 3.83 ppm. Notice also that in structure 120, the C-He bond is oriented to allow overlap of the carbonyl ~* orbital with incipient negative charge. This allignment is needed to assure the appropriate stereoelectronics for the enolate forming step that is to be utilized in each of the efforts described to append ring C to the [5.3.1 ] core structure. {a} DIBAL-H 94
{b)'" t-BuPh;SiCl
/
I
t
~'"'/"v~
~- ( ~q,s k
OP
)
{a}
0 3.
-78 *C
. . . (b} . Me2S (7o%}
(77%1
l19a, P - H l19b, P = SIPh2Bu-t
i
~ OslPh2Bu't
120 Scheme 12. Oxidative cleavage leading to the [5.3. I] ring system.
o,s
120, R= CCH3I-OCH2CH20-}
The enolate of enone 94 was alkylated regioselectively using 4-iodo-1to afford a 2.5"1 mixture of diastereomers 121a and 121b in 77% yield. The mixture of diastereomers were separated and the major product was carried forward. Decoupling
(tert-butyldiphenylsiloxy)butane
228
LITTLE and OTF
O 1
LDA, I{CH2)40"rBDPS r
-78 ~ to RT {77%} 2.5: I. 1 2 1 a : 1 2 1 b
121a (~-H) 121b {~-H}
94 II
I
II
IIII
III
III
and NOE experiments were used to determine that the ct-alkylated material 121a was the major product. Saturation of Ha produced enhancements in the vicinal syn and anti methylene protons, Hb (8%) and Hr (5%), respectively. The key result was that He showed a 5% enhancement, suggesting that it was on the same face of the molecule as Ha. Molecular mechanics calculations were in full accord, consistently placing 121a at a slightly lower energy than its 13-alkylated stereoisomer 12lb.
,,../~~ /'
\ \ '_JL/'""\
He
.
/
~--~NOE ""
O OTBDPS 121a Fig. (11). Overhauser effects.
Enone 121a was reduced with DIBAL-H and the resulting allylic alcohol protected using benzyl bromide to afford 122 in an 80% yield overall. The major diastereomer was oxidatively cleaved and the silyl group removed, thereby affording alcohol 123 in 70% yield (two steps). Conversion to the corresponding iodide 124 was accomplished using a one step procedure involving the addition of 123 to a stirred solution of triphenylphosphine, iodine and imidazole in THF/MeCN. The stage was set for intramolecular alkylation; a single product 125 formed immediately (70%) when iodide 124 was added to LDA at-78 ~ Unfortunately, deprotonation and cyclization occurred from Ca rather than Cb. Similar results were obtained independent of the counterion (e.g., NaHMDS,
BIOACTIVE NATURAL PRODUCTS
229
OBn
\ i~~/..,,,\ '"V
I. DIBAL-H
>
~
II ."
2. BnBr {80~
"',1|~
O~
<~b
OP 122
121a. with P = TBDMS
I. 0 3, DMS 2. TBAF {70%)
I
k I"'T "~..
pOBn
123
KHMDS). While the tricyclic core of 125 corresponds to that desired, the substituent-placement is incorrect. II,.
,. ~
Bn
Ph3P. 12
""II~
II,'
a,,
0
,.,.._ v
imidazole (90%)
0
<',
' '>
HO 124
123
~OBn
t
I LDA 170%1
I • 'OBn llt
125
230
LITTLE and OTT
A simple solution to the problem would be to eliminate the unwanted deprotonation at Ca thereby leaving only the opportunity for cyclization to occur in the desired sense. To do so we elected to maintain the carbonyl oxidation state at Ca by protecting the ketone as the corresponding dioxolane 126. In this manner, the oxidative cleavage of the double bond in 126 leads to an eight-membered ring devoid of an abstractable proton at Ca (see 127). Alkylation, should it occur, is thereby forced to occur in the desired sense.
Q
, IOl
I
I
III
II
II III
I
I
I
Ull
II
While conceptually simple, this approach appeared flawed when
ozonolytic cleavage of the ~ bond in the model substrate 129 failed miserably [46]. Fortunately, ruthenium tetraoxide, generated in situ from excess sodium periodate and 10 mol % ruthenium dioxide, worked splendidly [47]. After 10 minutes the reaction was complete, and the dione 130 was isolated in an 80% yield. o
Ill,.u
,v
94 5% TsOH, HOCH2CH2OH Phil. reflux (65%}
:
Oa, DMS
.......
,
_
CH3CN-COI4-H20
(80%1 Scheme 13. Ruthenium tctroxidr as an important alternative to ozonolysis.
BIOACTIVE NATURAL PRODUCTS
231
Buoyed by these results, we moved to the more demanding target structure, 92. It proved advantageous to use the mixture of diastereomers 121a,b in the ketalization step, since the use of either of the pure forms met with epimerization. Several conditions were examined, but the original 5 mol% TsOH proved optimal (50% ketal 126, 20% recovered starting material). Each isomer independently afforded the same 2.5" 1 ratio of isomeric ketals, suggesting that the 2.5" 1 ratio obtained in the initial alkylation step (94 to 121) reflected a thermodynamic distribution.
0
,,,..
\_-6
HOCH2CH2 OH Phil. TsOH. reflux
121a,b
126 I 1. RuO 2. NaIO4 (80%) 2. TBAF (95%)
nllo..~ 127
The critical ruthenium tetroxide oxidative cleavage of the g bond in 126 occurred rapidly and with consistently good yields (70-80%). However, it was important to carefully monitor the reaction to avoid overoxidation. Removal of the silyl ether with TBAF afforded a mixture of alcohols 127 (2.5"1, still reflecting the mixture resulting from the alkylation step) which could be separated by column chromatography. The major diastereomer 127a was oxidized to provide aldehyde 131, a substrate seemingly well-suited for the use of an intramolecular aldol condensation to complete the addition of the third ring [48]. When a methanolic solution of 131 was heated gently in the presence of a variety
232
LITTLE and O'I'F
of equilibrating bases (e.g., KOH; K2CO3; Na2CO3, NaOH), it was smoothly transformed into a single UV active product in a 70% yield. Characterization revealed that it did not correspond to the desired adduct 132, but instead to enal 133, the product resulting from closure of the aldehyde enolate onto the ketone carbonyl, rather than the reverse. 0
o::::on( 127a
) 131
t
~
base
0/-.7
t ,
0
"|ll''/~ 133
Scheme
14. Aldol condensation charts its own course.
At this point, it appeared that enolate formation toward the pro C3 carbon (see 92 for numbering) might not be a viable proposition. Given a choice, either it did not occur in that direction or if it did, then the enolate equilibrated to an alternative form that led to 125 in the case of the attempted alkylation of iodide 124, or to 133 in the attempted aldol cyclization of 131. We eventually elected to explore the intramolecular alkylative cyclization of keto iodide 134. To this end, mesylation of 127a followed by displacement with NaI led to 134 (80%). Treatment of this iodide with excess LDA in THF at-78 ~ followed by warming to room temperature, produced a single product. Workup, isolation, and characterization indicated that the desired adduct 92 had indeed been produced, and in a reasonable 63% yield.
BIOACTIVE NATURAL PRODUCTS
a,,..
233
O I. MsCl, NEt 3 ,
,
2. Nal, a c e t o n e
{8o%} I
134
127a
5 eq LDA THF -78 ~ (63%)
~
, II
0
H
*.
92
Molecular mechanics calculations place the t r a n s fused B,C ring system at considerably lower energies than its cis counterpart. The literature suggests that base-induced cis-to-trans isomerization is easily accomplished [49]. So, to ensure that the t r a n s ring fusion had been obtained, 92 was treated with K O B u - t / t - B u O H (8 h, room temp.)[49a]. The starting material was recovered unchanged, strongly supporting the notion that isomerization did not occur and that 92 does indeed possess the requisite stereochemistry. SUMMARY A functionalized taxol analog 92 has been synthesized from THF (Scheme 15). Substantial quantities of diazene 96 were synthesized in a convenient eight step sequence. The regioselective diyl trapping reaction, 96 to 95, consistently produced high yields of the key intermediate on scales >20 grams. A regioselective oxyphenyselenation reaction added the phenylselenyl unit to the bridgehead carbon of 95 and proved critical in efforts to isomerize the initially formed disubstituted double bond, and ultimately leading to enone 94. Cleavage of the tetrasubstituted olefin provided the [5.3.1] ring system present in taxoids. A regioselective
234
LITTLE and OTT
intramolecular alkylation served to append the side chain and form the core A-B-C ring system present in the natural products. This first generation synthesis of the taxol | skeleton provides ample opportunity to add key functionality in our ongoing second generation efforts designed to produce bioactive materials.
dlyl trap
., (8 steps)
~ |
|
~
r
0 N /
98
(3 steps) "
('"'
'"'
o
- " _",. ~---0
Scheme 15. Overview of route used to access analog 92.
It is now a relatively simple matter to selectively assemble either bridged or linearly fused tricyclie systems. The chemistry just described illustrates one of the materials toward which we have applied the methodology. In the next section, we focus upon its application to another important natural product, viz., the challenging anticancer agent, aphidieolin (89).
Aphidicolin (89) Aphidicolin (89) is a diterpenoid tetraol produced by the mold Cephalosporium aphidicola. In 1972, Hesp and co-workers reported its isolation and structure [50]. Aphidieolin (89) displays marked activity against Herpes simplex type I virus in cultures of human embryonic cells, as well as antifeedant properties. Furthermore, aphidicolin (89) exhibits antitumor activity in the C6 mouse colon and B 16 mouse melanosarcoma screens and has been shown to inhibit the growth of leukemic T- and Blymphocytes [51]. Such results have stimulated interest in clinical investigations of the effectiveness of 89 against human tumors.
BIOACTIVE NATURAL PRODUCTS
235
Development of 89 as an antitumor agent has been limited by its poor water solubility. However, reports of enhanced antitumor activity associated with the more water-soluble compounds such as aphidicolin17-glycinate HCI salt (135) and 16-fluoroaphidicolin (136) have revived interest in aphidicolin (89) and its analogs as potential therapeutic agents [52]. The potent activity of aphidicolin (89) is presumed to arise through its activity as a specific reversible inhibitor of DNA polymerase-a [51 ]. R2N I
al ~16
8
H
aphldlcolin (89}; R l = OH, R2 = H aphidicolin-17-glycinate HCI salt (135); R l - O H , R 2 = COCH2NH2-HCI
.
,~
HO
~
OH
16-flouroaphidicolin (136); R l = F, R2 = H
Fig.(12). Aphidicolin (89) and derivatives.
These biological properties along with its structural complexity have made 89 an attractive synthetic target. Aphidicolin (89) possesses an interesting array of ring systems, including both spiro and fused substructures, as well as the ever-challenging vicinal quaternary stereogenic centers [53]. The first two syntheses were reported independently by Trost and by McMurry in 1979. Many followed, including the first enantioselective synthesis reported by Holton in 1987. As is portrayed in Scheme 16, Fukumoto's approach to aphidicolin (89) used an intramolecular Diels-Alder reaction to both form and simultaneously fuse rings A and B onto a pre-existing CD unit (139 to
140) [54]. The route mirrors that conceptualized by this research in the early 1980's, the major difference being that in our route, an intramolecular diyl trapping reaction was to be utilized to assemble the [3.2.1] core (see Scheme 17). Thereafter, an intramolecular Diels-Alder reaction was planned in much the same manner as that implemented by the Fukumoto group. Nearly as soon as we realized that the diyl trapping reaction could be used to construct the [3.2.1 ] unit, aphidicolin (89) emerged as a target structure of interest. The problem, however, was that for many years it was not possible to use the diyl trapping reaction to form the bridged materials selectively, or in substantial quantity. That we are now able was in no small part motivated by the desire to synthesize the natural product [21.
236
LITTLE and OTF
~
~
O
0 H
{CH2}20H
o H
v
HO 137
139
138 ~ O
I 220 ~
140 Scheme 16. The Fukumoto route to aphidicolin (89).
We set our sights on a plan designed to converge with Fukumoto's Diels-Alder triene precursor, viz., with keto alcohol 138. We reasoned that if the intramoleeular diyl trapping reaction could be used to synthesize the bisketal 143, then conversion to 138 should be accomplished by oxidative cleavage of the lr bond to afford diol 142, selective protection of the primary alcohol, and the insertion of unsaturation between Cs and C:3. The latter operation is designed to allow use of both of the epimers that result from the diyl eyeloaddition step, as the Cs stereogenie center is removed when the sp 2 hybridized center is created. A least hindered side delivery of dihydrogen to the A-8,13 7r bond ought to position the Cs side chain on the alpha face, as needed. Removal of the alcohol protecting group and the selective conversion of the side chain ketal to a methyl ketone ought to afford 138, our convergent point with Fukumoto. To test the proposed route we capitalized upon our ability to use the intramoleeular diyl trapping reaction to synthesize 95, the mono-ketal analog of 143. Ozonolytie cleavage of the C-C 7r bond of 95 followed by reduction with NaBH 4 afforded diol 145 in an 85% yield. The primary alcohol was selectively protected as a silyl ether, and the secondary alcohol oxidized with PCC to provide ketone 146 as a 1:1 mixture of diastereomers.
BIOACTIVE NATURAL PRODUCTS
"~
237
~o
~o o o.
" , t ~ OH
9
:
HO
HO
HO
142
141 89
o~O I
/-t~, N 144
143
Scheme 17. The diyl trapping route to aphidicolin (89).
i(O,,..
/
b. N~BH4
Co
\
|,,,~.....~OH ,,..X.-"5~
Co
95
1
OH
145
I
II
2. PCC
/
\
i,,..~0 ,,..X.--"g~
Co
1
OTBDI~
146
III
Methodology developed by Ireland and coworkers was the first applied to insert a double bond between carbons 8 and 13 [55]. Unfortunately, treatment of enol phosphate 147 with lithium in liquid ammonia afforded a complex mixture of products. Though signals were present in the vinyl region of the IH NMR spectrum, none of the purified materials corresponded to the desired product 148.
238
LITTLEand 0 1 T
(a) LDA (b) (EtO)2POCI
,,.._
, 0
147
148
/~ LI/N]-I3
,,,,. ~ . ~ ~ . ~ ~ . . ~ O P
~.~0 I
148
II
The Shapiro reaction appeared to offer a reasonable alternative protocol. To this end, ketone 146 was converted to the corresponding tosylhydrazone 149 in reasonable yield (70%, in addition to recovered starting material). Both diastereomers of 149 were then subjected to a variety of standard, literature procedures [56]. In no case was product observed, only recovered starting material. Careful examination of the i,,..
..
O
NNHTs
i
TsNHNH2 D. EtOH
( | ~ ~ ~ ~ H ~
(70%)
PO
149
146, P = TBDPS
0~,0
n-BuLl, TMEDA reflux (85%)
/Ill,.
,i
compare (~O " ~
'
H
138
OH
PO
(a) dilmlde ~
TsOH TBAF (75%) OH 3 steps 150
Scheme 18. Convergencewith Fukumoto; a model system.
148
BIOACTIVENATURALPRODUCTS
239
literature reveals that the Shapiro reaction is, in fact, not very effective when there is branching adjacent to the carbonyl. We were, however, able to achieve the desired transformation by using TMEDA as the reaction solvent. Thus, at-78 ~ five equivalents of n-BuLi were added to a stirred solution of tosylhydrazones 149 in TMEDA. The reaction mixture was then refluxed for 8 h and upon workup, the desired olefin 148 was isolated in an 85% yield. It is important to note that both diastereomers were converted to olefin 148. This contrasts with our taxol | (90) related effort wherein only one of the diastereomers produced in the diyl cycloaddition proved effective in the oxyphenylselenation process. The sequence to 150 was completed as planned, by a least hindered exo face reduction of the olefin in 148 using diimide generated in situ, and deprotection of the ketal and silyl groups. OVERVIEW While the sequence just outlined has been carried out using the monoketal 95, rather than the bisketal 143, we believe that it will also be applicable to the latter. In fact, we have managed to utilize the intramolecular diyl trapping reaction to synthesize 143 and have, in preliminary experiments, demonstrated that it is possible to selectively convert the exocyclic ketal to a methyl ketone 151, as is needed to synthesize aphidicolin (89) [57]. At this time we are perfecting our route to diazene 144 and anticipate that the natural product is finally within our grasp.
CHaCN, reflux 12 h (80%)
N//N
143 144
PPTS, acetone, H20 ~ 0
~ [90%1
151 I
I
Ill
I
LITTLE and OTr
240
ACKNOWLEDGEMENTS We are exceptionally grateful to both the National Institutes of Health (National Cancer Institute), and the National Science Foundation for their support of our research programs. REFERENCES
[1] [2] [31
[4] [5] [6]
[7] [8] [91
[10] [11]
[12] [13] [14] [15] [16] [17]
[18] [19]
Paquette, L. A.; Doherty, A. M. Polyquinane Chemistry Springer-Verlag: Berlin, 1987. (a) Little, R. D. Chem. Rev. 1986, 86, 875. (b) Little, R. D. Chem. Rev. 1996, 96, 93. Little, R. D. In Comprehensive Organic Synthesis, Trost, B. M., Fleming, I., Eds.; Pergamon: Oxford, 1991; Vol. 5, Chapter 3.1, and references therein. Little, R. D.; Bukhari, A.; Venegas, M. G. Tetrahedron Lett. 1979, 305. (a) Stone, K. J.; Little, R. D. J. Org. Chem. 1984, 49, 1849. (b) McLoughlin, J. I.; Little, R. D. J. Org. Chem. 1988, 53, 3624. (a) Little, R. D.; Carroll, G. L. J. Org. Chem. 1979, 44, 4720. (b) Meehan, S.; Little, R. D. J. Org. Chem. 1997, 62, 3779. (c) Rastetter, W. H. J. ,4m. Chem. Soc. 1976, 98, 6350. (d) Gassman, P. G.; Schenk, W. N.J. Org. Chem. 1976, 42, 918. (e) Schwaebe, M. K.; Little, R. D. Electrochemica Acta, 1997, 42, 2201. Little, R. D.; Muller, G. W. J. Am. Chem. Soc. 1981, 103, 2744. (a) Little, R. D; Venegas, M. G. Org. Synth., Coll. Vol. VII ; Wiley: New York, 1990, 56. (b) Little, R. D.; Bregant, T. M. In Encyclopedia of Reagents for Organic Synthesis, Paquette, L. A., Ed.; Wiley: New York, 1995; Vol. 1, p. 572. With a two-carbon tether connecting the diyl to the diylophile, evidence strongly supports a stepwise mechanism, even when the diylophile is activated by an electron withdrawing group. Campopiano, O.; Little, R. D.; Petersen, J. L. J. Am. Chem. Soc. 1985, 107, 3721. Hudlicky, T.; Kutchan, T.; Wilson, S. R.; Mao, D. T. J. Am. Chem. Soc. 1980, 102, 6351. Little, R. D.; Higby, R. G.; Moeller, K. D. J. Org. Chem. 1983, 48, 3139. (a) Kunimoto, T.; Umezawa, H. Biophys. Acta 1974, 298, 513. (b) Ishizaka, M.; Iinuma, H.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1972, 25, 320 Kupka, J.; Anke, T.; Giannetti, B. M.; Steglich, W. Arch. Microbiol. 1981, 130, 223. (b) Giannetti, B. M.; Steffan, B. Steglich, W. Tetrahedron 1986, 42, 3587. (c) Steglich, W. Pure Appl. Chem. 1981, 53, 1233. (a) Van Hijfie, L.; Little, R. D. J. Org. Chem. 1985, 50, 3940. (b) Van Hijtte, L.; Little, R. D.; Petersen, J. L.; Moeller, K. D. J. Org. Chem. 1987, 52, 4647. Stone, K. J.; Little, R. D. J. Am. Chem. Soc. 1985, 107, 2495. Kissel, C. L.; Rickbom, B. J. Org. Chem. 1972, 37, 2060. (a) Lipshutz, B. H.; Parker, D. A.; Kozlowski, J.; Nguyen, S. L. Tetrahedron Lett. 1984, 25, 5959. (b) Lipshutz, B. H.; Wilhelm, R. S.; Kozlowski, J. Tetrahedron 1984, 40, 5005. (c) Lipshutz, B. H. ,4cc. Chem. Res. 1997, 30, 277. Koreeda, M.; Mislankar, S. G. J. Am. Chem. Soc. 1983, 105, 7203. Schuda, P. F.; Heimann, M. R. Tetrahedron 1984, 40, 2365.
BIOACTIVE NATURAL PRODUCTS
[20] [21] [22] [23] [24]
[25] [26]
[27]
[28] [291 [301 [31] [32]
[33]
[34]
241
(a) Danishefsky, S.; Zamboni, R.; Kahn, M.; Etheredge, S. J. J. Am. Chem. Soc. 1980, 102, 2097. (b) Danishefsky, S.; Zamboni, R.; Kahn, M.; Etheredge, S. J. J. Am. Chem. Soc. 1981, 103, 3460. Little, R. D.; Carroll, G. L.; Petersen, J. L. d. Am. Chem. Soc. 1983, 105, 928. Kaisan, M.; Sheikh, Y. M.; Durham, L. J.; Djerassi, C.; Tursch, B.; Daloze, D.; Braekman, J. C.; Losman, D.; Kadsson, R. Tetrahedron Left. 1974, 2239. Birch, A. M.; Pattenden, G. Tetrahedron Lett. 1982, 23, 991. Freisleben, W. Angew. Chem. 1963, 72, 576. Stembach, D. D. In Strategies and Tactics in Organic Synthesis, Lindberg, T. Ed.; Academic Press: San Diego, 1989; Vol. 2, Chapter 12. Platz, M. S.; Berson, J. A. J. Am. Chem. Soc. 1976, 98, 6743. (a) Beckwith, A. L. J.; Ingold, K. U. In Rearrangements in Ground and Excited States; deMayo, P., Ed.; Academic Press: New York, 1980; Vol.l, Chapter 4 and references therein. (b) Lusztyk, J.; Maillard, B.; Deycard, S.; Lindsay, D. A.; Ingold, K. U. J. Org. Chem. 1987, 52, 3509. Little, R. D.; Losinski-Dang, L.; Venegas, M. G.; Medic, C. Tetrahedron Lett. 1983, 24, 4499. Berson, J. A. In Diradicals; Borden, W. T., Ed.; Wiley: New York, 1982; Chapter 4. (a) Masjedizadeh, M. R.; Dannecker-Doerig, I.; Little, R. D.; J. Org. Chem. 1990, 55, 2742. (b) Little, R. D.; Masjedizadeh, M. R.; Moeller, K. D.; DanneckerDoerig, I. Synlett 1992, 107. Masjedizadeh, M., Postdoctoral Research Report, UCSB. (a) Taxane Anticancer Agents; Georg, G. I.; Chert, T. T.; Ojima, I.; Vyas, D. M.; Eds.; American Chemical Society: San Diego, 1995; Vol. 583. (b) Nicolaou, K. C.; Dai, W. M.; Guy, R. K. Angew. Chem. Int. Ed. Engl. 1994, 33, 15. (c) Swindell, C. S. Org. Prep. Proced. Int. 1991, 23, 465. (a) Nicolaou, K. C.; Zang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Claibome, C. F.; Renaud, J.; Couladouros, E. A.; Paulvannan, K.; Sorensen, E. J. Nature 1994, 357, 630. (b) Nicolaou, K. C.; Nantermet, P. G.; Ueno, H.; Guy, R. K.; Couladouros, E. A.; Sorensen, E. J. J. Am. Chem. Soc. 1995, 117, 624. (c) Nicolaou, K. C.; Liu, J. J.; Yang, Z.; Ueno, H.; Sorensen, E. J.; Claibome, C. F.; Guy, R. K.; Hwang, C. K.; Nakada, M.; Nantermet, P. G. J. Am. Chem. Soc. 1995, 117, 634. (d) Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Nantermet, P. G.; Claibome, C. F.; Renaud, J.; Guy, R. K.; Shibayama, K. J. Am. Chem. Soc. 1995, 117, 645. (e) Nicolaou, K. C.; Ueno, H.; Liu, J. J.; Nantermet, P. G.; Yang, Z.; Renaud, J.; Paulvannan, K.; Chadha, R. J. Am. Chem. Soc. 1995, 117, 653. (f) Holton, R. A.; Somoza, C.; Kim, H. B.; Liang, F.; Biediger, R. J.; Boatman, D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. S.; Liu, J. H. J. Am. Chem. Soc. 1994, 116, 1597. (g) Holton, R. A.; Kim, H. B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boatman, D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. S.; Liu, J. H. J. Am. Chem. Soc. 1994, 116, 1599. (h) Masters, J. J.; Link, J. T.; Snyder, L. B.; Young, W. B.; Danishefsky, S. J. Angew. Chem. Int. Ed. Engl. 1995, 34, 1723. (i) Wender, P. A.; Badham, N. F.; Conway, S. P.; Floreancig, P. E.; Glass, T. E.; Houze, J. B.; Krauss, N. E.; Lee, D. S.; Marquess, D. G.; McGrane, P. L.; Meng, W.; Natchus, M. G.; Shuker, A. J.; Sutton, J. C.; Taylor, R. E.; J. Amer. Chem. Soc. 1997, 119, 2757. Ott, M. M.; Little. R. D. d. Org. Chem. 1997, 62, 1610.
242
[35]
[36] [37]
[38] [39]
[40]
[41] [42] [43] [44]
[45] [46] [47] [48]
[49]
[50] [5~] [52] [531
LITTLE and 01T The corresponding dimethyl ketal is too labile to be useful, particularly in large scale operations. Its relatively facile hydrolysis complicates the diyl cycloaddition since the diazene-ketone leads preferentially to the linearly fused rather than the desired bridged adduct. The ethylene glycol ketal provided an ideal solution to the problem. Nystr0m, J. E.; McCanna, T. D.; Helquist, P.; Amouroux, R. Synthesis 1988, 56. The sequence leading to 99 was originally explored and developed by Mr. Scott Meehan of UCSB. The use of DMSO allowed the chemistry to be conducted at room temperature, made the process very convenient to conduct, and led to significantly improved yields. Parikh, J. R.; Doering, W. E. J. Am.Chem. Soc. 1967, 89, 5505. The identity of these materials was confirmed by conversion to and comparison of the spectral properties with with the known ketones; see reference 30. (a) Grieco, P. A.; Nishizawa, M.; Marinovic, N.; Ehmann, W. J. J. Am. Chem. Soc. 1976, 98, 7102. (b) Mehta, G.; Murthy, A. N. J. Org. Chem. 1987, 52, 2875. Raucher, S. J. Org. Chem. 1977, 42, 2950. Reich, H. J. J. Org. Chem. 1974, 39, 428. (a) Sharpless, K. B.; Lauer, R. F. J. Org. Chem. 1974, 39, 429. (b) Reich, H. J.; Wollowitz, S.; Trend, J. E.; Chow, F.; Wendelbom, D. F. J. Org. Chem. 1978, 43, 1697. In principle, both stereoisomers 95a and 95b can be converted to the same tetrasubstituted olefin. Unfortunately, and despite much effort, only 95a proved useful in the oxyphenylselenation step. We thank Dr. Andre D'Avignon of Washington University for performing the HMQC-TOCSY experiments. (a) Blechert, S.; Muller, R.; Beitzel, M. Tetrahedron 1992, 48, 6953. (b) Galatsis, P.; Manwell, J. J. Tetrahedron 1995, 51,665. (a) Carlsen, P. H. J.; Katsuki, T.; Martin, V, S,; Sharpless, K. B. J. Org. Chem. 1981, 46, 3936. (b) Mehta, G.; Krishnamurthy, N. J. Chem. Soc., Chem. Commun. 1986, 1319. (a) Marshall, J. A.; Greene, A. E. J. Org. Chem. 1972, 37, 982. (b) Marshall, J. A.; Greene A. E., Ruden, R. Tetrahedron Lett. 1971, 855. (r Marshall, J. A.; Greene A. E. Tetrahedron Lett. 1971, 859. (d) Heathcock, C. H.; Trice, C. M.; Germroth, T. C. J. Am. Chem. Soc. 1982, 104, 6081. (e) Crimmins, M. T.; Gould, L. D. dr. ,4m. Chem. Soc. 1987, 109, 6199. (a) Blechert, S.; Kleine-Klausing, A. Angew. Chem., Int. Ed. Engl. 1991, 30, 412. (b) Magnus, P.; Ujjainwalla, F.; Westwood, N.; Lynch, V. Tetrahedron Lett. 1996, 3 7, 6639. Bundret, K. M.; Dalziel, W.; Hesp, B.; Jarvis, J. A.; Neidle, S. J. Chem. Soc., Chem. Commun. 1972, 1027. Pedrali-Noy, G.; Belvedere, M.; Crepaldi, T.; Focher, F.; Spardari, S. Cancer Res. 1982, 42, 3 810. Hiramitsu, T.; Mouri, A.; Suzuki, H. (Nippon Mektron Ltd.). Japan Patent Kokai Tokkyo Koho JP 5-310621. (a) Trost, B. M.; Nishimura, Y.; Yamamoto, K.; McElvain, S. S. J. Am. Chem. Soc. 1979, 101, 1328. (b) McMurry, J. E.; Andrus, A.; Ksander, G. M.; Musser, J. H.; Johson, M. A. J. Am. Chem. Soc. 1979, 101, 1330. (r Corey, E. J.; Tius, M. A.; Das, J. J. Am. Chem. Soc. 1980, 102, 1742. (d) Ireland, R. E.; Godfrey, J. D.; Thaisrivongs, S. J. Am. Chem. Soc. 1981, 103, 2446. (e) van Tamelen, E. E.; Zawacky, S. R.; Russell, P. K.; Carlson, J. G. J. Am. Chem. Soc. 1983, 105, 142.
BIOACTIVE NATURAL PRODUCTS
[54] [551 [561
[57]
243
(f) Bettolo, R. M.; Tagliatesta, P.; Lupi, A.; Bravetti, D. Helv. Chim, Acta. 1983, 66, 1922. (g) Tanis, S. P.; Chuang, Y. H.; Head, D. B. Tetrahedron Lett. 1985, 26, 6147. (h) Holon, R. A.; Kennedy, R. M.; Kim, H. B.; Kraft, M. E. J. Am. Chem. Soc. 1987, 109, 1597. (i) Rizzo, C. J.; Smith, III, A. B. J. Chem. Soc., Perkin Trans. 1991, 1, 969. (j) Tanaka, T.; Murakami, K.; Okuda, O.; Inoue, T.; Kuroda, T.; Kamel, K.; Murata, T.; Yoshino, H.; Imanishi, T.; Kim, S. W.; Iwata, C. Chem. Pharm. Bull. 1995, 43, 193. Toyota, M.; Nishikawa, Y.; Fukumoto, K. Tetrahedron Lett. 1995, 36, 5379. Ireland, R. E.; Pfister, G. Tetrahedron Lett. 1969, 2145. (a) Dauben, W. G.; Lorber, M. E.; Vietmeyer, N. D.; Shapiro, R. H.; Duncan, J. H.; Tomer, K. J. Am. Chem. Soc. 1968, 90, 4762. (b) Chamberlin, A. R.; Stemke, J. E.; Bond, F. T. J. Org. Chem. 1978, 43, 147. (c) Stemke, J. E.; Chamberlin, A. R.; Bond, F. T. Tetrahedron Lett. 1976, 34, 2947. (d) Lipton, M. F.; Shapiro, R. H. J. Org. Chem. 1978, 43, 1409. Unpublished research with Dr. Joachim Dickhaut, UCSB; postdoctoral research report, 1994.
This Page Intentionally Left Blank
Atta-er-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22
245
9 2000 Elsevier Science B.V. All rights reserved
BIOACTIVE FUNGAL NATURAL PRODUCTS THROUGH CLASSIC AND BIOCOMBINATORIAL APPROACHES Z H I - D O N G JIANG* AND Z H I Q I A N G AN
Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge MA, 02139-1562 ABSTRACT" The kingdom of fungi is an important source of bioactive natural products, which have been a driving force in the development of modem pharmaceutical industry. Fungal natural products have provided revolutionary pharmaceuticals against various diseases, and have provided unique and inspirational chemicals for innovative drugs. Fungi are essentially an untapped source of drugs in spite of many remarkable therapeutic agents discovered from them so far. Of approximately 1.5 million species of fungi, to date only about 70,000 species have been described. Among these 70,000 species, only a small fraction can be isolated from nature and fermented in laboratory media for drug screening. The vast majority of slow-growing and unculturable fungi have received little attention for drug discovery due to technical limitations. Therefore, the potential for discovering bioactive agents from slow-growing and unculturable fungi is even greater than that from the species that we have explored. With the maturing technology of genetic engineering, it is now possible to express genes from unculturablr organisms in laboratory strains for secondary metabolism study. Today, mankind is not only still facing the challenge to treat untamed diseases, but is also fighting newly recognized diseases, and diseases that once were subdued but are developing resistance to the current therapeutic regimes. In this review, we will discuss recent developments and progress of several important fungal metabolites and their derived products as examples of drugs produced by natural isolates. We will also discuss the current progress of biocombinatorial drug discovery by genetic engineering approaches, as well as possibilities and strategies of exploring genetic diversity from unculturable fungi for drug discovery. The vast fungal kingdom, which consists of an estimated 1.5 million species, is of interest to the pharmaceutical industry for its production of many important secondary metabolites. Since more than one half century ago, when the fungal metabolite penicillin was first partially purified and used for treating bacterial infections, bioactive fungal metabolites have strongly influenced the development o f the modern pharmaceutical industry. Mevinolin, cyclosporin A, and [3-1actam antibiotics are examples of revolutionary pharmaceuticals that have a fungal origin. In addition, the diverse and unique chemical structures of fungal metabolites have served as an important source o f inspiration for structural motifs to synthetic chemists [1-2]. Fungal metabolites o f various biosynthetic origins have produced breakthrough pharmaceutical and agricultural products during the last
246
ZHI-DONG JIANG AND ZHIQIANG AN
decade. In this review, we describe recent progress of several fungal metabolites of pharmaceutical and agricultural importance. This by no means includes all the structures, rather we aim to demonstrate the potential of discovering new chemical structures from fungal sources and the value of fungi as an important source of compounds. Readers are encouraged to study recent reviews and databases that have extensively covered fungal natural products [3-5, 133]. In spite of the remarkable therapeutic agents discovered so far, fungi are essentially an untapped source of active metabolites. Only a small fraction of the fungal taxa can be and have been fermented in laboratory media for drug discovery. In this chapter, we also briefly discuss the potential of using biocombinatorial approaches to tap into the genetic diversity of fungi for drug discovery. FUNGI Fungi are nutrition-absorptive eukaryotic organisms found in every ecological niche. The kingdom Fungi comprises more than 1.5 million species of organisms [6]. It is difficult to generalize the characteristics of fungi due to the tremendous ecological, physiological, and morphological diversity within the fungal kingdom. The classification of fungi is consistently evolving as new information emerges related to fungal evolution and systematics. Currently, there are four phyla in the fungal kingdom: Chytridiomycota,Zygomycota,Ascomycota,and Basidiomycota. Figure 1 illustrates the current classification scheme for fungi. i. . . . Basidiomycota '-Ascomycetes I
Fun~---
I_
I
Ascomycota -----
Zygomycota Chytridlomycota
Deuteromycetes
Fig. (1). Phylogenetic Relationships of Major Groups in the Fungi Kingdom.
Chytridiomycetes is the only class in the phylum Chytridiomycota. Fungi in this group produce motile cells at some stage in their life cycle. Chytrids are present in both aquatic habitats and soils. This group of fungi has not been widely used for natural products screening because they are extremely small and difficult to isolate and culture. Since chytrids occur in very competitive ecological niches, access to this group of fungi might yield interesting secondary metabolites. The phylum Zygomycota comprises two classes: Zygomycetes and Trichomycetes. Many fungi in the class Zygomycetes produce a thick-
BIOACTIVE FUNGAL NATURAL PRODUCTS
247
walled resting spore known as zygospore. Species of Zygomycetes can be isolated from a variety of ecosystems. Some Mucorales fungi have been extensively used for production of various secondary metabolites, but in general, this group of fungi has not been well explored for natural products drug discovery. Fungi in the class Trichomycetesare obligately associated with living arthropods. Because they can not be easily cultured, they have not been subjected widely to drug discovery. Fungi in the phylum Ascomycota are grouped into two, classes: ascomycetes and deuteromycetes. Ascomycetes produce asci, which are saclike structures containing sexual ascospores. Deuteromycetes are asexual ascomycetes. Fungi in Ascomycota occur in a broad range of ecosystems and consist of species greatly diverse in morphology and habitat. Ascomycetes are well known and extensively investigated for their ability to produce a large number of secondary metabolites. Many of these metabolites are effective therapeutics. Drugs developed from natural products isolated from ascomycetes and other fungi are summarized in Table 1. Table 1.
Some of Fungal Significance
Metabolites
with Pharmaceutical
Producing organism
Fungal metabolite
Related industrial products
,, ,,,,,i
,
,
and Agricultural
i
,
i
Peniciilins
Penicillium sp.
Penicillin antibacterial agents
Cephalosporin C
Cephalosporium acremonium
Cephalosporin antibacterial agents
Mevinolin
Aspergillus sp.
Lovastatin, simivastatin, lipitor and other HMG-CoA inhibitors
Cyclosporins
Tolypocladium inflatum
Cyclosporin A, immunosuppresant
Mycophenolic Acid
Penicillium sp.
CellCept, immunosuppresant
Griseofulvin
Penicilllium griseofulvum
Griseofulvin, antifungal agent
Strobilurins
Strobilurus tenacellus
Amistar and kresoxim methyl, agricultural fungicides
Fusidic Acid
Fusidium coccineum
Fusidic acid, antibacterial agent
Ergot alkaloids
Claviceps purpurea
Ergotamine for migraine Ergonovine for obstetrics
Gibberellins
Fusarium moniliforme Fusarium graminearum
Zearalenone ,
ill
Gibberellins, plant growth hormone Zearalenone, growth promoter in cattle ,,,,,
The fungi in the phylum Basidiomycota are called basidiomycetes, which are distinguished from other fungi by the production of sexual basidiospores. Basidiomycetes consist of fungi commonly known as mushrooms, puffballs, rusts, and smuts.
248
ZHI-DONG JIANG AND ZHIQIANG AN
Antifungal Agents Lipopeptides The number of cases of systemic fungal infections has grown in recent years due to the increased number of immune compromised individuals including patients with acquired immunodeficiency syndrome (AIDS), and patients receiving such therapeutics as anti-neoplastic agents, immunosuppressants, or broad-spectrum antibiotics. Currently available chemotherapies for systemic fungal infections such as amphotericin B and fluconazole have limited uses because of their toxicity and/or insufficient therapeutic efficacy. Although more advanced liposomal formulations of amphotericin B have significantly improved its efficacy and reduced its toxicity, reports of fungal resistance to this drug are increasing [7-9]. New antifungal drugs with improved activity are greatly needed. Fungi have been a very productive source of potential antifungal agents and have yielded two classes of promising cyclic peptides with two novel mechanisms of antifungal activity. Echinocandins and pneumocandins, produced by Aspergillus species and Zalerion arboricola respectively, belong to the same class of cyclic lipopeptides whose characteristic long chain acyl groups connect to the cyclic peptide ring through an amide bond. As fungicidal agents, they inhibit ~-l,3-glucan synthase and prevent the formation of this class of glucan, an essential component of the fungal cell wall in many fungi [ 101 1]. Echinocandins are active in animal models against Candida sp., Pneumocystis carinii, and some Aspergillus sp.. They are not active against Cryptococcus sp. which contain mostly o~-glucan [ 12]. Two derivatives of echinocandin and pneumocandin, L-743872 by Merck and LY-303366 by Eli Lilly, are currently in clinical trials [127].
Aureobasidins Aureobasidins, produced by Aureobasidium pullulans, are another class of cyclic peptides [13-15]. Aureobasidin A, the most abundant component, is a fungicidal agent with strong activities against Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum. Its fungicidal activities in mice with candidiasis are more effective than fluconazole and amphotericin B, and it is well tolerated by mice [ 15]. The molecular target for aureobasidin A was identified recently as inositol phosphorylceramide synthase (IPC synthase), an enzyme that catalyzes a key step in sphingolipid biosynthesis, which is essential for yeast growth and viability. Aureobasidin A tightly binds to IPC synthase with an IC50 of 0.2 nM [16].
BIOACTIVE FUNGAL NATURAL PRODUCTS
249
Strobilurins and Oudemansins
In the agricultural antifungal area, two novel fungicides have been developed from the structural motif of strobilurins and oudcmansins. Strobilurins and oudemansins are fungicidal fungal metabolitcs isolated from Strobilurus tenacellus, Oudemansiella mucida, and several other basidiomycetes, as well as the ascomycete Bolinea lutea [17-20]. Strobilurin A is potently active against a wide range of plant pathogenic fungi through inhibiting the cytochrome bcl complex [21-23]. However, the producing fungi S. tenacellus is resistant to strobilurin A by a mutation of single amino acid at position 127 in its cytochrome bc 1 [24].
CH 3
CH3
Aurcobasidin A
\
Ho/
HO
F
o
~N-x / o
o ~ O H
~Ott -
-
Echinocandin
B
250
ZHI-DONG JIANG AND ZHIQIANG AN
~
CH 3
0
~
Strobilurin A
Me
O
Amistar (Azoxystrobln)
CN
CH3
IOCH3 0 Oudemansln A
Kresoxin methyl
The potent in vitro antifungal activities of strobilurin A were not maintained in the field due to the labile nature of its triene system. Several agricultural firms are engaged in synthetic efforts to produce stable and more potent analogues as potential products. In 1996, two products, amistar and kresoxim methyl, were approved as agricultural fungicides in the U.S. after more than a decade of research and development.
Anti-hypercholesterolemia Agents H M G - C o A Inhibitors
During the past decade, the most important development in the field of fungal natural products may have been the discovery and successful commercialization of HMG-CoA reductase (3-hydroxy-3-methylglutaryl CoA reductase) inhibitors. Their huge success in the pharmaceutical market reflects the fact that coronary heart disease has become one of the top killers in the industrialized nations (in the U.S. alone, more than 600,000 people die of heart disease each year) [25]. Hypercholesterolemia has been identified as an important cause for this disease. In the cholesterol biosynthetic pathway, HMG-CoA reductase is a rate-limiting
BIOACTIVE FUNGAL NATURAL PRODUCTS
251
enzyme that catalyzes the condensation of acetoacetyl-CoA with acetylCoA to form 3-hydroxy-3-methylglutaryl-CoA. Since Endo and his colleagues discoved compactin, a specific HMG-CoA reduetase inhibitor, this biological target has been a focus of international hypercholesterolemia research for more than 20 years, and there is no sign that interest in developing new inhibitors is waning [26]. After compactin was first isolated from Penicillium citrinum [27-28], several derivatives of compactin were isolated from Monascus and other filamentous fungi including Aspergillus, some species of Penicillium, Phoma, and Trichoderma [29]. The clinically important metabolite mevinolin was isolated independently by two groups of scientists at Sankyo and Merck from Monascus ruber and Aspergillus terreus, respectively [30-31 ]. The biosynthesis of mevinolin in Aspergillus terreus involves two polyketide chains, C 18 and C4, each synthesized from acetate units. The two methyl groups at 6 position and on the side chain were derived from methionine. Propionate was not incorporated [34]. Pravachol is a 6~-hydroxy acid form of compactin and can be produced by microbial transformation [32]. Pharmacologically, the active form of mevinolin is the ~l-hydroxyacid form; the lactone form is inactive. Chemical modification of the ester side chain revealed that its stereochemistry was not important but aeyl moiety was essential. Removal of the side chain or side chain ether analogues resulted in marked loss of potency [33]. Extensive SAR studies have led to the development of simvastatin, a 2,2-dimethylbutyrate derivative of mevinolin. Simvastatin, with the dissociation constant Ki of 0.2 nM, is about 3 times more potent than mevinolin and 11 times than pravachol and has been clinically proven to be more effective than either mevinolin or pravachol.
~
Rl
R2
O
Mevlnolln
CH 3
I R2
I O
CH3
Compactln
H
RI~~" 0
252
ZHI-DONGJIANG AND ZIlIQIANG AN
H0 ~ , ~
H O~ COOH H
H Paravachol
COOH
u ,m
CHa
0 Llpltor (atorvastatln)
The discovery of mevinolin, the first HMG-CoA reductase inhibitor approved to market, is a good example of how dramatic and competitive it can be to develop an innovative drug. Although Merck was the first pharmaceutical company to market a drug in this class, Merck was not the first to test this class of compounds in the clinic. In 1976, researchers at Sankyo first isolated compactin (mevastatin), which entered Phase I clinical trials in Japan and several other countries in 1978. It was a promising agent to significantly reduce the cholesterol levels with very few serious acute side effects. A year later, scientists at Merck isolated a related compound, mevinolin, which was put to Phase I clinical trials in early 1980, only to be suspended shortly thereafter. Prompting the suspension was the news that compactin caused intestinal lymphomas in 50 percent of the dogs treated with the agent. However, in 1984, Merck restarted the Phase I clinical trials with mevinolin after several physicians discovered that the drug dramatically reduced the cholesterol levels in patients whose severe hypercholesterolemia did not respond to existing therapies. Remarkably, mevinolin caused very few side effects. In November 1986, mevinolin was approved by the Federal Food and Drug Administration for marketing. In the meantime, scientists at Sankyo
BIOACTIVE FUNGAL NATURAL PRODUCTS
253
discovered another HMG-CoA reductase inhibitor, pravachol, which was subsequently licensed to Bristol-Meyers Squibb. Interestingly, pravachol was initially detected as a urinary metabolite of compactin in dogs and was the 6-~-hydroxy-l]-hydroxyacid form of compactin. Pravachol possessed cholesterol-reducing activity similar to mevinolin and was approved for marketing in the U.S. in 1991 [35]. However, the pharmaceutical industry's race for a slice of the huge market for HMG-CoA reductase inhibitors did not stop there. It was recently estimated that the worldwide HMG-CoA inhibitor market will reach $13 billion in the next few years [36]. This vast potential market for HMG-CoA reductase inhibitors has prompted large synthetic efforts to generate clinically more effective compounds. Studies have shown that the hydronaphthalene structure of natural inhibitor served only as a holder of the lactone and therefore could be replaced with other structural templates. One such compound produced by varying the templates is lipitor. Clinically, under the defined conditions, lipitor is more effective by several parameters in reducing lipids level. In three separate studies, lipitor has been shown to be superior to mevinolin, pravachol, and simvastatin in lowering the triglycerides by significant margins [37-39]. In 1996, nine years after mevinolin was first marketed, Parke-Davis introduced lipitor, a new synthetic HMG-CoA inhibitor developed from mevinolin structural motif, and it is quickly becoming one of the best selling drugs [40].
Squalene Synthase Inhibitors Squalene synthase is an enzyme catalyzing the formation of squalene from farnesyl diphosphate which is a committed step in the cholesterol biosynthetic pathway. Therefore, squalene synthase is considered a better target than HMG-CoA reductase because farnesyl pyrophosphate, a downstream product of HMG-CoA reductase, is needed for prenylation of proteins and for the biosyntheses of ubiquinone and dolichol (Fig. 2). Before squalestatins and zaragozic acids were discovered, a number of squalene synthase inhibitors were synthesized that showed respectable inhibitory potencies in vitro, but none were successful in animal testing [41]. It was the discovery of squalestatins and zaragozie acids that renewed interest in this biological target, and at picomolar potencies they were the most active inhibitors of squalene synthase. Squalestatins and zaragozir acids were isolated from several fungi including Phoma sp. C2931, Sporormiella intermedia, and Leptodontium elatius. Squalestatins and zaragozic acids possess the unusual structural feature of a highly functionalized bicyclic core with three earboxylic groups. Zaragozic acids differ from one another by varying side chains and long chain acyl groups [42-44].
254
Z H I - D O N G J I A N G AND Z H I Q I A N G AN
.oo~.
'~oo~
"
"
R2 Squalestatln 1 R l
CH 3 |
Squalestatln 2 R l
=
Squalestatln 3 R l
~ i O
=
|
Rl
-
Z a r a g o z l c acid C
Rl
=
R2
_
/o.
= l O
R2
-
H2C
R2
_-
H2C
R2
=
H2C
H
%CH 3
O Rl
=
0
H
Z a r a g o z l c acid B
R2 !
., o...
Z a r a g o z l c acid D
=
=
]
OH
BIOACTIVE FUNGAL NATURAL PRODUCTS
255
Acetyl CoA
.~etoaeetyl CoA
HMG CoA
Isopentenyl tRNA
~tatin
IIMGCoAReductase Mevalonle Acid
i I !
Isopentenyl Pyrophosphate ~
FamesylaUon of P r o t e l n s ' q [ - - - ' - - F a m e s y l P y r o p h o s p h a t e ~
Sualene Synthase ~
~
Dlmethylallyl
- - - ~
Prophosphate
Ublqulno a n d Dollchol
Squalestatins
Squalene
Lanosterol
! I I
Cholesterol Fig.
(2).
Cholesterol Biosynthesis and Utilities of Isoprenyi Intermediates.
Biosynthetically, squalestatin 1 was derived from mixed precursors. The major portion of the molecule was formed from two polyketide chains made of acetate units. The other portions were derived from benzoic acid (or phenylalanine), succinate, and methionine. Five of the oxygens were derived from atmospheric oxygen; the oxygens at the two ester carbonyls were derived from acetate (Fig. 3). Based upon this biosynthetic pathway, several biosynthetic analogues were isolated through feeding
256
Table 2.
ZHI-DONG JIANG AND ZHIQIANG AN
Biosynthetic Analogs of Squalestatin through Precursor Feeding
/
/ o~__~ ....\
\ o
oil,.
oH
H
O
O
C
~
R il
H O O C ~ X-" o il
,=
,
OH
COOH ,
i
ICso(ng/ml)
|',
I
0.2 Squalestatln I R = H 2 C - ~ 0.4
F Analogue I R = H 2 C ~
____/
0.3
F
Analogue!! R = H z C - ' ~ 0.2 AnalogueIII R = H 2 C - ~ ~
F 0.4
AnaloguelV R = H 2 C ~ 0.2 AnalogueV R =
H2C
~S 0.1
AnalogueVI R - H2C~'ff~
experiments. Several of them were shown to be as potent as squalestatin 1 [45-48] (Table 2, Fig. 3).
BIOACTIVE FUNGAL NATURAL PRODUCTS
#
i~b-.-
,"-'k ,. *
ox,
2"o t OH
O
O"
0 ~~ t " " ' ~ " 6 OH
H~176
257
.
COOH
Benzoic acid Acetate Succlnate NH 2 Methyl of methlonlne *,~
S
COOH
Fig. (3). The Biosynthetic Precurosrs of Squalestatin A.
Recently, zaragozic acids were shown to inhibit Ras farnesyltransferase, which involves posttranslational famesylation of all Ras proteins. Zaragozic acids blocked Ras processing in Ras transformed tumor cell lines at concentrations as low as 10 nM. Therefore, they potentially have anticancer applications [49].
Immunosupressive Agents Cyclosporin, Rapamycin, and FK506 Cyclosporin A, an immunosuppressant well known for its clinical property in organ transplantation [131, 132], was initially isolated as an antifungal agent from several fungi, including Tolypocladium inflatum [50]. Rapamycin and FK506, relatively new immunosuppressive agents used in the clinic, are metabolites of Streptomyces sp. Recently, it was revealed that the three agents had intriguing mechanisms of biological activities. Chemically, cyclosporin A is a cyclic peptide of eleven amino acid residues, very different from FK506 and rapamycin. FK506, although slightly smaller, resembles rapamycin, and their structures are partly identical. Surprisingly, it is FK506 and cyclosporin A that share almost
258
ZHI-DONG JIANG AND ZHIQIANG AN
\
"[ T
" H301-]~"f
[it
z
Y ,
O
~
pH3y
~,,.~
O
T -T ~
'11111~ '0
H
Cyclosporln A
~
H,
CH3 O
I H
OCH3
OH
H3 O
H $,~
J~
H3Co O
HO H3C_ft,./
~"~0
CH3
O"
~
"Cl 13
"OCH3
~H
v .~
"vOCH3
q,,
FK 506
Rapamycln
identical biological effects, not rapamycin and FK506. Furthermore, FK506 and rapamycin first bind to the same cytosolic binding protein, FKBP12, to form two different complexes, while cyclosporin A binds to a distinctly different protein, cyclophilin A. However, cyclosporin Acyclophilin A complex and FK506-FKBP12 complex inhibit the same target (calcineurin), while rapamycin-FKBP 12 complex inhibits a different target called FRAP. Both FKBP12 and cyclophilin A are peptidylproline cis-trans isomerases and are potently inhibited by FK506 and cyclosporin A respectively with subnanomolar IC50 values. The small molecules must bind their binding proteins to form the complexes that are specific inhibitors to calcineurin and FRAP; the small molecules alone
BIOACTIVE FUNGAL NATURAL PRODUCTS
259
exert no effects on calcineurin. Cyclosporin A and FK506 are highly specific inhibitors of their respective binding proteins [51-56]. Mycophenolic acid
The study of mycophenolic acid (MPA) has a long history. MPA was first observed in 1896 [57] and its structure determined in 1952, which was confirmed by a X-ray study in 1972 [ 129]. A wide range of biological activities were observed in MPA including antifungal, antiviral, and antitumor [58-59]. In the 1970s, attempt was made to develop this compound as an antitumor drug, but it was later withdrawn due to its toxicities [60]. In 1972, scientists at Eli Lilly firmly elucidated the mode of action of MPA to be a potent inhibitor of inosine monophosophate dehydrogenase (IMPDH) with the dissociation constant Ki values in the nanomolar range against IMPDH(s) from several biological sources [61 ]. MPA selectively inhibits the de novo pathway of guanosine nucleotide synthesis in vivo,. Because the proliferation of T- and B-lymphocytes require the de novo synthesis of purines, whereas other cell types can utilize salvage pathways, MPA selectively inhibits the proliferative responses of T- and B-lymphocytes to both mitogenic and allospecific stimulation. To develop MPA as an immunosuppressant, the prodrug mycophenolate mofetil was synthesized to improve the pharmacokinetic profile of MPA [62]. When mycophenolate mofetil is administered orally, it is rapidly absorbed and metabolized to MPA, the active metabolite. In 1996, one hundred years after MPA was discovered, mycophenolate mofetil was approved by the FDA for renal transplant rejection. The development of this drug shows how a compound that may be toxic in one disease area may be useful in other therapeutic areas.
Ho•H3
H
\ /0
0
Mycophenollc Acid
O
CH3
Mycophenolate Mofetil
CH 3
260
ZHI-DONG JIANG AND ZHIQIANG AN
A n t i c a n c e r Agents
llludin S and Hydroxymethylacylfulvene Illudins are unique sesquiterpenes produced by the fungus Omphalotus illudens [63-65] The compounds are extremely toxic and have caused poisoning when Omphalotus is mistaken for edible mushrooms [66-67].
OH .-
HO~'"
OH .~
]
9
CH2OH
HO~,"
O
O
llludln S
llludln M
2OH
0
9
HO~'" 0 Hydroxymethylacylfulvene
~176
0 Dehydrotlludtn M
llludin S and illudin M are reactive cytotoxic compounds that alkylate thiols through Michael addition reaction to form stable aromatic adducts [68-70] (Fig. 4). The antitumor activities of these two compounds were studied in a variety of rodent tumor models in the 1980s when they were found to be potent anticancer agents. However, because their cytotoxicity was not highly selective, illudins were not safe to use as anticancer agents [71]. Evidently, the ttl3-unsaturated ketone and cyclopropylmethyl carbinol were the key features of illudins required in the alkylation reaction. But in order to make illudins safer, analogues had to be less reactive to thiols and more selective to tumor cells. A series of compounds was prepared that resulted in the discovery of hydroxymethylacylfulvene, which caused complete tumor regression in metastatic lung carcinoma (MV 522) in xenograft animals and exhibited outstanding activities against breast, colon, and skin cancers. This compound entered Phase I clinical trials in 1995 [72-74].
BIOACTIVE FUNGAL NATURAL PRODUCTS
261
- H2
_OH ".,, HO ~'"
CH2OH
H+~tt~'~~SR
"CH20 H
OH OH
"%' H20H OH
SR
Fig. (4). Reaction of thiols with llludin S.
Fumagillin and A GM-14 70 Angiogenesis is a complex morphogenic process that forms new blood vessels. It is a rare event in healthy individuals and is a tightly regulated essential process involved in the normal growth and wound healing. However, in a variety of pathological states such as solid tumor growth, psoriasis, diabetic retinopathy, and ophthalmic diseases, it becomes an uncontrolled process [75]. The growth of solid tumor relies heavily on the formation of new blood vessels to supply nutrients to the tumor. Therefore, angiogenesis inhibitors can potentially be used as antitumor agents. One such compound is fumagillin, a well-known fungal metabolite [76] whose antiangiogenesis activity was discovered serendipitously in Folkman's lab when the producing organism, Aspergillus fumigatus, contaminated the endothelial cell culture in his lab [130]. The antiangiogenesis activity of fumagillin was identified at Tekeda in the late 1980s. Another natural product, ovalicin, structurally similar to, but more stable than fumagillin, exhibited similar bioactivity, and was synthesized enantioselectively by Corey's group[128]. There are several in vivo assay models for angiogenesis, including the shell-less chicken chorioallantoic membrane (CAM) assay, the cornea assay, and the endothelial cell assay. The CAM assay is most frequently used assay method because it is easy to use and less costly. In this assay, an inhibitor's effect on the shell-less chicken embryo culture can be observed directly, although quantitating the result is subjective. The mouse cornea assay is widely favored; because the cornea is an avascular tissue, any blood vessel development unequivocally demonstrates the lack of antiangiogensis activity [77-79]. However, all of these in vivo assay
ZIII-DONG JIANG AND ZHIQIANG AN
262
models share a common shortcoming" their low throughput is a major impediment to large-scale screening efforts. Fumagillin is a potent angiogenesis inhibitor that inhibits endothelial cell proliferation in vitro, ncovasculation in CAM assay, and tumor angiogenesis in the mouse [80]. At l0 ug, it exhibited 75% inhibition of blood vessel formation in the CAM assay [81-82]. In the late 1980s, Takeda Chemical Industries undertook synthetic effort to discover analogues of fumagillin that were more stable and more potent. As a result, AGM-1470 (formerly TNP-470) was synthesized to inhibit in vitro the proliferation of human umbilical vein endothelial cells at 50 times the potency of fumagillin. AGM-1470 reduces the growth rate of Lewis Lung carcinoma and B 16 melanoma in mice. In 1997, the molecular target of AGM-1470 and ovalicin was elucidated by Griffith and Su to be the bifunctional enzyme methioninc aminopcptidasc (type 2) (MctAP2) [83]. AGM-1470 potently and specifically inhibits MctAP2 with ICs0 values of 1.0 nM by covalcntly binding to the protein. AGM-1470 neither inhibits the type 1 methionine aminopeptidase (MetAP1) nor affects the other function of MctAP2, inhibition of elF-2oc phosphorylation. This finding suggests that MetAP2 may play an important role in the proliferation of endothelial cells and therefore may become a highthroughput screening target for new antiangiogenic agents.
O CH3
0 -CH3
!
"../H 9
-
1 .,~
_
"~ OH 0
O Fumagfllln
0
AGM-1470 -0 .CH3
y
"~ 0 Ovalicin
BIOACTIVE FUNGAL NATURAL PRODUCTS
263
Tapping Fungal Diversity for Drug Discovery Using Genetic Engineering Even though fungi are a proven source of drugs, they have been barely explored for drug discovery. Of approximately 1.5 million species, to date only about 70,000 species have been described. Of the 70,000 described species, only a small fraction has been screened for drug discovery; fungi that have been selected for drug screening are usually those that can be easily isolated from nature and easily cultured and maintained in laboratory media. However, culturable fungi constitute only a small portion of the fungal world. In contrast, unculturable and slow-growing fungi have received little attention for drug discovery due to technical limitations of studying them in the laboratory. No doubt many new agents will continue to be discovered from culturable fungi, but there is a clear need to expand the potential fungal drug source to include unculturable organisms. It is certain that novel metabolites exist in tmculturable and slow-growing fimgi. Since fungal secondary metabolites are encoded by genes, these genes can be cloned from unculturable fungi and introduced into fast-growing heterologous hosts. The heterologous hosts will then express the secondary metabolite-encoding genes from unculturable fungi to synthesize novel secondary metabolites. Recent studies have shown that genes involved in secondary metabolite biosynthesis in microorganisms are often clustered. For example, the genes involved in the biosynthesis of 13lactam antibiotics from three different eukaryotic organisms (Penicillium chrysogenum, Cephalosporium acremonium, A. nidulans) and two prokaryotes (Nocardia lactamdurans, Streptomyces clavuligerus) are all clustered. The three genes required for melanin biosynthesis of the filamentous fungus Alternaria alternata are clustered within a 30 kb fragment of genomic DNA. The genes required for the production of the polyketide antibiotics frenolin and nanaomycins by Streptomyces roseofulvus are clustered within a 10 kb DNA fragment. The entire set of actinorhodin biosynthetic genes from Streptomyces coelicolor is clustered within a 26 kb DNA fragment. More recently, genes involving sterigmatocystin biosynthesis in A. nidulans were defined within a 60 kb DNA fragment. The cyclic peptide siderophore biosynthetic genes in Ustilago maydis are also clustered (Sally Leong, personal communication). Based on this evidence, it is possible that a gene cluster for a particular secondary metabolite will be included when large pieces of DNA from unculturable fungi, as exemplified by cosmid clones (35-45 kb), yeast artificial chromosome clones (> 100 kb), and bacterial artificial chromosome clones (> 100 kb), are introduced into a recipient laboratory strain. The recipient strains for expressing genes from unculturable fungi should be easy to manipulate genetically; numerous fungi can serve as
264
ZHI-DONG JIANG AND ZHIQIANG AN
recipient hosts. The availability of multiple-recipient laboratory strains increases the chance for heterologous expression of foreign genes. One of the assumptions underlying this genetic approach is that genes or clusters of genes from unculturable fungi can be expressed in laboratory strains because transcription and translation control sequences from one organism often function in closely related organisms, and sometimes even in distantly related organisms [97,120]. For example, the fungal transformation vector pAN7-1 [118], which has the Escherichia coli hygromycin phosphotransferase gene (hph)under the control of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter and trpC terminator, efficiently confers hygromycin B resistance to a variety of fungal species. More than 40 fungal species have been transformed with this vector, ranging from the basidiomycetes Schizophyllum commune [115] and Laccaria laccata [84] to various ascomycetous species [97]. The A. niger glucoamylase gene promoter functioning in U. maydis [121] is another example of an ascomycete promoter that functions in a basidiomycete. Even genes from plants have been shown to be transcribed in Saccharomyces cerevisiae. An example of this is the maize storage protein zein gene [108]. A few Aspergillus genes have been isolated by complementation of S. cerevisiae mutant strains [119]. All this evidence suggests that genes of foreign origin may be expressed in laboratory strains. However, one must select a donorrecipient combination cautiously; not all transcription control sequences from one fungus function in other fungal hosts [ 121 ]. Several strategies, such as mRNA analysis, reporter gene analysis, and complementation of auxotrophic markers, can be applied to test gene expression. In summary, by exploring genetic diversity from unculturable fungi and by creating biocombinatorial diversity, we improve the likelihood of discovering novel secondary metabolites. Because the recipient strains are fast-growing, industrial organisms, once novel-drug producing transformants are identified, scale-up fermentation for commercial production can be quickly implemented.
Synthesizing Unnatural Natural Products Using Biocombinatoriai Approaches Recent advances in molecular genetics of secondary metabolite biosynthesis have made it possible to develop a whole new concept of drug discovery known as biocombinatorial production of synthetic natural products. The promise of this genetic engineering/chemistry hybrid approach for developing novel drugs and recent progress in the combinatorial biosynthesis of novel bacterial polyketides have led scientists to explore the potential of novel biocombinatorial fungal secondary metabolites as therapeutic agents. The use of fungi as sources of natural genes and as hosts for expressing engineered chimeric genes is
BIOACTIVE FUNGAL NATURAL PRODUCTS
265
especially timely. Until now, most work in this area has focused on bacteria, yet fungi are known to be prolific producers of biologically active secondary metabolites. The pathways of several classes of fungal secondary metabolites, such as polyketides and non-ribosomal peptides, are suited for biocombinatorial manipulations. Here we use the genetic engineering of bacterial polyketides as an example to illustrate the concept and challenges of this approach. We will also speculate the advantage and potential of engineering fungal polyketides and other secondary metabolites. Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and 13-ketoaeyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of I]-carbons, e.g., ~-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. Aromatic PKSs (or type II) are composed of several separate, largely monofunctional proteins, whose active sites are used iteratively for the assembly and functional-group manipulation of the polyketide chain. At least 13 different sets of aromatic polyketide PKSs have been cloned from Streptomyces and Saccharopolyspora species [100]. Best studied among all PKSs is the PKS for the benzoisochromanequinone antibiotic actinorhodin [109, 113, 114]. Genes for actinorhodin biosynthesis are designated actI-VIl. ActI encodes 3 different active sites: KS, AT, and chain-length-determining factor (CLF). ActllI encodes KR. ActI and actlII constitute the minimum PKS. Actll is responsible for transcriptional regulation of the act genes and for actinorhodin export. ActlV-VII encode several post-synthetic modifying functions, e.g. cyclization (VII), aromatization, and subsequent chemical tailoring. Other aromatic PKSs share the same basic architecture with minor structural differences [98, 100]. Molecular genetic analysis of PKS genes has confirmed earlier biochemical and chemical findings that the structural diversity of
266
ZHI-DONG JIANG AND ZHIQIANG AN
polyketides is a result of the different numbers and types of acyl units involved [109]. This body of research also supports the idea that novel polyketides can be produced by manipulating the sequence and specificity of enzyme-mediated reduction, dehydration, cyclization, and aromatization [89, 95, 99-101, 104-106, 112-114, 117, 126]. The era of rational design of novel antibiotic structures was ushered in by early successes synthesizing complex polyketides. The modular PKSs for complex polyketides contain a unique active site for each enzymecatalyzed reaction in the pathway, giving rise to final structures that are determined by the numbers and types of active sites. Donadio et al. demonstrated in the early 1990s that analogues of the erythromycin polyketide backbone could be generated by eliminating active sites within the PKS [90-92]. By repositioning a chain-terminating cyclase domain from the C-terminus of module 6 of DEBS3 to the C-terminus of module 2 of DEBS 1, Cortes et al. were able to construct a multienzyme unit that catalyzed only the first two rounds of polyketide chain extension [89]. The mutant produced a triketide lactone structure without any trace of erythromycin, the wild-type polyketide, indicating premature chain termination and cyclization. By expressing the entire DEBS gene cluster in a heterologous host, substantial quantities of 6-deoxyerythronolide B, the aglycone of the macrolide antibiotic erythromycin, were produced [101]. In contrast, aromatic PKSs contain a single set of iteratively used active sites, although basic organization of the two types of PKS is similar. Their characteristics make it difficult to predict the structure of the polyketide that will be produced after a PKS gene is modified. Great advances in research on the combinatorial biosynthesis of novel aromatic polyketides have been made by Khosla, Hopwood, and their collaborators. Hybrid aromatic polyketides have been generated by transferring partial or complete biosynthetic gene clusters between different polyketide producers and screening for new structures. For example, mederrhodin A, a novel aromatic polyketide, was made by a medermycin-producing Streptomyces strain transformed with the actVA gene, that strain normally produces only native mederrhodin [99]. This approach of generating novel polyketides was largely empirical, and required the activity of enzymes for late tailoring steps. During the last few years, tremendous progress has been made in the understanding of genetic programming of aromatic PKSs; consequently, the rational design of novel aromatic polyketides is also advancing. Several novel compounds have been generated by constructing and expressing recombinant PKSs in Streptomyces sp. [95, 106, 112-114]. Based on their work and the work of others, McDaniel et al. summarized 6 strategies for rational design of novel aromatic polyketides. They are chain length, ketoreduction, cyclization of the first ring, first ring aromatization, second ring cyclization, and additional cyclization.
BIOACTIVE FUNGAL NATURAL PRODUCTS
267
Fungi, filamentous fungi in particular, are an especially rich source of polyketides [ 116]. To date, nucleotide sequences have been reported for 7 fungal PKS genes, all of which encode type I PKSs. The C. heterostrophus PKS1 gene is responsible for the biosynthesis of T-toxin [124]. The MSAS1 gene ofPenicillium urticae [123] and P. patulum [85] encodes 6-methylsalicylic acid synthase (MSAS), which catalyzes synthesis of the tetraketide 6-methylsalicylic acid, the precursor of a mycotoxin, wA gene ofAspergillus nidulans encodes a PKS of 1,986 amino acids, whose product is a component of green pigment; pksA of A. parasiticus encodes a polypeptide required for the production of a yellow pigment and for biosynthesis of aflatoxin B-l, a potent carcinogen [88]; pksL1 of A. parasiticus encodes 2,109 amino acids and is required for production of several related aflatoxins and their biosynthetic precursors [93]; pksST ofA. nidulans encodes a polypeptide of 2,181 amino acids required for the production of the polyketide-derived mycotoxin sterigmatocystin [125]; PKS1 of Colletotrichum lagenarium encodes a polypeptide of 2,187 amino acids required for production of the pentaketide precursor of melanin [ 122]. Similar principles and roles for genetic engineering of bacterial polyketides could be applied to combinatorial synthesis of fungal polyketides and other fungal secondary metabolites, such as nonribosomal peptides, but specific protocols need to be developed for fungi since fungal gene regulation and structure are very different from bacteria. For example, fungal genes contain nontranslated DNA known as introns. The concept of making new metabolites by chimeric genes is rather simple and obvious, but the challenge of constructing a number of chimeric gene clusters is great. This approach, though very promising, has yet to be proven practically. CONCLUSION As the pharmaceutical and agricultural industries continue their endeavor to find small molecular agents for a variety of diseases, natural products have become an important source of bioactive compounds. Many natural products have been used directly or have provided structural templates for synthetic analogues for these diseases. Economically, natural products and their related compounds account for a significant portion of worldwide pharmaceutical sales. Among the billion-dollar drugs for 1995, approximately 40% were small molecule natural products or drugs that contained structural motifs of natural products. Recent advances in molecular biology and genetics and their application to biocombinatorial synthetic natural prodcuts will generate unprecedented novel natural products. New separation and detection technologies will accelerate the process of discovering drugs from natural products. With the help of these novel approaches, natural products will play a even greater role as a
268
ZHI-DONG JIANG AND ZHIQIANG AN
source chemical diversity for the pharmaceutical and agricultural industries. ACKNOWLEDGEMENT
The authors gratefully acknowledge Ms. Louise Gachet for editing this manuscript. REFERENCES
[1] [z]
Cragg, G.M.; Newman, D.J.; Snader, K.M.J. Nat. Prod., 1997, 60, 52. Beese, K. In Phytochemical Diversity, Wrigley, S.; Hayes, M.; Thomas, R.; Chrystal, E. Ed.; The Royal Society of Chemistry: Cambridge, UK, 1997, pp. 129-140. [3] (a) Frisvad, J.C.; Thrane, U. In Chromatography of Mycotoxins, Betina, V. Ed.; Elsevier: Amsterdam, 1993, pp. 253. (b) Jong, S.C.; Gantt, M.J. ,4 TCC Catalogue of Fungi~Yeasts, 17th ed. With an index to the industrial applications. American Type Culture Collection, Rockville, MD, 1987. [4] (a) Jong, S.C.; Donovick, R. Adv. Appl. Microbiol., 1989, 34, 183. (b) Gloer, J. In Developments in Industrial Microbiology Series, Gullo, V.P.; HunterCevera, J.C.; Cooper, R.; Johnson, R. Ed.; Society of Industrial Microbiology: 1993, Vol. 33, pp. 1-12. [5] (a) Demain, A.L. Ciba Found. Symp., 1992, 171, 3. (b) Cole, R. J.; Cox, R. H. Handbook of Toxic Fungal Metabolites, Academic Press: New York, 1981. [6] Hawksworth, D. L.; Rossman, A. Y. Phytopathology, 1997, 87, 888. [7] Kelly, S.L,; Lamb, D.C.; Kelly, D.E.; Manning, N.J.; Loeffler, J.; Hebart, H.; Schumacher, U.; Einsele, H. FEBS Lett., 1997, 400, 80. [8] Nolte, F.S.; Parkinson, T.; Falconer, D.J.; Dix, S.; Williams, J.; Gilmore, C.; Geller, R.; Wingard, J.R. Antimicrob. Agents Chemother., 1997, 41, 196. [9] Sandven, P.; Nilsen, K.; Digranes, A.; Tjade, T; Lassen, J. Antimicrob. Agents Chemother., 1997, 41, 1375. [10] Mizoguchi, J.; Saito, T.; Mizuno, K.; Hayano, K. J. Antibiotics, 1977, 30, 308. [ll] Baguley, B.C.; Rommele, G.; Gruner, J.; Wehrl, W. Eur. J. Biochem., 1970, 97, 345. [12] James, P.G.; Cherniak, R.; Jones, R.G.; Stortz, C.A.; Reiss, E. Carbohydr. Res., 1990, 198, 23. [13] Takesako, K.; Ikai, K.; Haruna, F.; Endo, M.; Shimanaka, K.; Sono, E.; Nakamura, T.; Kato, I. Yamaguchi, H. J. Antibiotics, 1991, 44, 919. [141 Yoshikawa, Y.; Ikai, K.; Umeda. Y.; Ogawa, A.; Takesako, K.; Kato, I.; Naganawa, H. J. Antibiotics, 1993, 46, 1347. [15] Takesako, K.; Kuroda, H.; Inoue, T.; Haruna, F.; Yoshikawa, Y.; Kato, I.; Uchida, K.; Hiratani, T.; Yamaguchi, H. J. Antibiotics, 1993, 46, 1414. [16] Nagiec M.M.; Nagiec E.E.; Baltisberger, J.A.; Wells, G.B.; Lester, R.L.; Dickson, R.C.J. Biol. Chem., 1997, 272, 9809. [17] Musilek, V. British Pat. 1965, GB 1163910.
BIOACTIVE FUNGAL NATURAL PRODUCTS
[18] [19]
[20] [El]
[22]
[23] [24]
[25]
[26] [27] [28] [29]
[3o] [31] [32] [33]
[34] [35] [36] [37] [38]
[39] [40]
[41]
[42]
[43] [44] [45] [46]
269
Vondracek, M.; Capkova, J.; Slechta, J.; Benda, A.; Musilel, V.; Cudlin, J. British Pat., 1967, GB 1181892. Bauerle, J.; Anke, T. Planta Med., 1980, 39, 195. Anke, T.; Besl, H.; Mocek, U.; Steglich, W. J. Antibiotics, 1983, 36, 661. Becker, W.F.; yon Jagow, G.; Anke, T.; Steglich, W.; FEBS Lett., 1981, 132,
329. Von Jagow, G.; Becker, W.F. Bull. Mol. Biol. Med. 1982, 7, !. Von Jagow, G.; Gribble G.W.; Trumpower, B.L. Biochemistry 1986, 25, 77578O. Kraiczy, P.; Haase, U.; Gencic, S.; Flindt, S.; Anke, T.; Brandt, U.; Von Jagow, G. Eur. J. Biochem., 1996, 235, 54. Manson, J.E.; Tosteson, P.H.H.; Rider, P.M.N. Eng. J. Med., 1992, 21, 1406. Endo, A.; Hasumi, K. Nat. Prod. Report, 1993, 541. Endo, A.; Kuroda, M.; Tsujita, Y. J. Antibiotics, 1976, 29, 1346. Endo, A.; Kuroda, M.; Tanzawa, K. FEBS Lett., 1976, 72, 323. Juzlova, P.; Martinkova, L.; Kren, V. J. Industrial Microbiol., 1996, 16, 163. Endo, A.; J. Antiobiotics, 1979, 32, 852. Alberts, A.W.; Chen, J.; Kuron, G. Proc. Natl. ,4cad. Sci. USA, 1980, 77, 3957. Serizawa, N.; Serizawa, S.; Nakagawa, K.; Furuya, K.; Okazaki, T.; Terahara, A. J. Antibiotics, 1983, 36, 887. Sliskovic, D.R.; Roth, B.D. In Biological Inhibitors, Choudhary, M.I. Ed.; Harwood Academic Publishers: 1996, pp.87-160. Moore, R.N.; Bigam, G.; Chan, J.K.; Hogg, A.M.J. Am. Chem. Soc., 1985, 107, 3694. Scherer, F. Pharmaceutical R & D: Costs, Risks and Rewards, Government Printing Office: 1994, pp. 74-75. O'Reilly, B. Fortune, 1997, October 13. Bertolini, S.; Bon, G.B.; Campbell, L.M.; Famier, M.; Langan, J.; Mahla, G.; Pauciullo, P.; Sirtori, C.; Egros, F.; Fayyad, R.; Nawrocki, J.W. Atherosclerosis, 1997, 130, 191. Dart, A.; Jerums, G.; Nicholson, G.; d'Emden, M.; Hamilton-Craig, I.; Tallis, G.; Best, J.; West, M.; Sullivan, D.; Braes, P.; Black, D.; Am. J. Cardiol. 1997, 80, 39. Alaupovic, P. Heinonen, T.;I Shurzinske, L. Black, D.M. Aeherosclerosis, 1997, 133, 123. B W Health Wire, October, 23, 1997. Abe, I.; Tomesch, J.C.; Wattanasin, S.; Prestwich, G.D. Nat. Prod. Reports, 1994, 279. Dawson, M.J.; Farthing, J.E.; Marshall, P.S.; Middleton, R.F.; O'Neill, M.J.; Shuttleworth, A.; Stylli, C.; Tait, R.M.; Taylor, P.M.; Wildman, H.G.; Buss, A.D.; Langley, D.; Hayes, M.V.J. Antibiotics, 1992, 45, 639. Bergstrom, J.D. et al, Proc. Natl. Acad. Sci. U S d, 1993, 90, 80. Tanimoto, T.; Hamano, K.; Onodera, K.; Hosoya, T.; Kakusaka, M.; Hirayama, T.; Shimada, Y.; Koga, T.; Tsujita, Y. J. Antibiotics, 1997, 50, 390. Chen, T.S.; Petuch, B.; MacConnell, J.; White, R.; Dezeny, G.; Arison, B.; Bergstrom, J.D.; Colwell, L.; Huang, L.; Monaghan, R.L.J. Antibiotics, 1994, 47, 1290. Carol, A.J.; Sidebottom, P.J.; Cannell, R.J.P.; Noble, D.; Rudd, B.A.M., J. Antibiotics, 1992, 45, 1492.
270
[47] [48]
[49] [50] [51] [521
[531 [541 [551 [56] [57]
[58] [59]
[60] [61] [62] [63]
[64] [65] [66] [67] [68] [69] [70] [711 [72] [73] [74] [75] [76]
ZHI-DONG JIANG AND ZHIQIANG AN
Cannel, R.J.P.; Dawson, M.J.; Hale, R.S.; Hall, R.M.; Noble, D.; Lynn, S.; Taylor, N.L.d. Antibiotics, 1994, 47, 247. Tanimoto, T.; Hamano, K.; Onodera, K.; Hosoya, T.; Kakusaka, M.; Hirayama, T.; Shimada, Y.; Koga, T.; Tsujita, Y.J. Antibiotics, 1997, 50, 390. Qian, Y.; Sebti, S.M.; Hamilton, A.D. Biopolymers, 1997, 43, 25. Ruegger, A.; Kuhn, M.; Lichti, H.; Loosli, H.-R.; Huguenin, R.; Quiquerez, C.; Von Wartburg, A. Helv. Chim. Acta, 1976, 59, 1075. Rahfeld, J.-U.; Schierhom, A.; Mann, K.; Fisher, G. FEBS Lett., 1994, 343, 65. Wiederrecht, G.; Brizuela, L.; Elliston, D.; Sigal, N.; Siekierka, J. Proc. Acad. Sci. USA, 1991, 88, 1029. Tropschug, M.; Barthelmess, I.; Neupert, W. Nature, 1989, 342, 953. Liu, J.F.; Lane, J.D.; Friedman, J.; Weissman, I.; Schreiber, S.L.; Cell, 1991, 66, 807. Liu, J.; Albers, M.; Wandless, T.J.; Luan, S.; Alberg, D.A.; Belshaw, P.J.; Cohen, P.; MacKintosh, C.; Klee, C.B.; Sehreiber, S.L. Biochem., 1992, 31, 3896. Brown, E.J.; Albers, M.W.; Shin, T.B.; Ichickawa, K.; Keith, C.T.; Lane, W.S.; Schreiber, S.L. Nature, 1994, 369, 756. Go sio, B. Rivista Igiene Sanita Publica Ann., 1896, 7, 825. Noto, T.; Sawada, M.; Ando, K.; Koyama, K. d. Antibiotics, 1969, 22, 165. Ando, K.; Suzuki, S.; Tamura, G.; Arima, K. J. Antibiotics, 1968, 21, 649. Sweeney, M.J.; Gerzon, K.; Harris, P.N.; Holmes, R.E.; Poore, G.A.; Williams, R.H. Cancer Research,1972, 32, 1795. Sweeney, M.J.; Hoffman, D.H.; Esterman, M.A. Cancer Research, 1972, 32, 1803. Nelson, P.H.; Gu, C-L.L.; Allison, A.G.; Eugui, E.M.; Lee, W.A. US Patent 4,753,935, 1985. Nakanishi, K. Nature, 1963, 197, 292. McMorris, T.C.; Anchel, M.J.d. Am. Chem. Soc., 1963, 85, 831. Nakanishi, K.; Ohashi, M; Tada, M.; Yamada, Y. Tetrahedron, 1965, 21. 123 !. Maretic, Z.; Russell, F.; Golobic, V. Toxicon, 1975, 13, 379. French, A..L.; Garrettson, L.K. Clin. Toxic., 1988, 26, 81. McMorris, T.C.; Kelner, M.J.; Wang, W.; Moon, S.; Tactic, R. Chem. Res. Toxicol. 1990, 3, 574. Tanaka, K.; lnoue, T.; Tezuka, Y.; Kikuchi, T. Chem. Pharm. Bull., 1996, 44, 273. McMorris, T.C.; Kelner, M.J.; Wang, W.; Estes, L.A.; Montaya, M.A.; Taetle, R.J. Org. Chem., 1992, 5 7, 6876. Kelner, M.J.; McMorris, T.C.; Beck, W.T.; Zamora, J.M.; Tactic, R. Cancer Res., 1987, 47, 3186. MacDonald, J.R.; Muscoplat, C.C.; Dexter, D.L.; Mangold, G.L.; Chen, S.F.; Kelner, M.J.; McMorris, T.C.; Von Hoff, D.D. Cancer Res., 1997, 57, 279). McMorris, T.C.; Kelner, M.J.; Wang, W.; Yu, J.; Estes, L.A.; Taetle, R. d. Nat. Prod, 1996, 59, 896. McMorris, T.C.;Kelner, M.J.; Wang, W.; Diaz, M.A.; Estes, L.A.; Taetle, R. Experientia, 1996, 52, 75. Folkman, J. Adv. Cancer Res., 1985, 43, 175) (Folkman, J.; Klagsbrun, M. Science, 1987, 235, 442. Tarbell, D.S.; Carman, R.M.; Chapman, D.D.; Huffman, K.R.; McCorkindale, N.J.d. Am. Chem. Soc., 1960, 82, 1005.
BIOACTIVE FUNGALNATURALPRODUCTS
[77]
[78] [79]
[80] [81] [82] [83] [84]
[85] [86]
[871 [88] [89] [90] [91]
[92] [93] [94] [95] [96]
[97]
[98] [991
[100] [~0~] [~021 [103] [104] [105]
271
Langer. L.; Conn, H.; Vancanti, J., Maudenschild, C.; Folkman, J.; Proc. Natl. Acad. Sci., USA,, 1980, 77, 4331. lngber, D.E.; Folkman, J. J. Cel. Biol., 1989, 109, 317. Zimrin, A.B.; Villeponteau, B.; Maciag, T. Biochem. Biophys. Res. Commun. 1995, 213, 630. Ingber, D.E.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.; Breem, H.; Folkman, J. Nature, 1990, 348, 555. Folkman, J.; Fujita, T.; Ingber, D.; Kanamaru, T. U.S. Patent, 1992, 5135915. lngber, D.E.; Folkman, J. d. Cel. Biol., 1989, 109, 317. Griffith, E.C.; Su, Z.; Turk, B.E.; Chen, S.; Chang, Y.; Wu, Z.; Biemann, K.; Liu, J.O. Chem. Biol., 1997, 4, 461. Barrett, V.; Dixon, R. K.; Lemke, P. A. Appl. Microbiol. Biotechnol., 1990, 33, 313. Beck, J.; Ripka, S.; Siegner, A.; Schiltz, E.; Schweizer, E. Eur. J. Biochem., 1990, 192, 487. Bibb, M. J.; Sherman, D. H.; Omura, S.; Hopwood, D. A. Gene, 1994, 142, 31. Brown, D. W.; Yu, J.-H.; Kelkar, H. S.; Fernandes, M.; Nesbitt, T. C.; Keller, N. P.; Adams, T. H.; Leonard, T. J. Proc. Natl. Acad. Sci. USA, 1996, 93, 1418. Chang, P. K.; Cary, J. W.; Yu, J.; Bhatnager, D.; Cleveland, T. E. Mol. Gen. Genet. 1995, 248, 270. Cortes, J.; Wiesmann, K. E. H.; Roberts, G. A.; Brown, M. J. B.; Staunton, J.; Leadlay, P. F. Science, 1995, 268, 1487. Donadio, S.; Katz, L. Gene, 1992, 111, 51. Donadio, S.; MeAlpine, J. B.; Sheldon, P. A.; Jackson, M. A.; Katz, L. Proc. Natl. Acad. Sci. USA, 1993, 90, 7119. Donadio, S.; Staver, M. J.; McAlpine, J. B.; Swanson, S. J.; Katz, L. Science, 1991, 252, 675. Feng, G. H.; Leonard, T. J. J. Bacteriol., 1995, 177, 6246. Fernandez-Moreno, M. A.; Martinez, E.; Boto, L.; Hopwood, D. A.; Malpartida, F. d. Biol. Chem., 1992, 267, 19278. Fu, H.; McDaniel, R.; Hopwood, D. A.; Khosla, C. Biochemistry, 1994, 33, 9321. Hawksworth, D. L.; Rossman, A. Y. Phytopathology, 1997, 87, 888. Hondel, C. A. M. J. J. v. d.; Punt, P. J.; Gorcom, R. F. M. V. Heterologous gene expression in filamentous fungi. In More gene manipulations in fungi, J. W. Bennett, and L. L. Lasure, Eds.; Academic Press, Inc.: San Diego, 1991, pp. 396-428. Hopwood, D. A.; Khosla, C. In Secondary metabolites: their function and evolution, C. F. Symposium, Ed.; Chichester: Wiley, 1992, Vol. 17, pp. 88112. Hopwood, D. A.; Malpartida, F.; Kieser, H. M.; Ikeda, H.; Duncan, J.; Fujii, I.; Rudd, B. A. M.; Floss, H. G.; Omura, S. Nature, 1985, 314, 642. Hutchinson, C. R.; Fujii, I. Annu. Rev. Microbiol.,1995, 49, 201. Kao, C. M.; Katz, L.; Khosla, C. Science, 1994, 265, 509. Katz, L.; Donadio, S. dnnu. Rev. Microbiol., 1993, 47, 875. Keller, N. P.; Adams, T. H. SAAS Bull Biochem. Biotechnol., 1995, 8, 14. Khosla, C.; Ebert-Khosla, S.; Hopwood, D. A. Mol. Microbiol., 1992, 6, 3237. Khosla, C.; McDaniel, R.; Ebert-Khosla, S.; Torres, R.; Sherman, D. H.; Bibb, M. J.; Hopwood, D. A. d. Bacteriol., 1993, 175, 2197.
ZHI-DONG JIANG AND ZHIQIANG AN
272
[1o6] Kim, E.; Cramer, K. D.; Shreve, A. L.; Sherman, D. H. J. Bacteriol., 1995, 177, 1202.
[107] Kimura, N.; Tsuge, T. Journal of Bacteriology, 1993, 175, 4427. [los] Langridge, R.; Eibel, H.; Brown, J. W. S.; Feix, G. The EMBO Journal, 1984, [109] [110] [Ill] [112] [ll3] [114]
IllS] [116]
[117] [118] [119] [120] [121] [1221 [123] [124] [125] [126] [1271 [1281 [1291 [130] [131] [1321 [133]
3, 2467. Malpartida, F.; Hopwood, D. A. Nature, 1984, 309, 462. Martin, J. F. J. Ind. Microbiol., 1992, 9, 73. Mayorga, M. E.; Timber lake, W. E. Mol. Gen. Genet., 1992, 235, 205. McDaniel, R.; Ebert-Khosla, S.; Fu, H.; Hopwood, D. A.; Khosla, C. Proc. Natl. Acad. Sci. USA, 1994, 91, 11542. McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. Science, 1993, 262, 1546. McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. Nature, 1995, 375, 549. Mooibroek, H.; Kuipers, A. G. J.; Sietsma, J. H.; Punt, P. J.; Wessr J. G. H. Mol. Gen. Genet., 1990, 222, 4 I. O'Hagan, D. The polyketide metabolites, J. Mellor, Ed.; Ellis Horwood: Chichester, 1991. Pieper, R.; Luo, G.; Cane, D. E.; Khosla, C. Nature, 1995, 378, 263. Punt, P. J.; Oliver, R. P.; Dingemanse, M. A.; Pouwr P. H.; Vanden Hondel, C. A. M. J. J. Gene, 1987, 56, 117. Rambosek, J.; Leach, J. CRC Crit. Rev. Biotech., 1987, 6, 357. Romanos, M. A.; Scorer, C. A.; Clare, J. J. Yeast, 1992, 8, 423. Smith, T. L.; Gaskell, J.; Berka, R. M.; Yang, M.; Henner, D. J.; Cullen, D. Gene, 1990, 88, 259. Takano, Y.; Kubo, Y.; Shimizu, K.; Mise, K.; Okuno, T.; Furusawa, I. Mol. Gen. Genet., 1995, 249, 162. Wang, I.; Reeves, C.; Gaucher, G. M. Can. J. Microbiol., 1991, 37, 86. Yang, G.; Rose, M. S.; Turgeon, B. G.; Yoder, O. C. Plant Cell., 1996, 8, 2139. Yu, J.; Leonard, T. J. J. Bacteriol., 1995, 177, 4792. Yu, T.; Hopwood, D. A. Microbiology, 1995, 141, 2779. Krishnarao, T.V.; Galgiani, J.N. Antimicrob. Agents Chemther., 1997, 41, 1957. Coey, E.J.; Guzman-Perez, A.; Noe, M.C.J. Am. Chem. Soc., 1994, 116, 12109. Harrison, W.; Shearer, H.M.M.; Trotter, J. J. Chem. Soc. Perkin II, 1972, 1542. Ingber, D.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.; Brem, H.; Folkman, J. Nature, 1990, 348, 555. Borel. J. F. Pharmacol. Rev., 1990, 41, 259. Thomson, A.W.; Whiting, P.H.; Simpson, J.G. Agents Actions, 1984, 15, 306. Pearce, C. Adv. Appl. Microbiol., 1997, 44, 1.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22 9 2000 ElsevierScience B.V. All rights reserved
273
THE CHEMISTRY OF 2-AMINOCYCLOPENTANECARBOXYLIC ee
ACID
ee
FERENC FULOP
Institute of Pharmaceutical Chemistry, Albert Szent-GyOrgyi Medical University, H-6701 Szeged, POB 121, Hungary, ABSTRACT: The syntheses, transformations and some of the biological features of 2aminocyclopentaneearboxylie acid are reviewed. The (IS,2R) enantiomer (cispentaein) was recently isolated from different natural sources, c i s - 2 - A m i n o e y c l o p e n t a c e e a r b o x y l i c acid is also a component of the antibiotic amipurimyein. The paper discusses the syntheses of the racemic compounds, resolutions of the racemates and enetioselective syntheses of the title compounds. The transformations to heteroeyelic compounds, applications in peptide syntheses, and biological effects are reviewed.
INTRODUCTION [~-Amino acids, although of less importance than their o~-analogues, are also present in peptides and different heterocycles, and their free forms and derivatives exhibit interesting pharmacological effects [ 1]. A number of syntheses and transformations have been performed on their stereoisomeric alicyclic analogues (e.g. 1-3). Until recently, the investigations were mainly of academic interest since no naturallyoccurring compounds were known. Among the 13-amino acid derivatives of cycloalkanes, one of the most exciting is (1R,2S)-2-aminocyclopentanecarboxylic acid (cispentacin), an antifungal antibiotic, recently isolated independently by two Japanese groups from Bacillus cereus [2] and Streptomyces setonii [3]. cis-2-Aminocyclopentanecarboxylic acid (cis-2-ACPC) is a component of the antibiotic amipurimycin reported by Goto et al. Amipurimycin (4) contains a nucleic base attached to the anomeric carbon of a branched-chain deoxy sugar. The chain is extended by a dipeptide containing the cis-2ACPC moiety. Amipurimycin, isolated from S t r e p t o m y c e s novoguineensis, is strongly active both in vitro and in vivo against Pyricularia oryzae, responsible for rice blast disease. It is also active in vitro against Alternaria kikuchiana and Helminthosporium sigmoideum var. irregulare [4-13]. Although the absolute configuration of the cis-2ACPC moiety has not been determined, it is probably similar to that of naturally-occurring cispentacin.
274
FERENC FIDLOP
~iOOH 1
~ vC
H~.
- NO H20 H
2
COOH
~ 8
NH2
oon NH
H O_
H0
H~O H
N~
NH2
4
Chart 1.
Besides the pharmacological importance of the alicyclic I]-amino acids, they can be used as building blocks for the preparation of modified (unnatural) analogues of biologically active peptides. By insertion of an alicyclic 13-amino acid in place of an t~-amino acid of a naturally-occurring pharmacologically active peptide, the activity or the effect can be modified. By means of such an exchange, the stability of the natural peptides can be increased. The difference in the ring size allows modification of the conformations of the peptides. Such investigations are applied for determination of the fine structures of receptors. Due to the natural occurrence and the novel biological activity, interest in investigations of alicyclic 13-amino acids has been aroused. A number of new enantioselective syntheses have been developed and protected by patents [ 14-26]. The writing of this review was prompted by the renewed interest in the title compound 1. The primary focus was its synthesis and some of its transformations. Besides 1, other alicyclic [3-amino acids, such as cis- and trans-2-aminocyclohexanecarboxylic acid (2), 2,3-diendo- and 2,3-diexo-3-aminobicyclo[2.2.1 ]heptane-2-carboxylic acid (3) and some of their partially unsaturated analogues and derivatives will be mentioned. The biological properties of these and related compounds will also be discussed. CISPENTACIN. ISOLATION AND CHARACTERIZATION In 1989-90 two Japanese research groups independently isolated a simple, unique [3-amino acid having the chemical structure (-)-(1R,2S)-2-ACPC (5). In the course of antifungal screening, cispentacin was isolated from the culture broth of a Bacillus cereus strain by Oki et al. [2, 27]. In parallel with that investigation, Hashimoto et al. isolated an identical substance
2-AMINOCYCLOPENTANECARBOXYLIC ACID
275
from Streptomyces setonii No.7562 [3, 28]. The isolation and purification were in both cases based on ion-exchange chromatography. C,O O H
Chart 2.
S
NH2
Cispentacin has an amphoteric character; it is readily soluble in water, slightly soluble in methanol, and insoluble in acetone or ethyl acetate. It gives a positive ninhydrin test. Its accurate mass spectrum shows that cispentacin has the molecular formula C6HIINO2. The most important spectral data are as follows: IR (KBr) cm -l, 295, 2870, 2680, 2550, 2200, 1645, 1550, 1415, 1335, 1310, 1170, 1070, 840 [2]. ~H NMR (400 MHz, D20) ~5, 1.70-1.89 (4H, m), 2.04-2.15 (2H, m), 2.87 (1H brq, J = 6.2 Hz), 3.73 (1H, brq, J = 6.2 Hz) [2]. ~3C NMR (100 MHz, D20) ~5, 22.0 (t), 28.8 (t), 30.2 (t), 48.4 (d), 53.7 (d), 181.7 (s) [2]. The absolute configuration was first described from a derivative. Cispentacin was treated with 1-(3-methylaminopropyl)-3-ethylcarbodiimide to give the corresponding [3-1actam, which exhibited a positive Cotton effect at 214 nm, indicating the (1R,2S) configuration of the antibiotic [2]. The absolute configuration was later proved by X-ray diffraction of its phenylalanine derivative [28]. A simple HPLC method was developed for the separation and identification of the (1S,2R), (1R,2S), (1S,2S) and (1R,2R) enantiomers of 2-ACPC by using pre-column derivatization with the chiral derivatizing reagents 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (Marfey's reagent) and 2,3,4,6-tetra-O-acetyl-~-D-glucopyranosyl isothiocyanate [29]. Analogue and homologue derivatives can also be detected by this method [30-33]. The two enantiomers of cispentacin can be determined in rat urine by reverse-phase HPLC after derivatization with Marfey's reagent [34]. Physicochemical data such as dielectric increments [35], partial molar volumes [36] and protonation and complex formation constants [37, 38], were earlier determined for racemic cis- and trans-2-ACPC. SYNTHESES OF 2-AMINOCYCLOPENTANECARBOXYLIC ACID
Syntheses of the Racemic Compound Racemic cis-2-ACPC was first prepared by Plieninger and Schneider, by Hofmann degradation of cis-2-carbamoylcyclopentanecarboxylic acid 10
276
FERENCFOLOP
with sodium hypobromite [39]. The synthesis of cis-cyclopentane-l,2dicarboxylic anhydride 9, necessary for the preparation of carboxamide 10, starts from the ethyl tetracarboxylate 6 by sodium ethylate ring closure, followed by hydrolysis and decarboxylation. In acetic anhydride, isomerization and ring closure take place, resulting in the cis-anhydride 9 (Scheme 1) [40, 41]. Anhydride 9 can be prepared alternatively by the reaction of acetic anhydride with cis-cyclopentane-1,2-dicarboxylic acid 8, prepared from cyclopent- 1-ene- 1,2-dicarboxylic acid [42, 43]. OOEt COOEt Na, EtOH COOEt COOEt 6
~ ~OOEt H2SO4 ~._ ~ i ( ~--COOEt ~.~--COOEt AcOH, H20,A COOEt
COOH OOH
7 A I Ac20
~COOH "~NH2 11 Scheme
_.~ H~ degrad.
~COOH ~'CONH2 I0
NHaOH ~'~ 9
O
1.
Ammonolysis of alicyclic anhydrides followed by Hofmann degradation is one of the most frequently used methods for the synthesis of alicyclic 13-amino acids, since many anhydrides are commercially available cheap substances produced by Diels-Alder addition of maleic anhydride and the corresponding dienes [44]. By this method, cis- and trans-2-aminocyclohexane- and -cyclohexenecarboxylic acids, cis-2aminocycloheptanecarboxylic acid [45], 3-endo-aminobicyelo [2.2.1 ]heptane-2-endo-carboxylic acid, 3-endo-aminobicyclo[2.2.1]hept-5ene-2-endo-carboxylic acid and some analogue ]3-amino acids have been prepared [44, 46-48]. For partially unsaturated alicyclic compounds, a modified Hofmann degradation is used, sodium hypochlorite being applied to avoid bromine addition to the double bond [48, 49]. Since Hofmann degradation affords a crude product containing a large amount of inorganic derivatives, ion-exchange chromatography is an excellent technique for desalting. The reaction of chlorosulfonyl isocyanate (CSI) and cyclopentene at -78 ~ yielded 2-chlorosulfonyl-2-azabicyclo[3.2.0]heptan-3-one (13), which was transformed to azetidinone 14 with potassium bisulfite. The
2-ANIN l OCYCLOPENTANECARBOX ACI YLD IC
277
resulting 13-1actam was hydrolysed with concentrated hydrochloric acid to give the cis amino acid hydrochloride 15 (Scheme 2) [43]. Although the free amino acid 11 was prepared from 15 on treatment with a large excess of silver oxide [43], ion-exchange chromatography was later found to be more suitable [2], use of the expensive silver oxide thereby being avoided.
(~ 12
CSI ,,
~
o
~ (
NaHSO3
(
NS02CI
13
14
a
~ C NO HO 2H 11 Scheme2.
I' C(iiilC . 15
~ C ONOHEt2 16
With ethanolic hydrogen chloride, the 13-1actam 14 gave the ethyl ester 16 [501. The 1,2-dipolar cycloaddition of chlorosulfonyl isocyanate to different cycloalkenes has become a well-known route for the synthesis of cycloalkane-fused 13-1actams, and for alicyclic 13-amino acids, after hydrochloric acid treatment. The addition takes places regio- and stereospecifically, in accordance with the Markovnikov orientation rule [51-55]. In this manner, a number of homologue and analogue alic.yclic [3amino acids have been prepared, such as c i s - 2 - a m l n o - 2 methylcyclopentanecarboxylic acid [56], cis-2-amino-2-methylcyclohexanecarboxylic acid [56], (1R*,2S*,4S*)2-amino-4-tert-butylcyclopentanecarboxylic acid [55, 58], 3-exo-aminobicyclo[2.2.1]heptane-2-exocarboxylic acid [59, 60] and 3-exo-aminobicyclo[2.2.1 ]hept-5-ene-2-exoearboxylie acid [61 ]. The following relatively long procedure also results stereospecifically in the racemic cis amino acid 11. The commercially available 5-hexen-1-ol (17) was transformed into the aldehyde 18 by Collins oxidation, and subsequent N-benzylhydroxylamine treatment gave the nitrone 19. Cyclization resulted only in the cis-fused isoxazolidine 20. Catalytic hydrogenolysis with Pd/C in acetic acid was effected by cleavage of the NO bond and removal of the protecting benzyl group (Scheme 3). Boc protection, Jones oxidation and removal of the protecting group gave the
278
FERENCFOLOP
desired 11 [62]. Although the yields of the individual steps are high, the overall yield of this process is rather low in consequence of the length of the process.
<
c
17
~'NH2
20 CHePh
Pd/C,H2
19
ox~~ B o
~ C_O O % o n e x
II
Scheme
CH2Ph
-~~
~COOH
-
~~@~O-
18
23
22
o. --.2c(~
c
21
3.
trans-2-ACPC (27) can be obtained selectively in a moderate yield by Michael addition of ammonia to cyclopent-l-enecarboxylic acid (26)at 150-170 oC in an autoclave [39]. The key intermediate 26 can be prepared by many different methods. Scheme 4 depicts two different ways. From keto ester 24, by reduction, followed by hydrolysis and thermal water elimination, 26 was prepared in high overall yield. Another good method for the preparation of 26 is by the hydrolysis of 1cyclopentenecarbonitrile (29), obtained by the dehydration of cyclopentanone cyanohydrin (28) [40].
COOEt
24
0
<
o. 28
Scheme
.COOEt
,COOH
26
~..,
NCHO02H
27
4.
In the Michael addition to 26, Connor and Ross reported the isolation of the cis counterpart 11 besides the main trans acid 27 [63]. Later, there was no mention of the formation of the cis product [40, 41 ]. The present author has repeated the Michael addition process several times, but has never observed the presence of cis-2-ACPC.
2-AMINOCYCLOPENTANECARBOXYLIC ACID
279
Various methods have been applied for the reduction of alicyclic 13enamino esters, but these reactions in general gave diastereomeric mixtures [64-69]. For example, the sodium triacetoxyborohydride reduction of enamino ester 30 (R = Ph, CH2Ph) resulted in about 10% trans isomer 32 besides the main cis product 31 [69].
~
,COOEt
NaHB{OAc)3
~ C O O E t
,
~
~-
.
COOEt
+
NHR
NHR
30
~
"% NHR
31
32
C h a r t 3.
A number of substituted analogues of 2-ACPC have been synthetized by different routes [70-77]. T a b l e 1.
P h y s i c a l D a t a on S o m e 2 - A C P C R a c e m a t e D e r i v a t i v e s
,COR l
~ N H R R1
R2 i
i,,i
2 Reference
Configuration
Mp (~
i
,
i
i
OH
H
202-204
cis
[391
OH
H
2O2
c~
[43]
OH
H
223-224 201
9
OH
H
OH
H
OH
H
.
......
cis
[40]
I
cis
[62]
I
cis
[43]
..
L
|
210.212 a . . . . .
i
.,
240
j
trans
,,-
OH
|
[39] .
239-240
H
.. [
.
.
.
.
.
.
.
.
.
.
trans
[40]
cis
[28]
cis
'[50]
i
i
OMe
H
120.123 a .
OEt
.
.
.
.
.
H
.
.
.
.
j
|
40.41 b ,
OH
CH2Ph
146-149
OH
COOBut
161-162
OH
COOBut
oil
COOCH2Ph
180-183
|
,
trans
[111]
cis
[41]
cis
[62]
trans
[41 ]
!i ~
l
[
.
.
.
.
.
.
.
.
.
.
.
.
.
|
.
OH .
.
.
OEt
Ph
OEt
Ph
oil .
.
.
.
.
.
OEt
.
,
oil
aHCI salt. bBoiling point at 0.02 mm Hg.
.
.
.
.
.
.
[69]
trans
[691
.
.
|
[69, I I I] ,,
leans .
!
a
cis
!
CH2Ph i
-,
cis
.,.
OEt
.
!
oil
CH2Ph
.
|
oil ,,
.
,,
|
.
.
. . . . . . . . . . . . . . . . . .
[69, Ill]
280
FERENC F~LOP
Resolutions of the Racemates Diastereomeric Salt Formation
The first resolution of racemic cis-2-ACPC was reported only after the isolation of natural cispentacin. Konishi et al. resolved racemic cis-2benzoyloxycarbonylaminocyclopentanecarboxylic acid by fractional crystallization of the salt formed with (+)-dehydroabietylamine. After repeated crystallizations from acetonitrile, the enantiomers were recovered by treatment with alkali and deprotection by catalytic hydrogenation [2]. Yamazaki et al. resolved the Boo-protected cis racemate [78]. Treatment of the racemic cis-2-ACPC with di-tert-butyl dicarbonate resulted in the N-Boo-protected derivative, which was mixed with (-)ephedrine. The resulting salt was fractionally crystallized from the ethyl acetate/diethyl ether solvent system. Treatment with sodium hydrogensulfate and removal of the Boc group with trifluoracetic acid (TFA) gave the (+)-(1S,2R) enantiomer. By means of diastereomerie salt formation, c i s - a n d t r a n s - 2 aminocyclohexanecarboxylic acids and 2,3-die n d o - 3 aminobicyclo[2.2.1 ]hept-5-ene-2-carboxylic acid were also separated [79-
83].
Diastereomeric Pair Formation
Kawabata et al. [28] resolved methyl (+)-N-(Boe-L-phenylalanyl)-cis-2aminocyclopentanecarboxylate by fractional crystallization from ethyl acetate. The phenylalanine derivative was prepared from the methyl ester of racemic cis-2-ACPC (11) by acetylation with N-Boc-L-phenylalanine using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC). Edman degradation of the separated isomers, followed by deprotection, acid hydrolysis and desalting with anion-exchange resin, gave enantiomerically pure (1S,2R)- and (1R,2S)-2-ACPC. The absolute configurations were proved by X-ray diffraction of the L-phenylalanine derivatives [28]. The enantiomers of trans-2-ACPC were separated by Yamazaki et al. [78]. The Boo-protected racemic amino acid was acylated with R-(+)-o~methylbenzylamine by use of a mixed anhydride (Scheme 5). The diastereomers 34 and 35 were separated by silica gel column chromatography after removal of the acid-labile Boc group. The absolute configuration was proved by X-ray diffraction of 35. The amino acid enantiomers 36 and 37 were obtained after strong acid treatment of 34 and 35, and anion-exchange desalting of the product [78].
2 - A M I N O C Y C L O P E N T A N E C A R B O X Y L I C ACID
~
281
COOH
.oCOOH
~ 1 S.,
NIl
NHBoc
A
NIt 2
33 (trans)
36
34
I. TFA O
Me
9
O
Me
Ph NttB~
~
2R NH a
34,35 (trans)
".,,NH 2 37
35
S c h e m e 5.
A very similar method was used for the separation of the c is carboxamide enantiomers 40 and 41 (Scheme 6). After protection of racemic c i s - 2 - A C P C , the Z-amino acid 38 was treated with isobutyl chloroformate, and the anhydride formed was reacted with (+)-(R)-o~methylbenzylamine, resulting in the diastereomeric mixture of 39. The protecting group of 39 was removed by hydrogen bromide treatment and, after neutralization, the diastereomers 40 and 41 were separated by flash chromatography [84]. ,COOH 1. C1COOBu i
f
:2. (R)-H2NCH(Me)Ph
NHZ
38 (c/s)
1
Ph
N
NHZ
39 (e/s)
~
/
1. H B r 2. N a O H 3. F l a s h c h r o m a t o g r a p h y
Me Ph
2 40
S c h e m e 6.
N ~
"'~
"~176 2
41
282
FERENC F1DLOP
Enzymatic Resolutions
There is only one method for the direct enzymatic separation of ethyl 2aminocyclopentanecarboxylate enantiomers [85]. Some other enzymatic processes which separate various precursors of 2-ACPC will also be discussed in this section. Kanerva et al. resolved ethyl esters of ten alicyclic 13-aminocarboxylic acids by lipase catalysis in organic solvents. The resolutions were based on acylation of the amino group at the R-stereogenic centre with various 2,2,2-trifluoroethyl esters. From the cis and trans racemic esters 42, all four enantiomers of 2-ACPC were prepared. The absolute configurations of 43 and 44 were proved by transformation to the known 2-ACPC enantiomers by hydrolysis and subsequent desalting with an anionexchange resin [85].
~
COOEt
lipase PS
~__
~
C
OOEt +
,,
NH2
CICH2COOCH2CF3
-
C I ~
S ~NH2
42
43
NH~"'..~ 44
Chart 4.
A highly selective ring opening of 6-azabicyclo[3.2.0]hept-3-en-7-one (45) was achieved by using a whole cell preparation of ENZA-1 (Rhodococcus equi) [20, 86, 87]. The hydrolysis selectively gave the amino acid 46, which was converted directly to the ester acetamide 47. The recovered lactam 48 was reduced to 49. This was hydrolysed to 5, which displayed a similar optical rotation to that of natural cispentacin (Scheme 7). It is worthy of mention that ENZA-1 exhibited only poor hydrolytic activity towards (+)-49.
v
ENZA-1 [~ 4s
"eNH2 46
r
48 Scheme 7.
49
47 ~CONHOH
2-AMINOCYCLOPENTANECARBOXYLIC ACID
283
A highly enantioselective enzymatic acylation was observed on Nhydroxymethylated 13-1actam 50, which was prepared from (+)-14 with paraformaldehyde by sonication in tetrahydrofuran [88]. Lipase AKcatalysed butyrylation with vinyl butyrate gave the readily separable azetidinones 51 and 52. Hydrolysis of 51 and 52 resulted in the 2-ACPC hydrochlorides 53 and 54, respectively (Scheme 8) [88].
Cj_ o
lipase AK
0
1 5 ~ ~ SR
OH vinylbutyrate
N ~ . ~ ~
I_.+) so
OHOn
ultrasound
Sl
IHC
52
IHO
~CONOH2.HHc I 1+_} 14
Scheme
53
54
8.
Theil et al. developed a method for chemoenzymatic synthesis of both enantiomers of cispentacin [89]. trans-2-Hydroxymethylcyclopentanol, obtained by the sodium borohydride reduction of ethyl 2oxocyclopentanecarboxylate, was monosilylated with tertbutyldimethylsilyl (TBDMS) chloride to afford 55. Lipase PS-catalysed transesterification with vinyl acetate in tert-butyl methyl ether furnished the ester 56 and the alcohol 57. The deacetylated 58 was obtained by the Mitsunobu reaction with phthalimide, triphenylphosphine and diethyl azodicarboxylate (DEAD) to furnish the cis oriented 59 with inversion of configuration (not retention as mentioned in the original article) (Scheme 9). Desilylation, Jones oxidation and subsequent deprotection with aqueous methylamine gave the (1R,2S) enantiomer 5 [89]. The (1S,2R) enantiomer was prepared by the same route from the silyl alcohol 57. The Mitsunobu reaction is also the basic step in the synthesis of 65. The starting homochiral hydroxy ester 62 is readily available by yeastmediated reduction of ethyl 2-oxocyclopentanecarboxylate. Treatment of 62 with hydrazoic acid under Mitsunobu conditions resulted in inversion to the azide 63, which was hydrogenated in the presence of di-tert-butyl
284
FERENC FOLOP
dicarbonate to give 64. Ester hydrolysis was effected with lithium hydroxide, resulting in 65 [90]. ~ D T B D M S
.__
- C L"'"IkOTBDMS
~OTBDMS ~"R""OAc
55
~
~
OH
5G
0
phthalimide TBDMS , DEAD ~._
%"'- "'OH
PPh3
57
OH
OTBDM8 AcOH
"-
58
nes
(•••COOH ~..x.~.~
coo.
ox.
NH2 S
61
Scheme 9.
O i.ii~oo~ 62 Chart
,,~oo~
,,coo~,
63
...,,coo,,
64
6S
5.
COOMe 'C OOMe
.q
HSCH2CHaSH SnCl4
_
Q 70
Scheme
10.
_
~COOMe
C~~....coo,."~ C,~~....coo, ~~^
67
G6
o
~l)COOMe
_
~COOMe
C~~'"eNHBoc 69
TPA
68
2-AMINOCYCLOPENTANECARBOXYLIC ACID
285
EnantioselectiveSyntheses For synthesis of the trans N-Boo enantiomer 70, use was made of the chiral ketone 66, which is readily available by enzymatic resolution [91 ]. The ketone moiety was protected as the thioketal, and selective monohydrolysis then gave the monoacid 68. Curtius rearrangement of 68 with diphenylphosphorylazide (DPPA) and triethylamine (TEA) in tertbutanol gave the Boo-protected amine. Desulfurization with Raney nickel furnished the methyl ester 70 (Scheme 10). The (1S,2S) trans enantiomer was prepared analogously, starting from the enantiomer of ketone 66 [91 ]. An elegant enantioselective synthesis was described by Konoshu and Oida [62] Nitrone 72, prepared from the ehiral aldehyde 71, with (R) absolute configuration, underwent intramolecular cycloaddition and yielded isoxazolidine 73 with high diastereoselectivity (73:74 = 15:1). The cycloadduct 73 was transformed into natural cispentacin 5 in four steps (Scheme 11). The reaction conditions were the same as for the synthesis of racemic 2-ACPC (see Scheme 3).
U ~o.
~ '~o.
_
CH2Ph
CH2Ph
CH2Ph
73 ~
72
74
u
S
76
H
75
S c h e m e 11.
Michael addition of a chiral amine to a cycloalkenecarboxylie acid derivative controls the new stereogenic centre [92, 93], and is therefore suitable for enantioselective syntheses of 2-ACPC. The following reactions are based in practice on the high diastereofacial control. Addition of chiral lithium (S)-(a-methylbenzyl)benzylamide to tertbutyl 1-cyclopentenecarboxylate (77)at-95 oC, followed by quenching with 2,6-di-tert-butylphenol, gave 78 in 98% diastereomeric excess. 78 was debenzylated catalytically and, after acidic hydrolysis and desalting on Dowex resin, afforded pure cispentacin 5 (Scheme 12) [94].
FERENCI~LOP
286
,COOBu' - -
t
H2 Pd/Cr"- ~
COOBut NH2
77
80
I. HCI 2. Dowex
78
I
KOBut
S COOBut
<~COOH
'N ~Ph
5
NH2
Me~"[~ ph Scheme
"/9
12.
The method provides a possibility for enantioselective synthesis of the (1 S,2S) trans enantiomer, since 78 was easily isomerized to its C-1 epimer 79 in 98% diastereomeric excess [94]. The addition was repeated with the (R)-(cx-methylbenzyl)benzylamide enantiomer to give adduct 81. This was epimerized at C-1 to 82, which was deprotected in the same way as the stereoisomer, affording 83 with (1R,2R) absolute configuration [95]. .,~COOBu'
O Chart
6.
~
COOBut
MI~ . Ph
MI~,.." Ph
81
82
83
The above method was also applicable for enantioselective synthesis of the cis and trans-2-aminocyclohexanecarboxylic acid enantiomers [95]. Trans-ACPC with (1S,2S) configuration was prepared in an elegantly short pathway (Scheme 13) by Enders et al. By addition of the chiral ammonia equivalent lithiated ( S ) - ( - ) - 2 - m e t h o x y m e t h y l - 1 trimethylsilylaminopyrro-lidine (TMS-SAMP) to co-halide-substituted enoate 84, followed by Michael-initiated ring closure (MIRC), 85 was formed with 96-98% diastereoselectivity (Scheme 13). After desilylation,
2-AMINOCYCLOPENTANECARBOXYLIC ACID
287
reductive N - N bond cleavage and hydrolysis of the ester and desalting with ion-exchange chromatography, 36 was obtained [96-98a,b].
COOBu' ,
Br
I.TMS-SAMP, nBuLi,_78oc 2. HMPA,-78~ 3. NaHCO3 ,
,,
9
~,~COOBu' "/l N~ SiMe | N 85
I SiO EtOAc
~ ,COOBu' ~
N ~ ,CO H 2OH
I. Raney-Ni,Ha 2. HCI 3. Dowex ,,,
36
86 S c h e m e 13.
This method could additionally be applied for the synthesis of trans-2aminocyclohexane- and -cycloheptanecarboxylic acid enantiomers [96, 97]. Chiral ~-enamino ester 87 was prepared from 2-oxocyclopentanecarboxylates and (R)-a-methylbenzylamine [69, 99]. Reduction of 87 with sodium triacetoxyborohydride led to the amino ester 88 with diastereoselectivities of 85% (R=Et) and 67% (R=CH2Ph). After separation of the cis and trans isomers of 88 (R=CH2Ph), the benzyl groups were removed by hydrogenolysis, giving directly pure 89 with (1S,2R) configuration [99].
~,COOR
<~
NH ~
Chart 7.
87
Ph
@iCOOR "" Me "~
~ 88
~..)~%COOH "*NH2
Ph 89
Various enantioselective syntheses of homologue and analogue derivatives of 2-ACPC have been described, for example in refs. [ 100109].
288
FERENC I~LOP
TRANSFORMATIONS A number of simple transformations of cis- and trans-2-ACPC have been carried out, under practically the same conditions as for o~-amino acids. For esterification, the best procedure is the use of thionyl chloride and absolute alcohol at -10 oC (see e.g. refs. [55, 58]). Lithium aluminium hydride reduction can be performed on either the acid or the ester 90, or on their N-acyl derivatives 91, in diethyl ether or tetrahydrofuran, which results in excellent yields of the corresponding amino alcohols 92 and 93, respectively (Scheme 14) [40, 110, 111 ]. Reductive alkylation with oxo compounds and sodium borohydride afford N-alkyl-substituted derivatives 94 [58, 111 ].
NH2 ~ 92
~ N 90
R2
91 H ~rrl~R2 93
C(coo. 3. NHR
NCS
NH 2
94 96
96
Scheme 14.
The N-unsubstituted carboxamide 95 (R=H) can be prepared if the ester 90 is allowed to stand for about a week at room temperature in methanol containing 25% ammonia [58, 112]. N-Substituted derivatives of 95 were prepared from Boc- or Z-protected amino acids with mixed anhydride methods, using isobutyl chloroformate and the corresponding amine [78, 84, 113-115]. The amino group in 90 (cis and trans, R I=Et) was readily transformed to a thiocyanate group (96) with thiophosgene in the presence of triethylamine [116].
2-AMINOCYCLOPENTANECARBOXYLIC ACID
Table 2.
Physical Data on Some 2-ACPC Enantiomer Derivatives
~ |,,,
,,
CNOHRRI2
,1,111
RI ,,
289
R2
,
j,,,
,
OH
ii
,,
H
,
Configuration [
Mp (~ 179-182
Ir,2s
195-196
1a125.7.3(c=0.5 ' H20)
,
,
OH
Optical rotation
Ref.
i
, F
i [2] |
IR,2S
[al2D0-9.S (c=l, H20 ) [a1~o.8.9
. . . . .
i DIP I
OH
199-200
IR,2S
OH
not given
IR,2S
OH
227
IR,2S
[al2DS.9.1 (c=1.23, H20)
[621
OH
194-197
IR,2S
[a12DS-8.8 (c-I, H20 )
[94,9 5]
not given
IR,2S
[a]~o.7.5 (c=l ' H20)
[89]
OH
not given
IR,2S
[a12oo.9.6 (c=l" H20)
[85]
OH
156-157a
IR,2S
1a1~~
(c=l, H20 )
[281
OH
155-156a
IR,2S
I~l~~
(c=l, H20)
[85]
OH
182-184
IS,2R
lal~ s +8.9 (c=0.5, H20 )
[21
OH
198-199
IS,2r
[a]~ ~ +8.9 (c=l, H20 )
[28]
OH
not given
IS,2R
lc~l~~ +8.2 (c=2.3, H20 )
[99]
OH
not given
IS,2R
[a]~~ +8.5 (c=l, H20)
[89]
OH
156-157 a
IS,2R
1a1~o+6.5 (c=l ' H20)
[2s]
OH
221
]s,2s
[~l~~
(c-I, H20 )
[7s]
OH
218-221
IS,2S
la] 2~ +62.3 (c-I, H20 )
[85l
OH
224-226
IS,2S
OH
212-214
IR,2R
OH
OH
I
i
, i
H ,,,
,,
,
150-155 a
[aiD-8
[281
H20 )
[861
H20 )
(c--l,H20)
[a1~5-50.7(c---0.75, H20 )
IR,2R ,
( c = l ,
[tXI2D2 +81.2 (r
[a1~~
H20)
(c=l '
|
,
,
[97] [781
[951
FERENC FOLOP
290
(Table 2). contd .....
R1
Mp (~
R2
Optical r o t a t i o n
Configuration
~ Ret'.
i |
i
,,
OEt
H
,,~'~
,,
168.169 a
IR,2S
i
,,
[tq~~
(c=l, EtOH)
l i
[ss]
OEt
69.71 a
IS,2S
[=12D~ +6t.8 (c=l, EtOH)
OBut
oil
IR,2S
[~12DS.5.6 (c=0.63, CHCI3)
[951
oil
IR,2S
[c~]~)2 + 108 (c=0. I, MeOH)
[84]
oil
IS,2R
[o~]~D2 +124 (c=0.1, MeOH)
[841
HN~Ph =.
HN~Ph t
i!
,,
[851
OH
COOBut
oil
IR,2S
OH
COOBu t
syrup
IR,2S
OH
COOBut
not given
OH
COOBut
OH
[62]
[oq~2 -29.6 (c=1.56, CHCl3)
.....
[a]~5-69.7 (c=0.9, MeOH)
[91]
IS,2R
[(x]22 +47.7 (c=1.5, MeOH)
[78]
syrup
IS,2R
[Ot]2D2 +64.9 (c=1.3, MeOH)
[91]
COOBut
117=120
ts~s
OMe
COOBut
73-74
IR,2R
OMe
COOBut
66-67
IS,2S
[(x]2D2 +44.6 (c=1.3, CHCI3)
OEt
COOBut
56-58
IS,2S
[0t]2Ds +41.4 (c=0.96, CHCI3)
OEt
COCH2CI
53-54
IS,2R
OEt
COCH2CI
49-51
IR,2R
not given
IS,2R
[0t]2D~ +101 (c=6, EtOH)
[99]
not given
IS,2R
[(xl2D0 +73 (c=2, EtOH)
[99]
OEt
]~/[e [
HN~Ph |
[90]
[a]2Ds +2.69 (C=1.02, CHCI3) [(z]~)2 -48.2 (c=1.2, CHCI3) I
[91]
I[91] [90]
[(x12D~
(c=l, EtOH)
[85]
[(x]~~
(c=l, MeOH)
[85]
g
OCH2Ph
HN~Ph ,,,,,,
aHC! salt.
I
2-AMINOCYCLOPENTANECARBOXYLIC ACID
291
Transformations to Heterocyclic Compounds The synthesis, stereochemistry and transformations of cyclopentane-, cyclohexane-, cycloheptane- and cyclooctane-fused 1,3-oxazines, 1,3thiazines and pyrimidines were recently reviewed, the syntheses starting from 2-ACPC [117] also being discussed. Accordingly, only the most important and most recently published transformations to heterocycles will be mentioned here. The reactions of amino esters 97 (RI=H, CH2Ph) with potassium cyanate afforded cis-cyclopenta[d]pyrimidine-2,4-dione 98, while those with potassium thiocyanate led to cis-2-thioxocyclopenta[d]pyrimidin-4one 99 (Scheme 15) [111, 118, 119]. 0
0
KOCN O
~
~
II
KSCN
NH
NI-IRl
~l
98
(
coo :t
S
97
99 0
-
.~
NHR2
if X =
A l
O
--~
~NHR I
1
100
102
103
HCI~ ifX=S 0
S
I Rl 101
Scheme 15.
When esters 97 (R~=H, CH2Ph) were reacted with phenyl or methyl isothiocyanate or isocyanate, adducts 100 were formed in excellent yields. Ring closure of 100 (RI=H) took place without difficulty when it was refluxed in dilute hydrochloric acid, resulting in pyrimidinones 101 and 102 [58, 111 ]. N-Benzyl-substituted derivatives of 100 could be cyclized to 101 and 102 by refluxing in ethanol containing 22% dry hydrogen chloride [ 111 ]. Further derivatives of 102 were prepared from carboxamide 103 (RI=H) by cyclization with 1,1'-carbonyldiimidazole [113].
292
FERENC FOLOP
It is noteworthy that the ring closures of 1,2-disubstituted cyclohexane, cycloheptane and cyclooctane derivatives revealed no appreciable differences in the reactivities of the cis and trans isomers in the formation of six-membered 1,3-heterocycles [117]. In contrast, striking differences were observed in the cyclizations of the cis and t r a n s cyclopentane derivatives. For instance, the above cyclizations to pyrimidinones, starting from the t r a n s counterparts, were unsuccessful. The attempted ring closure from 104 did not result in the cyclized products, but gave hydrolysed derivatives 105 and 106 [111].
COOEt
X
~.,oCOOH =
"~ N
NPh
if R ~ = H
105
NPh
,H,
,,
if R x =C H2Phv
NH~P
104
h
106
Chart 8.
A great number of 2-substituted pyrimidinones 108 have been synthetized by reaction of the corresponding 13-amino acid 107 (R=OH) or its derivatives (R=OEt, NHR) with ethyl benzimidates, or by reaction of orthoesters with carboxamide 107 (R=NH2). Of this set of compounds, the racemic 2-(m-chlorophenyl)-3,4a,5,6,7,7a-hexahydrocyclopenta [d]pyrimidin-4(3H)-one (CHINOIN 143) displayed excellent antiinflammatory activity [44, 46, 120, 121 ]. H
R
H
OEt
R1
NH 2 107
108
C h a r t 9.
The first step in the ring closure of carboxamide 107 (R=NH2) with imidates is the formation of an amidine intermediate, with subsequent nucleophilic attack of the imino group of the amidine on the carbonyl group; this takes places with loss of the carboxamide N-substituent [117]. This observation formed the basis of a simple synthesis of CHINOIN 143 enantiomers. From carboxamides 39 and 40 with ethyl mchlorobenzimidate, 109 and 110 were obtained in high enantiomeric purity. The absolute configurations were determined by hydrolysis of 109 and 110 to the corresponding amino acid, which was identified by HPLC [79].
2-AMINOCYCLOPENTANECARBOXYLICACID
293
Ph NH2 39
109
O
~176
(-q..L.H
Ph
N
r
%.'~",, f f " ~ ~ ~ l ,
~..,tNH2 40 Chart 10.
,'cl
110
It is interesting to note that the absolute configuration of the enantiomer with the higher anti-inflammatory activity [44, 46, 122] corresponds to that of cispentacin [79]. A number of further transformations of homologous alicyclic amino acids to give various heterocycles have been performed [44, 46, 123-145]. Alicyclic amino alcohols 111, obtained in a facile way from amino acids by lithium aluminium hydride reduction, are also useful starting substances for different heterocycles [ 117]. From 111 (cis or trans, R=H, Me), by reaction with phenyl isothiocyanate, thioureas 112 were prepared which, after a short refluxing in ethanolic hydrogen chloride, gave the c i s - a n d t r a n s -
T'O I
R 112
I
R ~
I. Mel
111
~
CH20
115
Cl
NPh [
R 113 Scheme 16.
R
114
Me
116
294
FERENC F1DLOP
cyclopenta[d][1,3]thiazines 113. When thioureas were stirred with methyl iodide, followed by treatment with alkali, 1,3-oxazines 114 were formed, by methyl mercaptan elimination (Scheme 16) [110]. Treatment of 111 with phosgene in the presence of triethylamine resulted in 1,3-oxazin-2ones 115 (X-O) [57], while treatment with carbon disulfide, followed by ethyl chloroformate, furnished the 2-thioxo derivatives of 115 (X=S) [57, 146]. With a mixture of formaldehyde and formic acid, 111 (cis, R=H) underwent ring closure and N-methylation, resulting in 1,3-oxazine 116 [147]. The trans counterpart failed to react, even during a much longer reaction time. The above striking differences were found earlier in connection with the N---~O acyl migration of c i s - a n d t r a n s - 2 - h y d r o x y m e t h y l cyclopentylamine. These reactions were studied very thoroughly by Bern~ith et al., who found that the rate constant of the N--~O acyl migration 117---~118 for the trans isomers was essentially lower than that for the cis counterparts [40, 148].
a
4-
.H + 107
118
Chart 11.
Stability differences between cis and trans cyclopentane-fused 1,3oxazines were also observed in the case of the ring-chain tautomeric mixtures 1 1 9 - 1 2 1 , obtained by reacting c i s - and t r a n s - 2 hydroxymethylcyclopentylamine with aromatic aldehydes [ 149]. The ring form of the trans derivatives is present in fairly low amount as compared with the cis compounds [149, 150]. N-Methyl substitution stabilizes the ring forms in both the cis and trans cases [149]. For a detailed discussion of the ring-chain tautomerism of alicyclic 1,3-amino alcohols, see, for example, refs. [ 150, 151 ]. H
H
oH
H
H 120
Chart 12.
H 119
H 120
2-AMINOCYCLOPENTANECARBOXYLIC ACID
295
A number of further transformations of homologue and analogue alicyclic amino alcohols have been performed, resulting in various heterocyclic compounds [ 117, 152-166].
Applications in Peptide Syntheses To investigate the antifungal activity of cispentacin derivatives, six dipeptide derivatives of (+)-cis-2-ACPC were synthetized. The syntheses were straightforward: the Z-protected (+)-cis-2-ACPC was activated with isobutyl chloroformate and treated with the corresponding amines, followed by deprotection to give the carboxamides 122 (Rl=Me, R2=H; RI=H, R2=Me; RI=CH2Ph, R2=H) [167]. The N-acyl derivatives 2 (Rl=Me, R2=H; RI=H, R2=Me; RI=CH2Ph, R2=H) were prepared from the methyl ester of (+)-cis-2-ACPC, which was acylated with Boo-amino acids, followed by hydrolysis and deprotection, to afford 123 [ 167]. 0
Rl
R2
~~~i~ ~~'~NH~'~COOH NH2 122
~~_Lrl R~I..,,R jNH2 2 OOH
123
C h a r t 13.
For further characterization of subsite $2' of both neutral endopeptidase (NEP) and aminopeptidase (APN), a new set of hydroxamate inhibitors containing cyclic 13-amino acids have been synthetized (Scheme 17). The tert-butyl esters of racemic trans-2-ACPC 124 (n=l) and cis- and trans-2aminocyclohexaneearboxylic acid 124 (n=2) were coupled with 3ethoxycarbonyl-2-benzylidenepropanoic acid (125) by treatment with dicyclohexyl carbodiimide (DCC). After alkaline hydrol.ysis, the second DCC coupling was performed with O-benzylhydroxylamme, giving 127, which was hydrolysed and deprotected to 128. The resulting diastereomers were separated by chromatography on silica gel. All the compounds 128 synthetized are highly efficient inhibitors of NEP and APN, exhibiting inhibitor activity in the same range [168]. A number of tetrapeptide analogues related to morphiceptin were synthetized in which the proline in the second position was replaced with cis- or trans-2-ACPC [41, 169, 170]. The synthetic scheme is shown in Figure 1. The diastereomers were separated by using preparative HPLC. By means of this pathway, the following peptides were prepared" Tyr(1R,2S)-ACPC-Phe-VaI-NH2, Tyr-(1S,2R)-ACPC-Phe-VaI-NH2, Tyr(1R,2S)-ACPC-Phe-D-VaI-NH2, Tyr-(1S,2R)-ACPC-Phe-D-Val-NH2, Tyr-(1R,2S)-ACPC-Phe-Pro-NH2, Tyr-(1S,2R)-ACPC-Phe-Pro-NH2 and
296
FERENC FULOP
~OOH (ell 2In
H~
+
,._
jCO OBut
~H~
COOEt 124
ttCPh
125
126 1. NaOH 2. PhCH2ONH2 DCC
,/~,~
COOBut
I ~ N H
~OH
2. H2Pd/C
~
N
H
~
NHOCH2Ph
HCPh 128
O
127
Scheme 17.
Tyr-trans-ACPC-Phe-Pro-NH2(a mixture of diastereomers). The absolute
,!r
configurations were determined via high-resolution (500 MHz) NMR and computer simulations [41, 169, 171 ]. )he
~CPC
Bo,
-~.k,;M 2 I"-~
ov~;
EDC IItOBt Boq
,~But 'r,
B(
Ir ,,
n
;
OCH2Ph
G:
9%,,i
- rl
TFATI
-------tq H 2
EDC/HOSt
Boc
H# Pd/C /But
~
ral
-------NH
2
~
2
TFA/CH~Cla ~r H
EDCI H O B t B(
BU t
---.----N H 2 T I N CI~CI=
TFA*H
Ir
-----NH
2
Fig, (I), Synthetic pathway for morphlceptln analogues.
Pharmacological screening demonstrated that the (1S,2R)-ACPC derivatives exerted activity at the p-receptor, but were inactive at the 8receptor. The (1R,2S)-ACPC derivatives were completely inactive at both
2-AMINOCYCLOPENTANECARBOXYLIC
ACID
297
the Ix and ~i-receptors. It was concluded from biological assays and from the conformational analysis that the (1S,2R)-ACPC analogues display the relatively large separation of the Tyr and Phe side-chain residues that is required for the I.t-opioid receptor activity of these molecules [169]. Eight analogues of the ~t-specific opioid peptide dermorphin were synthetized by replacing D-AIa 2 with stereoisomers of alicyclic 13-amino acids. The peptides were synthetized by solid-phase techniques involving Boc chemistry; the couplings were performed with DCC. The pharmacological investigations revealed that the insertion of the rigid ring structure instead of D-AIa 2 was unfavourable for both ~t and 5-binding activity [172]. The cyclic hexapeptide analogue of somatostatin c[Pro6-PheT-D-TrpL Lys9-Thr~~ ~l] displays high potency in inhibiting the release of gro.wth hormone. To investigate the structural role played by the bridging region, a series of cyclic hexapeptide analogues containing 2-ACPC as proline mimetics were synthetized [173]. The synthetic pathway is shown in Figure 2. In the final step, the diastereomers were separated by preparative HPLC. The absolute configurations were determined from the
~07
Ul'l
I OSUIl"h
1~2 HOBt TEA
Cbz
2 or
,,,,But
,I'
/l~c
iBut
~r
H 2/Pdl C
H
,,
,
,
, =,
OPPA
~r
Fig. (2). Synthetic pathway for somatostatin analogues.
298
FERENC FOLOP
lH NMR data [171, 173]. The following sequences were prepared: c[(1S,2S)-ACPC-Phe-D-Trp-Lys-Thr-Phe], c[(1R,2R)-ACPC-Phe-D-TrpLys-Thr-Phe], c[(1R,2S)-ACPC-Phe-D-Trp-Lys-Thr-Phe] and c[(1S,2R)ACPC-Phe-D-Trp-Lys-Thr-Phe]. The synthetized analogues contain a trans amide bond in the bridging region, which leads to the loss of binding activity [173]. After the discovery that L-aspartyl-L-phenylalanine methyl ester (aspartam) is about 200 times sweeter than sucrose, a large number of Laspartyl di- and tripeptides were synthetized. The conformation of a molecule with a sweet taste is described as possessing an "L shape" [78, 174]. Goodman et al. investigated the structure - taste relationships of Laspartyl dipeptides, preparing all four isomers of methyl L-aspartyl-2aminocyclopentanecarboxylate 131 [78]. The syntheses started from racemic cis and trans Boc-protected ACPC 129. After esterification with diazomethane, the methyl ester was coupled to N-benzyloxycarbonyl-~benzyl-L-aspartate [Z-Asp(OCH2Ph)OH], using N-hydroxybenzotriazole (HOBt) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC). Deprotection of 130 by hydrogenolysis yielded the diastereomeric pairs, which were separated by reverse-phase HPLC. The determination of the absolute configuration was based on NMR measurements, but was also proved by synthesis, starting from the enantiomers of 129. In agreement with the conformational studies and model calculations, the (1R,2R) and (1S,2R) ACPC derivatives have L-shaped conformations and a sweet taste. The (1S,2S) derivative has a bitter taste, while the (1R,2S) derivative is tasteless, as predicted [78]. Whereas a-amino acids can adopt the well-known a-helical motif of proteins, [3-peptides constructed from carefully chosen [3-amino acids can adopt a different, stable helical conformation defined by interwoven 14,COOH NHBoc 129
I. CHaN2 2. TFA
COOMe ~
3. Z-Asp(OCH~Ph)OH EDC, HOBt
NHAsp(OCH2Ph )
I
Z 130 I H~, Pd/C
COOMe
NHAsp 131 Chart 14.
2-AMINOCYCLOPENTANECARBOXYLIC ACID
299
membered-ring hydrogen bonds. It was reported recently that ]3-amino acids such as trans-2-ACPC and trans-2-ACHC can also be used to design 13-peptides with a very different secondary structure, a 12-helix. This demonstrates that, by altering the nature of the ~-peptide residues, one can exert rational control over the secondary structure [ 175,176]. BIOLOGICAL EFFECTS The biological activities of [3-amino acids were recently reviewed, but of the 2- ACPC derivatives only amipurimycin was mentioned [177]. Because of the tumour growth inhibitory activity of 1-ACPC, a number of amino acid derivatives of cycloalkane have been investigated, among them 2-ACPC. Significant anti-tumour activity was exhibited only by compounds closely related to 1-ACPC [ 178,179]. Various T-aminobutyric acid (GABA) analogues, among them cis- and trans-2-ACPC, were investigated as potential inhibitors of the GABA uptake in brain synaptosomes and in synaptic membrane vesicles [180182]. cis-2-ACPC was found to be a potent inhibitor of the GABA uptake in the synaptosomes. The trans counterpart proved to be half as potent as the cis isomer [182]. After the isolation of cispentacin, its antifungal activity was investigated thoroughly. Cispentacin demonstrated good therapeutic efficacy against a systemic Candida infection in mice following either parenteral or oral administration. It was also effective in a systemic infection with Cryptococcus neoformans and in both lung and vaginal infections with Candida albicans in mice [27, 28]. In the acute toxicity experiment in mice, cispentacin did not show any lethality at a dose of 1 mg/kg administered by the iv route or at 1.5 mg/kg following ip or oral administration [27]. The mechanism of the antifungal activity of cispentacin has also been studied [ 183-187]. When the antifungal activities of certain homologue and analogue derivatives of cispentacin were investigated, it was found that, among the [3-amino acids investigated, the five-membered ring derivatives displayed reasonable activities, whereas the cyclohexane and norbomane derivatives had no activity. Several dipeptides derivatives of cispentacin also exhibited potent anticandida activity [ 167]. SUMMARY AND CONCLUSIONS The syntheses and transformations of some of the biological features of 2aminocyclopentanecarboxylic acid (2-ACPC) are reviewed. The (1S,2R) enantiomer (cispentacin) was recently isolated from different natural sources, cis-2-ACPC is also a component of the antibiotic amipurimycitt The discussed [3-amino acids exhibit a wide range of pharmacological activities. Cispentacin exerts strong antibiotic and antifungal activities.
300
FERENC FIDLOP
The examples presented demonstrate that 2-ACPC has a high importance among the 13-amino acids. Although a number of reactions have been performed with these compounds, further transformations should be of interest, and their use as peptidomimetics is of high priority as concers the transformations. The biological activity reveals the unique position of 2-ACPC among 13-amino acids, but further studies are needed for a better understanding of the pharmacological effects. ACKNOWLEDGEMENTS
Financial support from OTKA and MKM (FKFP) is gratefully acknowledged. REFERENCES
[1]
Enantioselective Synthesis of fl-Amino Acids, Juaristi, E. Ed.; Wiley-VHC, New York, 1997; Cole, D.C. Tetrahedron 1994, 50, 9517. [2] Konishi, M.; Nishio, M.; Saitoh, K.; Miyaki, T.; Oki, T.; Kawaguchi, H. J. Antibiotics, 1989, 42, 1749. [3] Iwamoto, T.; Tsujii, E.; Ezaki, M.; Fujie, A.; Hashimoto, S.; Okuhara, M.; Kohsaka, M.; lmanaka, H.; Kawabata, K.; lnamoto, Y.; Sakane, K. J. Antibiotics, 1990, 43, 1. [4] Goto, T.; Toya, Y.; Kondo, T. Nucleic Acids Syrup. Ser., 1980, 8, 73 (Chem, Abstr., 1981, 94, 1855). [51 Goto, T.; Toya, Y.; Ohgi, T.; Kondo, T. Tetrahedron Letters, 1982, 23, 1271. [61 Knapp, S. Chem. Rev., 1995, 95, 1859 [71 Garner, P.; Ramakanth, S. J. Org. Chem., 1986, 51, 2609. [81 Garner, P.; Ramakanth, S. J. Org. Chem., 1988, 53, 1294. [9] Garner, P.; Park, J.M.J. Org. Chem., 1990, 55, 3772. [10] Casiraghi, G.; Colombo, L.; Rassu, G.; Spanu, P. J. Org. Chem., 1991, 56, 6523. [11] Czemecki, S.; Franco, S.; Valery, J.M.J. Org. Chem., 1997, 62, 4845. [12] Czemeeki, S.; Valery, J.M.; Wilkens, R. Bull. Chem. Soc. dpn., 1996, 69, 1. [13] Rauter, A.P.; Femandes, A.C.; Czemeeki, S.; Valery, J.M.J. Org. Chem., 1996, 61, 3594. [14] Oishi,N.; Maeno, S.; Kobayashi, M.; Sugiyama, H.; Toyoka, K. Jpn. Kokai Tokkyo Koho JP 63 83,004 (Chem. Abstr., 1988, 109, 165710). [15] Miyauehi, M.; Tsujii, E.; Ezaki, M.; Hashimoto, H.; Okuhara, M. Eur. Pat. Appl. EP 298,640(Chem. Abstr., 1989, 111, 152147). [161 Sugiyama, H.; Mori, K.; Maeno, S.; Muramatsu, N. Jpn. Kokai Tokkyo Koho JP 63,287,753 (Chem. Abstr., 1989, 110, 168095). [171 Kazuo, S.; Koji, K.; Yoshiko, I. Jpn. Kokai Tokkyo Koho JP 02 49, 758 (Chem. Abstr., 1990, 113, 40012). [is] Sakane, K.; Kawabata, K.; Inamoto, Y. Jpn. Kokai Tokkyo Koho JP 02,174, 753 (Chem. Abstr., 1991, 114, 24607).
2-AMINOCYCLOPENTANECARBOXYLICACID
301
Sadao, O.; Toshiyuki, K. Jpn. Kokai Tokkyo Koho JP 04,243,870 (Chem. Abstr., 1993, i 18, 80923). [20] Evans, C.T.; Roberts, S.M.; Sutherland, A.G. PCT Int. Appl. WO 92 18,477 (Chem. Abstr., 1993, 118, 168892). [211 Crowley, P.J.; Youle, D.; Heaney, S.P. PCT Int. Appl. WO 94 03,061 (Chem. Abstr., 1994, 120, 263835). [22] Crowley, P.J.; Heaney, S.P.; Lawson, K.R.; Youle, D. PCT Int. Appl. WO 95 07,022 (Chem. Abstr., 1995, 123, 77144). [23] Setsuo, H.; Miko, S. Jpn. Kokai Tokkyo Koho JP 06,321,950 (Chem. Abstr., 1995, 123, 83099). [24] Mittendorf, J.; Arold, H.; Fey, P.; Matzke, M.; Militzer, H.C.; Mohrs, K.H. Ger. Often. DE 4,400, 749 (Chem. Abstr., 1996, 124, 9443). [25] Crowley, P.J.; Lawson, K.R.; Mound, W.R. Brit. UK Pat. Appl. GB 2,291,872 (Chem. Abstr., 1996, 125, 10361). [26] Lawson, K.R.; Warrington, R.P. Brit. UK Pat. Appl. GB 2,290,540 (Chem. Abstr., 1996, 124, 288995). [271 Oki, T.; Hirano, M.; Tomatsu, K.; Numata, K.; Kamei, H. J. Antibiotics, 1989, 42, 1756. [28] Kawabata, K.; Inamoto, Y.; Sakane, K.; lwamoto, T.; Hashimoto, S. J. Antibiotics, 1990, 43, 513. [29] P6ter, A.; FUlSp, F. J. Chromatography A, 1995, 715, 219. [30] P~ter, A.; TOrOk, G.; Csom6s, P.; P6ter, M.; Bern6th, G.; FfllSp, F. J. Chromatography ,4, 1997, 761, 103. [3~] P~.ter, A.; T0rSk, G.; Csom6s, P.;. Kanerva, L.T.; FfllOp, F. J. Chromatography A, 1998, 797, 177. [32] P6ter, A.; TOr~k, G.; FfllOp, F. J. Chromatogr. Sci., 1998, 36, 311. [33] TOrSk, G.; P~,ter, A.; Ffll0p, F. Chromatographia, 1998, 48, 20. [34] Zanol, M.; Hermann, R.; Bernareggi, A.; Borgonovi, M.; Taglietti, E.; Zerilli, L.F. Boll. Chim. Farm., 1995, 134, 390 (Chem. Abstr., 1995, 123, 329111). [35] Edward, J.T.; Farrell, P.G.; Job, J.L.J. Am. Chem. Soc., 1974, 96, 902. [36] Shahidi, F.; Farrell, P.G.J. Chem. Soc., Faraday, 1978, 858. [37] Gaizer, F.; G0ndOs, G.; Gera, L. Polyhedron, 1986, 5, 1149. [38] Gaizer, F.; GOndOs, G.; Gera, L. Magy. Kdm. Foly., 1986, 92, 117. [39] Plieninger, H.; Schneider, K. Chem. Ber., 1959, 92, 1594. [40] Bern6th, G.; GSndSs, G.; M6rai, P.; Gera, L. `4cta Chim. `4cad. Sci. Hung., 1972, 74, 479. [41] Mierke, D.F.; NOflner, G.; Schiller, P.W.; Goodman, M. Int. J. Peptide Protein Res., 1990, 35, 35. [42] Takaya, T.; Yoshimoto, H.; Imoto, E. Bull. Chem. Soc. Jpn, 1967, 40, 2844. [43] Nativ, E.; Rona, P. Isr. J. Chem. 1972, 10, 55. [44] Bern6th, G.; FUlOp, F.; Sffsjer, G. Janssen Chim. Acta, 1991, 9, 12. [45] Bern6th, G.; G0nd0s, G.; Kov~cs, K.; Soh,'tr, P. Tetrahedron, 1973, 29, 981. [46] Bem6th, G. Bull. Soc. Chim. Belg., 1992, 123, 509. [47] Bern~th, G. Acta Chim. Hung.-Models in Chemistry, 1992, 129, 107. [48] St~jer, G.; Szab6, A.E.; FUI0p, F.; Bern6th, G.; Soh6r, P. J. Heterocyclic Chem., 1983, 20, 1181. [491 Bern6th, G.; St6jer, G.; Szab6, A.E.; FillSp, F.; Soh6r, P. Tetrahedron, 1985, 41, 1353. [50] Furrer, H. Chem. Ber., 1972, 105, 2780. [511 Rasmussen, J. K.; Hassner, A. Chem. Rev., 1976, 76, 389. [19]
302
[52]
FERENC I~LOP
Kamal, A.; Sattur, P. B. Heterocycles, 1987, 26, 1051.
[531 Paquette, L.A.; Kakihana, T.; Hansen, J.F.; Philips, J. C. J. Am. Chem. Soc., 1971, 93, 152. Parcell, R.F.; Sanchez, J. P. J. Org. Chem., 1981, 46, 5055. [551 Ftll/Sp, F.; BemAth, G.; Spitzner, R.; Mattinen, J.; Pihlaja, K. dcta Chim. Hung. - Models in Chemistry 1994, 131,435. [561 Szakonyi, Z.; Ftll0p, F.; Bem~tth, G.; Riddell, F. G. Tetrahedron, 1998, 54, 1013. [57] Bemhth, G.; Szakonyi, Z.; FUlOp, F.; Soh~tr, P. Heterocycles, 1994, 37, 1687. [581 Szakonyi, Z.; Ftll0p, F.; Bem~th, G.; Soh~tr, P. Heterocycles, 1996, 42, 625. [59] Moriconi, E.J.; Crawford, W.C.J. Org. Chem., 1968, 33, 370. [60] Moriconi, E.J.; Mazzocchi, P.H.J. Org. Chem., 1966, 31, 1372. [61] St~jer, G.; M6d, L.; Szab6, A.E.; FUlSp, F.; Bernfith, G.; Soh~tr, P. Tetrahedron, 1984, 40, 2385. [62] Konosu, T.; Oida, S. Chem. Pharm. Bull., 1993, 41, l 012. [63] Connors, T.A.; Ross, W.C.J.J. Chem. Soc., 1960, 2119. [64] Bemfith, G.; Kov,Scs, K.; Lfing K. L.: Acta Chim. ,4cad. Sci. Hung. 1970, 64, 183. [651 Borch, R. F.; Bernstein, M. D.; Durst, H. D." J. Am. Chem. Soc. 1971, 93, 2897. [66] Nakamura, S.; Karasawa, K.; Tanaka, N.; Yonehara, H.; Umezawa, H. d. Antibiotics, 1960, 13, 362 (Chem. Abstr., 1960, 57, 7115). [67] Murakami, M.; Suzuki, K.; Kang, J.W. Nippon Kagaku Zasshi, 1962, 83, 1226 (Chem. Abstr., 1962, 59, 13868). [68] Bernfith, G.; Gera, L.; G0ndOs, G.; Kovfics, K.; Janvfiri, E.; Sebesty~n, G.; Ecsery, Z.; Hermann, J. Ger. Often. 2,624,290 (Chem. Abstr., 1977, 87, 102009). [69] Bartoli, G.; Cimarelli, C.; Marcantoni, E.; Palmieri, G.; Marino, P. J. Org. Chem. 1994, 59, 5328 [70] Takahashi, T.; Kato, A.; Hirose, N. Yakugaku Zasshi, 1960, 80, 1440 (Chem. Abstr., 1960, 55, 5483). [71] Burrows, B.F.; Turner, W.B.J. Chem. Soc. (C), 1966, 255. [72] Polveche, M.; Bar, D.; Debaert, M.; Febvay-Garot, N. Bull. Soc. Chim. Fr., 1977, 995. [73] Oleksyszyn, J.; Gruszecka, E.; Kafarski, P.; Mastalerz, P. Monats. Chem., 1982, 113, 59. [74] Berthelot, P.; Debaert, M.; Cremieux, A.; Baghadi, N. II Farmaco, 1983, 38, 73. [75] Mittendorf, J.; Kunisch, F.; Matzke, M.; Militzer, H.C.; Endermann, R.; Metzger, K.G.; Bremm, K.D.; Plempel, M. Eur. Pat. Appl. EP 571,870 (Chem. Abstr., 1994, 121, 83988). [76] Nohira, H.; Kikegawa, K. Jpn. Kokai Tokkyo Koho JP 06/271527 (Chem. Abstr. 1995, 122, 132653) [771 Brown, T.H.; Harling, J.D.; Orlek, B.S. PCT Int. Appl. WO 95 26,327 (Chem. Abstr., 1996, 124, 145497). [78] Yamazaki, T.; Zhu, Y.F.; Probstl, A.; Chadha, R.K.; Goodman, M. J. Org. Chem., 1991, 56, 6644. [79] Armarego, W. L. F.; Kobayashi, T.: J. Chem Soc. (C), 1969, 1635. [80] Armarego, W. L. F.; Kobayashi, T. J. Chem Soc. (C), 1970, 1597; Armarego, W.L.F.J. Chem. Soc. (C), 1971, 1812. [54]
2-AMINOCYCLOPENTANECARBOXYLIC ACID
[81] [82] [83] [84]
[85] [86] [87]
[881 [89] [90] [91] [92] [93] [94] [95] [96] [97] [98]
[99]
[loo] [101] [102] [103] [104]
[105] [1061 [107] [10S] [109]
303
Nohira, H.; Ehara K.; Miyashita, A. Bull. Chem. Soc. Jpn., 1970, 43, 2230. Saigo, K.; Okuda, Y.; Wakabayashi, S.; Hoshiko, T.; Nohira, H. Chem. Lett., 1981, 857 Saigo, K.; Ogawa S.; Kiguchi, S.; Kashara A.; Nohira, H. Bull. Chem. Soc. Jpn., 1982, 55, 1568. Szakonyi, Z.; F016p, F.; Bern,Sth, G.; T6r6k, G.; P6ter, A. Tetrahedron" Asymmetry, 1998, 9, 993. Kanerva, L.T.; Csom6s, P.; Sundholm, O.; Bem~ith G.; FtllSp, F. Tetrahedron: Asymmetry, 1996, 7, 1705. Evans, C.; McCagur R.; Roberts, S.M.; Sutherland, A.G.; Wisdom, R. J. Chem. Soc., Perkin Trans. 1, 1991, 2276. Roberts, S.M. Pure Appl. Chem., 1992, 64, 1933. Csom6s, P., Kanerva, L.T.; Bern~ith, G.; F016p, F. Tetrahedron: Asymmetry, 1996, 7, 1789. Theil, F.; Ballschuh, S. Tetrahedron: Asymmetry, 1996, 7, 3565. Tilley, J.W.; Danho, W.; Shiuey, S.J.; Kulesha, I.; Swistok, J.; Makofske, R.; Michalewsky, J.; Triscari, J.; Nelson, D.; Weatherford, S.; Madison, V.; Fry, D.; Cook, C. J. Med. Chem., 1992, 35, 3774. N6teberg, D.; Branalt, J.; KvamstrOm, I.; Classon, B.; Samuelsson, B.; Nillroth, U.; Danielson, U.H.; Karl6n, A.; Hallberg A. Tetrahedron, 1997, 53, 7975. d'Angr J.; Desmaele, D.; Dumas, F.; Guingant, A. Tetrahedron: Asymmetry, 1992, 3, 459. Juaristi, E.; Garcia-Barradas, O. In Enantioselective Synthesis offl-Amino Acids, Juaristi, E. Ed.; Wiley-VHC, New York, 1997. pp. 139-150. Davies, S.G.; Ichihara, O.; Waiters, I.A.S. Synlett, 1993, 461. Davies, S.G.; Ichihara, O.; Lenoir, I.; Waiters, A.S.J. Chem. Soc., Perkin Trans. 1, 1994, 1411. Enders, D.; Wiedemann, J.; Bettray, W. Synlett, 1995, 369. Enders, D.; Wiedemann, J. Liebigs Ann., 1997, 699. (a) Enders, D.; Bettray, W.; Schankat, J.; Wiedemann, J. In Enantioselactive Synthesis of fl-Amino Acids, Juaristi, E. Ed.; Wiley-VHC, New York,1997. pp.187-210.; (b) Bartoli, G.; Cimarelli, C.; Dalpozzo, R.; Palmieri, G. Tetrahedron, 1995, 51, 8613. Cimarelli, C.; Palmieri, G.; Bartoli, G. Tetrahedron: Asymmetry, 1994, 5, 1455. Kobayashi, S.; Kamiyama, K.; Imori, T.; Ohno, M." Tetrahedron Lett. 1984, 25, 2557 Tamura, N.; Kawano, Y.; Matsushita, Y.; Yoshioka, K.; Ochiai, M." Tetrahedron Lett. 1986, 27, 3749 Kaiga, H.; Kobayashi, S.; Ohno, M. Tetrahedron Lett., 1988, 29, 1057. Kobayashi, S.; Kamiyama, K.; Ohno, M. J. Org. Chem., 1990, 55, 1169 Vanderplas, B.; Murtiashaw, C.W.; Sinay, T.; Urban, F.J. Org. Prep. Proc. Int., 1992, 24, 685. Grees, T.; Schweizer, W.B.; Seebach, D. Helv. Chim. Acta, 1993, 76, 2640. Elliott, R.P.; Hui, A.; Fairbanks, A.J.; Nash, R.J.; Winchester, B.G.; Way, G.; Smith, C.; Lamont, B.; Storer, R.; Fleet, G.W.J. Tetrahedron Letters, 1993, 34, 7949. Hayashi, Y.; Rohde, J. J.; Corey E. J. J. Am. Chem. Soc. 1996, 118, 5502 Voigt, K.; Lansky, A.; Noltemeyer, M.; de Meijere, A. Liebigs Ann., 1996, 899. Xu, D.; Prasad, K.; Repic, O.; Blacklock, T.J. Tetrahedron: Asymmetry, 1997, 8, 1445.
304
FERENC FOLOP
[110] FtliOp, F.; Csirinyi, G.; Bem/tth, G. Acta Chim. Hung., 1988, 125, 193. [111] Ftll6p, F.; Szakonyi, Z.; Bemfith, G.; Sohfir, P. d. Heterocyclic Chem., 1997, 34, 1211. [112] Pihlaja, K.; F01Op, F.; Mattinen, J.; Bem,Sth, G. Acta Chem. Scand, 1987, B41, 228. [113] Stfijer, G.; Sz/Ske-Molnfir, Z.; Bemfith, G.; Sohfir, P. Tetrahedron, 1990, 46, 1943. [114] GOndOs, G.; Gera, L.; Wittmann, G.; Balfispiri, L.; Kovfics, K. Acta Chim. Hung., 1987, 124, 187. [115] Bemfith, G.; Gera, L.; GOndSs, G.; Pfinovics, I.; Eesery, Z. Acta Chim. ,4cad. Sci. Hung., 1976, 89, 61. [116] FOlOp, F.; Palk6, M. unpublished results. [117] FtllOp, F.; Bemfith, G.; Pihlaja, K. Adv. Heterocyclic Chem., 1998, 69, 349. [118] Sohfir, P.; SzSke-Molnfir, Z.; Stfijer, G.; Bemfith, G. Magn. Reson. Chem., 1989, 27, 959. [119] Frimpong-Manso, S.; Nagy, K.; Stfijer, G.; Bernfith, G.; Sohfir, P. d. Heterocyclic Chem., 1992, 29, 22 I. [120] Bemfith, G.; Gera, L.; GOndOs, G.; Hermann, J., Szentivfinyi, M.; Eesery, Z.; Janvfiri E. Ger. Often. 2,643,384 (Chem. Abstr., 1977, 87, 168078). [121] Bernfith, G.; Gera, L.; GOndSs, G.; Eesery, Z.; Hermann, J.; Szentivfinyi, M.; Janvfiri, E. Australian 507, 798 (Chem. Abstr., 1981, 94, 175144). [122] /~cs, M.; Fogassy, E.; Faigl, F. Tetrahedron, 1985, 41, 2465. [123] Armarego, W.L.F.; Reeee, P.A.d. Chem. Soc., Perkin Trans. 1, 1974, 2313. [124] Kricheldorf, H.R. MakromoL Chem., 1974, 175, 3343. [125] Krieheldorf, H.R. Liebigs Ann. Chem., 1975, 1387. [126] Nohira, H.; Watanabe, K.; Ishikawa, T.; Saigo, K. Heterocycles, 1977, 7, 301. [127] Ribfir, B.; Petrovic, A.; GOnd6s, G.; Bemfith, G. Co,st. Struct. Commun., 1979, 8, 671. [128] Kapor, /i,.; Ribfir, B.; Argay, G.; K/dmfin, A.; Bern~tth, G. Cryst. Struct. Commun., 1980, 9, 343. [1291 Kapor, /k.; Ribfir, B.; Argay, G.; Kfilm,Sn, A.; Bemfith, G. Cryst. Struct. Commun., 1980, 9, 347. [1301 FOlOp, F.; Simon, K.; T6th, G.; Hermecz, I.; M6szfiros, Z.; Bemfith, G. d. Chem. Sot., Perkin Trans. 1, 1982, 2801. [131] Stfijer, G.; Szab6, A.E.; FtllSp, F.; Bemfith, G. Synthesis, 1984, 345. [132] Bemfith, G.; T6th, G.; FtllOp, F.; GOnd6s, G.; Gera, L. d. Chem. Sot., Perkin Trans. 1, 1979, 1765. [133] Stfijer, G.; Szab6, A.E., Pintye, J.; Bemfith, G.; Sohfir, P. d. Chem. Soc. Perkin, Trans. 1, 1985, 2483. [134] Stfijer, G.; Szab6, A.E.; F01Op, F.; Bemfith, G.; Sohfir, P. Chem. Ber., 1987, 120, 259. [1351 F018p, F.; Bem~th, G.; Pihlaja, K.; Mattinen, J.; Argay, G.; Kfilmfin, A. Tetrahedron, 1987, 43, 4731. [1361 Stfijer, G.; Szab6, A.E.; Bemfith, G.; Sohfir, P. d. Chem. Soc., Perkin Tram. 1, 1987, 237. [137] Bemfith, G.; Stfijer, G.; Szab6, A.E.; SzSke-Molnfir, Z.; Sohfir, P.; Argay, Gy.; Kfilmfin, A. Tetrahedron, 1987, 43, 1921. [~38] F01Op, F.; Pihlaja, K.; Mattinen, J.; Bemfith, G. Tetrahedron Lett., 1987, 28, 115.
2-AMINOCYCLOPENTANECARBOXYLIC ACID
305
[139] FtllOp, F.; Huber, I.; Szab6,/i,.; Bem,'tth, G.; Soh,fr, P. Tetrahedron, 1991, 47, 7673. [140] Huber, I.; Szab6, A.; FtilOp, F.; Bem~tth, G.; Soh~tr, P. Tetrahedron, 1992, 48, 4949. [141] FillOp, F.; Huber, I.; Bem~th, G.; T6th, G.; Pricken, A.; Pflegel, P. Pharmazie, 1990, 45, 109. [142] Canonne, P.; Akssira, M.; Dahdouh, A.; Kasmi, H.; Boumzebra, M. Tetrahedron, 1993, 49, 1985. [143] Akssira, M.; Dahdouh, A.; Kasmi, H. Bull. Soc. Chim. Belg., 1993, 102, 227. [144] G6nd6s, G.; Gera, L.; Dombi, G.; Bem,fth, G. Monats. Chem., 1996, 127, 1167. [145] St~jer, G.; Szab6, A.E.; Fillap, F.; Bem6th, G.; Soh~r, P. J. Heterocyclic Chem., 1984, 21, 1373. [146] FtilOp, F.; Csirinyi, G.; Bem~tth, G. Synthesis, 1985, 1149 [147] FtllOp, F.; Pihlaja, K.; Bem6th, G. Acta Chem. Scand., 1987, B41, 147. [148] Bem,Sth, G.; L,Sng, K.L.; GOndOs, G.; M,Srai, P.; Kovfics, K. Tetrahedron Lett., 1968, 4441. [1491 Ftil0p, F.; Pihlaja, K.; Mattinen, J.; Bem6th, G. Tetrahedron, 1987, 43, 1863. [~50] Ftll6p, F. Acta Chim. Hung.-Models in Chemistry, 1994, 131 697. [15~] Valters, R.E.; FtllOp, F.; Korbonits, D. Adv. Heterocyclic Chem., 1996, 66, 1. [152] Bem6th, G.; Kov6cs, K.; L6ng, K.L. Tetrahedron Lett., 1968, 2713. [~53] Bem~tth, G.; FtllOp, F.; Gera, L.; Hackler, L.; K,51m6n, A.; Argay, G.; SoMr, P. Tetrahedron, 1979, 35, 799. [1541 Gera, L.; Bem~th, G.; Soh~ir, P. Acta Chim. Hung., 1980, 105, 293. [155] St(0er, G.; Szab6, A.E.; FtilOp, F.; Bem6th, G.; Soh,Sr, P. Heterocycles, 1982, 19, 1191. [1561 FtllOp, F.; Bem6th, G.; Argay, Gy.; K/tlm6n, A.; Soh6r, P. Tetrahedron, 1984, 40, 2053. [1571 Bern6th, G.; Ftll0p, F.; K61m6n, A.; Argay, G.; Soh6r, P.; Pelczer, I. Tetrahedron, 1984, 40, 3587. [158] FUlOp, F.; St6jer, G.; Bem~tth, G.; SoMr, P. Tetrahedron, 1985, 41, 5159. [1591 Bem~tth, G.; FtilOp, F.; Csirinyi, G.; Szalma, S. Monats. Chem., 1987, 118, 503. [160] FtllOp, F.; L~z,~r, L.; Pelczer, I.; Bem6th, G. Tetrahedron, 1988, 44, 2992. [161] Bem,Sth, G.; FUlOp, F.; Soh,Sr, P. Tetrahedron, 1987, 43, 4359. [162] FiilOp, F.; Bem6th, G.; Csirinyi, G. Org. Prep. Proc. Int., 1986, 20, 73. [163] L~z~r, L.; FtllOp, F.; Bem~th, G.; K~tlm6n, A.; Argay, G. J. Heterocyclic Chem., 1991, 28, 1213. [164] Oksman, P.; Pihlaja, K.; Ftll0p, F.; Bem6th, G. Rapid Commun. Mass. Spectrom., 1995, 9, 615. [165] Pihlaja, K.; Mattinen, J.; FtllOp, F. Magn. Reson. Chem., 1996, 34, 998. [166] St~jer, G.; Szab6, E.A.; FtllOp, F.; Bem6th, G.; SoMr, P. Heterocycles, 1993, 36, 995. [167] Ohki, H.; Inamoto, Y.; Kawarata, K.; Kamimura, T.; Sakane, K. J. Antibiotics, 1991, 44, 546. [168] Xie, J.; Soleilhar J.M.; Renwart, N.; Peyroux, J.; Roques, B.P.; Fourni,Zaluski, M.C. Int. J. Peptide Protein Res., 1989, 34, 246. [169] Yamazaki, T.; Probstl, A.; Schiller, P.W. Goodman, M. Int. J. Peptide Protein Res., 1991, 37, 364.
FERENC FI~LOP
306
[170] Yamazaki, T.; Huang, Z.; Probstl, A.; Goodman, M. Proc. Eur. Pept. Syrup., 21 st 1990, pp.389-392. (Chem. Abstr., 1991, 115, 208553). [171] Yamazaki, T.; Goodman, M. Chirality, 1991, 3, 268. [172] Boz6, B.; Ftll6p, F.; T6th, G.K.; T6th, G.; Szllcs, M. Neuropeptides, 1997, 31, 367. [1731 Huang, Z.; Probstl, A.; Spencer, J.R.; Yamazaki, T.; Goodman M. Int. d. Peptide Protein Res., 1993, 42, 352. [1741 Kamphuis, J.; Lr F.; Tancredi, T.; Toniolo, C.; Temussi, P.A. Quant. Struct.-Act. Relat., 1992, 11,486. [175] Appella, D.H.; Christianson, L.A.; Klein, D.A.; Powell, D.R.; Huang, X.; Barchi, J.J.; Gellman, S.H. Nature, 1997, 387, 381. [176] Appella, D.H.; Christianson, L.A.; Karle, I.L.; Powell, D.R.; Gellman, S.H.d. Am. Chem. Soc., 1996, 118, 13071. [177] Tamariz, J. In Enantioselective Synthesis of fl-Amino Acids, Juaristi, E. Ed.; Wiley-VHC, New York, 1997. pp. 45-66. [178] Connors, T.A., Elson, L.A.; Haddow, A.; Ross, W.C.J. Biochem. Pharmacol., 1960, 5, 108. [179] Uehara, H.; Miyagawa, T.; Tjuvajev, J.; Joshi, R.; Beattie, B.; Oku, T.; Finn, R.; Blasberg, R. J. Cereb. Blood Flow Metab., 1997, 1239. [18o] Segal, M.; Sims, K.; Smissman, E. Br. J. Pharmacol., 1975, 54, 181. [1811 Nicoll, R.A. Br. J. Pharmacol., 1977, 59, 303. [1821 Early, S.L.; Michaelis, E.K.; Mertes, M.P. Biochem. Pharmacol., 1981, 30, 1105.
[1831 Mikami, Y.; Scalarone, G.M.; Kurita N.; Yazawa, K.; Miyaji, M. Nippon Ishinkin Gakkai Zasshi, 1992, 33, 355 (Chem. Abstr., 1993, 118, 76906). [184] Capobianco, J.O.; Zakula, D.; Coen, M.L.; Goldman, R.C. Biochem. Biophys. Res. Commun., 1993, 190, 1037. [185] Mikami, Y.; Scalarone, G.M.; Kurita, N.; Yazawa, K.; Miyaji, M. Jpn. J. Med. Mycol., 1992, 33, 355. [186] Naruse, N.; Yamamoto, S.; Yamamoto, H.; Kondo, S.; Masuyosih, S.; Numata, K.; Fukagawa, Y.; Oki, T. d. Antibiotics, 1993, 46, 685. [187] Jethwaney, D.; Hofer, M.; Khaware, R.K.; Prasad, R. Microbiology, 1997, 143, 397.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22 9 2000 Elsevier Science B.V. All rights reserved
307
STRUCTURE-ACTIVITY RELATIONS OF FLAVONOIDS AS ANTIOXIDANT AND PRO-OXIDANT COMPOUNDS P. COS, M. CALOMME, L. PIETERS, A. J. VLIETINCK and D. VANDEN BERGHE*
Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Antwerp, Belgium A B S T R A C T : Flavonoids are a group of naturally occurring polyphenolic compounds which possess a wide range of biological activities. The present review is focused on the antioxidant activity of five major groups of flavonoids, namely flavanones,
dihydroflavonols, flavones, flavonols, and flavanols. Four basic mechanisms of such action have been described and are applicable to flavonoids: (1) free radical scavenging activity, (2) quenching of singlet oxygen, (3) chelation of transition metals, and (4)
inhibition of enzymes. Related structure-activity relationships are discussed in detail for the first three mechanisms. Structure-antioxidant activity relationships often depend on assay procedures. Some of the techniques currently used in antioxidant research are briefly discussed. On the other hand, the pro-oxidant effects of some flavonoids are also summarized. INTRODUCTION
Flavonoids, a group of naturally occurring polyphenolic compounds, are widely distributed in fruits and vegetables and are therefore frequently consumed. Some flavonoids have been reported to possess interesting medicinal properties, including antiviral [1-2], antimutagenic [3-4], antilipoperoxidant [5-6], radioprotective [7-8], anti-complementary [9-10], and anti-inflammatory [11-12] activities. Several epidemiological studies suggest that a high consumption of flavonoids is inversely related to the risk of cardiovascular diseases [13-15]. These effects are believed to be related to the antioxidant properties of flavonoids. Consequently, a growing interest in the study of the antioxidant activity of flavonoids exists today. Four basic mechanisms of such action have been described and are applicable to fiavonoids: (1) free radical scavenging activity [16], (2) quenching of singlet oxygen [17], (3) chelation of transition metals [18], *to whom correspondence 'should be addressed, phone: (32) 3- 820 25 42; fax': t32) 3- 820 25 44'i e-mail:
[email protected]
308
V A N D E N B E R G H E et al.
and (4) inhibition of enzymes, such as lipoxygenase [19-20], cyclooxygenase [20], monooxygenase [21], xanthine oxidase (XO) [22], and phospholipase A2 [23-24]. The latter mechanism has already been extensively reviewed [25-26], therefore only the first three mechanisms will be discussed in detail. In vitro antioxidant activity of flavonoids has been recognized for a considerable time. Recently, an increasing number of studies have been published on structure-activity relationships of flavonoids as antioxidants [27-29] and the bioavailability of flavonoids [30-31]. Clearly, these are important issues in the investigation of the possible role of flavonoids as food antioxidants and as therapeutical agents. Many techniques currently used in antioxidant research are highly specialized and the obtained results depend often on the applied techniques. Therefore, some of the pitfalls in antioxidant research will be briefly discussed. In addition to antioxidant activities, it is also important to consider that some flavonoids were found to have pro-oxidant effects, possibly promoting oxidative damage to DNA, lipids, proteins, and carbohydrates [29, 32]. R E A C T I V E OXYGEN OXIDATIVE STRESS
SPECIES,
ANTIOXIDANTS
AND
Reactive oxygen species (ROS) is a collective term for oxygen-derived species, namely oxygen radicals and certain non-radicals that are oxidizing agents and/or easily converted into radicals (Table 1) [33-34]. A free radical is defined as any species capable of independent existence and containing one or more unpaired electrons [35]. Biological systems are exposed to ROS which have been formed endogenously or which are present in the environment. ROS can be generated in vivo by enzymes (e.g. XO, NADPH oxidase, ...), by auto-oxidation (e.g. adrenaline, dopamine, ...), by leakage of electrons from the mitochondrial electron transport chain, by the use of certain chemicals (e.g. doxorubicin, cigarettes, ...), by the catalytic action of free transition metals (e.g. Fe 2§ Cu § ...), and by radiation from the environment (e.g. radon gas, UV light, 9..) [35-36]. ROS are ubiquitously present in the environment, especially in the form of atmospheric ozone (03) and as antimicrobial compounds (HOC1 and H202) [35]. All aerobic organisms possess an antioxidant defense system to protect against ROS-mediated injury. A broad definition of an antioxidant is "any substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate" [37-38]. Oxidizable substrates include DNA, lipids, proteins, and carbohydrates. This definition emphasizes the importance of the damaged target studied and the source of ROS used when the in vitro antioxidant activity of a compound is studied [37]. The human antioxidant defense system consists of an enzymatic and a non-enzymatic system.
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
Table 1.
Reactive Oxygen Species (ROS) ,
IIII
I
309
,,
I
,,,
,
ROS
Symbol
Superoxide
02"-
Hydroxyl
OH*
Alkoxyl
RO* / LO*
Peroxyl
RO0* / LO0*
Hydroperoxyl
HO2"
,
,
Radicals
ii
i
i
Non-radicals
i
Hydrogen peroxide
H202
Hypochlorous acid
HOCI
Ozone
03
Singlet Oxygen
Io 2
Lipid peroxides ,
,,
,,
R = Organic molecule; L = Unsaturated lipid molecule
The enzymatic system includes enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), catalase, and others. SOD catalyzes the dismutation of 02 ~ at a rate constant 104 times higher than that for spontaneous dismutation at pH 7.4 (Eq. 1) [39]. 202*- + 2H +
SOD
H202 + 02
(Eq. 1)
Human cells have a manganese-containing SOD (Mn-SOD) in the mitochondria, whereas the copper- and zinc-containing SOD (Cu,ZnSOD) is primarily present in the cytosol [39]. Two enzyme systems exist to catalyze the breakdown of H202. Firstly, the enzyme catalase, which is located in the peroxisomes, converts H202 into H20 and 02 (Eq. 2) [35]. catalase 2H202 ~ 2H20 + 02 (Eq. 2) Secondly, the group of selenium-containing glutathione peroxidases uses H202 as an oxidant to convert reduced glutathione (GSH) to oxidized glutathione (GSSG) (Eq. 3) [35]. 2GSH + H202
GSHPx _ GSSG + 2H20 (Eq. 3)
The non-enzymatic antioxidants are classified into two groups, namely the endogenous and the dietary antioxidants. GSH, uric acid, and albumin
310
VANDEN BERGHE et aL
are some of the endogenous antioxidants, whereas vitamins C and E, 13carotene, and flavonoids represent some of the dietary antioxidants [4041]. In healthy individuals, the production of ROS is balanced with the antioxidant defense system. Oxidative stress is generated when the balance is in favor of the ROS (Fig. (1)) [39, 42]. Increased production of ROS and/or a depletion of antioxidant levels will cause oxidative stress. Most cells can tolerate a mild degree of oxidative stress, by the action of repair systems, such as DNA repair enzymes [43], phospholipid hydroperoxide GSHPx [44], and proteases that destroy abnormal proteins [45] and by up-regulating the antioxidant system by expressing oxidative stress genes [46]. ROS and oxidative stress are associated with a variety of human diseases, including Cancer [47-48], Parkinson's disease [49], and atherosclerosis [50-51 ]. ROS contribute to tissue damage in diseases such as rheumatoid arthritis [52] and cataract [53]. However, ROS are in most cases not considered as the major cause, but rather as a consequence contributing to the etiology of the disease [54-55]. There exists growing evidence that a high intake of vegetables and fruits can decrease the incidence of cardiovascular diseases [56-57] and cancer [58-61].
Fig. (1). Imbalance between reactive oxygen species and antioxidant defense system causes oxidative stress.
STRUCTURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS
311
Antioxidants present in such diets are most likely responsible for these effects or contribute to it [39]. Consequently, they can play an important role in the modem human diet. FLAVONOIDS Flavonoids are diphenylpropanes (C6-C3-C 6 configuration) and consist of a benzene ring (A) condensed with a six-membered ring (C), which is substituted with a phenyl group (B) (Fig. (2)) [62]. The individual carbon atoms of the A- and C-rings are numbered with ordinary numerals and those of the B-ring with primed numerals. At the 2-position of the C-ring flavonoids have a substituted phenyl group, whereas isoflavonoids have
( 0
0
Flavanonel
3'
f
Dlhydroflavonol
4' 5' 5
4
Basic flavonoid Structure
(
1 O Flavone
Fig. (2).
Flavanol
)
Structuraldiversityof flavonoids.
O Flavonol
312
VANDEN B E R G H E et al.
this phenyl group at the 3-position. The six-membered ring is fused with respectively a T-pyrone for flavonols and flavones, a dihydropyrone for dihydroflavonols and flavanones, and a dihydropyrane for flavanols [62]. Flavonols and dihydroflavonols differ from respectively flavones and flavanones by the presence of a OH group in the 3-position. Structural diversity of the flavonoids is possible by hydroxylation, methoxylation, glycosylation, etc. (Table 2) [63]. Hydroxylation and methoxylation of flavonoids often occur at positions 3, 5, 7, 3', 4', and 5', whereas the glycosides are most commonly located in positions 3 and 7. Glycosides contain sugars, such as glucose, galactose, rhamnose, xylose, and arabinose, although disaccharides or oligosaccharides also occur [63]. Table
2.
Some
Flavanone
i
Important
Flavonoids
and
Structures
7-Hydroxyflavanone
-
-
OH
.
Hespcrctin
-
OH
OH
OH
Naringenin
-
OH
OH
Dihydroflavonol ii
Flavanol
Taxifolin
Ill
Ill
OH
OH
OH
(+)-Catechin
~-OH
OH
OH
(-)-Epicatechin
a-OH
OH
OH
(-)-Epigallocatechin
a-OH
OH
OH
i
I
I
.
II
OCH 3 OH Illll
-
Ill I
OH
OH
-
OH
OH
-
OH
OH
Ill
-
OH
i
Flavone
.
-
i II
i
their
OH
OH
i
Apigenin
OH
OH
Chrysin
OH
OH
Diosmctin
OH
Luteolin
OH
HI
-
OH
OH
OH
OCH 3
OH
OH
OH
II
Flavonol
OH
-
OH
Galangin
OH
OH
OH
Kacmpfcrol
OH
OH
OH
-
OH OH
OH
I
Fisetin
-
-
OH
-
.....
Morin
OH
OH
OH
OH
Myricetin
OH
OH
OH
-
Quercetin
OH
OH
Quercitrin
OR!
OH
Rutin
OR 2
OH
....
R I = Rhamnosyl; R2 - Rutinosyl
OH
OH
OH
OH
OH
OH
-
OH
OH
OH
-
OH
OH
OH
-
. . . . . . . .
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
313
The flavonoids are a group of secondary metabolites widely distributed in the plant kingdom [63]. In 1984, more than 4000 flavonoids were identified in plants [64]. The major dietary sources of flavones are spices and pot herbs, such as parsley, rosemary, and thyme [65], whereas flavonols are predominantly found in onions, kale, broccoli, apples, berries and cherries, and in tea and red wine [66]. The flavanones are mainly restricted to citrus fruits [67], and flavanols are found in considerable amounts in tea, apricots, apples, and cherries [66]. In 1976, the daily dietary intake of flavonoids in the USA was estimated to be approximately 1 gram, of which about 170 mg - expressed as glycosides - or 115 mg - expressed as aglycones - were flavanones, flavones, and flavonols [67]. Recently, Hertog et al. calculated the Dutch intake of five major food antioxidant flavonoids, namely the flavonols quercetin, kaempferol, and myricetin and the flavones apigenin and luteolin [68]. The average dietary intake was estimated to be 23 mg/day with quercetin as the main compound [68]. In the Seven Countries Study, it was shown that the daily consumption of these five major food antioxidant flavonoids varied strongly between different countries, namely from 2.6 mg in West-Finland to 68.2 mg in Ushibuka, Japan [15]. Only a very limited number of studies are available on the bioavailability of flavonoids in humans. Mainly based on animal studies, Kfihnai concluded that there are two restrictions to the absorption of flavonoids from the intestine [67]. Firstly, dietary flavonoids having a 5,7,3',4' hydroxylation pattern are preferentially subjected to microbial degradation in the colon. Secondly, most flavonoids (with the exception of flavanols) are present in foods as [3-glycosides, and only flavonoids without a sugar molecule, the so-called aglycones, are thought to be absorbed. Since no [3-glycosidase activity is present in the gut or the intestinal wall, hydrolysis of the flavonoid ~-glycosides by the intestinal microflora is necessary for absorption. In addition, also the flavonoid aglycones are very susceptible to microbial degradation, and therefore only a marginal absorption of dietary flavonoids can be expected [66, 67]. Das demonstrated that oral administration of labeled 3 - 0 methylcatechin to three volunteers was followed by plasma peak levels within 2 hours after administration [69]. Hollman et al. studied the absorption of different glycosides of quercetin and the quercetin aglycone in nine healthy ileostomy subjects to avoid losses caused by colonic bacteria [30-31 ]. The absorption after oral administration decreased in the following order: 52% for quercetin glucosides from fried onions > 24% for quercetin aglycone > 17% for quercetin rutinoside [31 ]. Consequently, humans absorb considerable amounts of quercetin, but the absorption depends on the glycosidic nature of the flavonoid [31 ].
314
V A N D E N B E R G H E et as
ANTIOXIDANT ACTIVITY OF FLAVONOIDS
Free Radical Scavenging Activity Free radicals are reactive and therefore short-lived as a consequence of containing one or more unpaired electrons. However, there is still a considerable difference in their reactivities and lifetimes [70]. 02 ~ reacts quickly with only a few molecules, whereas OH" is so unstable that it reacts at the site of formation. ROO 9 and RO 9 have intermediate reactivities [40]. Their relatively short lifetimes and migration distances complicate the measurement of free radicals. Free radicals can be generated in vitro either enzymatically, e.g. by the xanthine/XO system, or nonenzymatically, e.g. by the action of transition metals or ionizing radiation. Flavonoids can interfere both with the formation and propagation reactions of free radicals [27]. To act as an antioxidant, a free radical scavenger should produce a more stable and therefore less harmful compound after reaction with a free radical. In general, flavonoids are excellent hydrogen- or electron-donors and the resulting flavonoid radical is relatively stable due to electron delocalization and intramolecular hydrogen bonding [71]. 02 ~ Scavenging Activity
02*- is a ROS produced in vivo by a number of enzyme systems, by autooxidation reactions, and by non-enzymatic electron transfers [33]. Hydroperoxyl radical (HO2o), which is much more lipid-soluble than O2o-, can be formed in acid medium by protonation of 02 ~ [35]. Many studies demonstrated the O2"- scavenging activity of flavonoids and tried to determine structure-activity relationships. In most studies either the phenazine methosulfate-NADH system [72-76] or the xanthine/XO system [16, 77-79] was used to produce 02"-. The non-enzymatic phenazine methosulfate-NADH system [80] generates a myriad of free radicals and other ROS, including OH', H202, and 02 ~ [81]. Consequently, this method is not recommended [35]. Another source of O2~ is the enzyme XO that catalyzes the oxidation of hypoxanthine and xanthine to uric acid. During re-oxidation of XO, molecular oxygen acts as electron acceptor, producing 02"- and H202 [82]. These reactions can be written as follows (Eq. 4-5) [78]: xanthine + 202 + H20 xanthine + 02 + H20
XO = uric acid + 202 ~ + 2H § (Eq. 4) XO ~ uric acid + H202 (Eq. 5)
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
315
It is important to consider that flavonoids can inhibit XO. Those containing C-5 and C-7 OH groups are especially involved, so that an overestimation occurs for the 02"- scavenging activity of these compounds [83-86]. This can be checked by measuring the formation of uric acid (spectrophotometrically or by HPLC) [38]. To detect O2"- most studies used the indirect NBT assay [16, 72-75]. In this method O2"-reduces the yellow dye nitro-blue tetrazolium (NBT 2§ to produce the blue formazan, which is measured spectrophotometrically at 560 nm [87]. Cotelle et al. [77, 79] and Cos et al. [78] investigated the O2"- scavenging activity of flavonoids using respectively chemiluminescence with lucigenin and the nitrite method. Only Sichel et al. studied the 02"- scavenging activity of flavonoids by a direct measurement of the reduction of the 02"- electron spin resonance (ESR) signal intensity, which was generated by the addition of acetone to H202 in alkaline medium [88]. This method has the disadvantage of using media with high and therefore with a nonphysiological pH. All studies conclude that an ortho-dihydroxy (catechol) structure in the B-ring is important for a high O2"- scavenging activity [ 16, 72, 74-79, 88]. Baumann et al. [72] and Huguet et al. [74] found that the scavenging activity of flavones and flavanones decreased in the following order: an ortho-dihydroxy function in the B-ring > a 4'-OH substitution > no OH groups in the B-ring. According to Huguet et al. [74], this was not the case for flavonols, since he found - in contrast to Baumann et al. [72] and Cos et al. [78] - that galangin was a better O2"- scavenger than quercetin. Huguet et al. also demonstrated that a resorcinol moiety in ring A increased the scavenging activity and that flavones were slightly more active than flavanones [74]. Cotelle et al. observed that the O2"- scavenging activity was favored by the presence of a 6-OH group [77, 79]. Interestingly, there is no unanimity about the role of the C-3 OH group regarding the 02"- scavenging activity. Sichel et al. [88] and Cos et al. [78] found a higher activity for flavonols compared to related flavones, whereas Huguet et al. [74] found the opposite. According to Baumann et al., there was no relationship between the presence of a C-3 OH group and the 02"scavenging activity [72]. Replacing the active OH groups by one or more methoxy groups decreased the activity [72, 74, 77], whereas the activity increased by introducing a gallate structure [76]. The presence of a sugar moiety attached to the flavonoid nucleus affects the activity. Aglycones were more active than their glycosides, but for quercetin and rutin (quercetin 3-rutinoside) contradictory results have been reported [73, 74, 88]. Yuting et al. [75] and Huguet et al. [74] described a higher activity for rutin, whereas Sichel et al. [88] demonstrated a higher activity for quercetin. Robak and Gryglewski [73] and Baumann et al. [72] observed no significant difference in activity between these compounds.
316
VANDEN B E R G H E et al.
OH" Scavenging A cavity The most common method to produce the highly reactive OH" is the socalled Fenton reaction, which was first observed by Fenton in 1894 (Eq. 6) [89]. Fe 2§ + H202 --> Fe 3§ + OH- + OH" (Eq. 6) However, there is still doubt whether some or even all of the OH" produced from the Fenton reaction may remain bound to the iron center, either as the FeOH 3+ (Eq. 7)or the FeO 2§ intermediate (Eq. 8) [90-91]. Fe 2+ + H202 "-~ FeOH 3§ + OH- (Eq. 7) Fe 2+ + H202 ~ FeO 2+ + H20 (Eq. 8) The Fenton reaction was used by Yoshiki et al. [92] and Puppo [93], whereas Husain et al. [94] generated OH" by UV photolysis of H202. It should be considered that several flavonoids chelate transition metals and therefore inhibit the production of OH- generated in the Fenton reaction. Different methods were applied to detect OH'. Husain et al. [94] spin trapped the OH" by 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and measured these radicals by the highly sensitive HPLC-electrochemical detection method [95]. The technique of spin-trapping involves the addition of a free radical to a diamagnetic compound (the spin-trap) to form a more stable radical adduct [96]. The intensity of chemiluminescence was observed by Yoshiki et al. [92] as a measure for OH. scavenging activity, whereas Puppo [93] detected the OH. either by the deoxyribose assay (see also section pro-oxidant activity of flavonoids) or by aromatic hydroxylation of phenylalanine. In aromatic hydroxylation OH-attack a specific detector, in this case phenylalanine, resulting in the production of three isomeric tyrosines. These hydroxylated products can be separated by HPLC equipped with a highly sensitive electrochemical detector [97]. Only a few studies are published concerning the OH- scavenging activity of flavonoids. In accordance with the 02 ~ scavenging activity, three studies reported an increase of the OH" scavenging activity with an increasing number of OH groups in the B-ring [92-94]. However, this was not confirmed by Hanasaki et al. [16] and Laughton et al. [32], who observed that e.g. quercetin could enhance OH" production and could therefore act as a pro-oxidant (see also section pro-oxidant activity of flavonoids). Apparently, the OH" scavenging activity varies with the method applied. The introduction of a C2-C3 double bond and/or a C-3 OH group had no significant effect on the activity [92, 94], whereas according to Husain et al. [94] the presence of a carbonyl function at C-4 appeared to play an important role. Husain et al. reported that the most
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
317
active flavonoids (myricetin and quercetin) were better OH" scavengers than ethanol and 1-butanol, but less active than DMSO [94].
RO0" Scavenging Activity ROO 9are formed in vivo during the major chain-propagation step in lipid peroxidation, but they can also be formed from DNA and proteins [37]. The ROO 9scavengers can be divided into water-soluble scavengers, e.g. scavengers of ROO 9resulting from DNA or proteins, and lipid-soluble scavengers, e.g. chain-breaking antioxidants [37]. Torel et al. [98] studied the ROO 9scavenging activity of flavonoids from the inhibition of the formation of hydroperoxide isomers during auto-oxidation of linoleie acid and methyl linoleate [99]. The level of hydroperoxides was evaluated by measuring the UV-absorption of conjugated dienes at 234 nm. The ROO ~ scavenging activity increased in the following orders" cateehin < quercetin < rutin = luteolin < kaempferol < morin for linoleic acid; rutin < catechin < morin = kaempferol for methyl linoleate [98]. A common method to generate ROO" is the use of azo-initiators (R-N=N-R), which decompose at a temperature-controlled rate, yielding molecular nitrogen and two carbon radicals (R~ (Eq. 9) [ 100]. The R ~ can react rapidly with molecular oxygen to produce ROO ~ (Eq. 10). R-N=N-R --) R~ + N2 + R ~ (Eq. 9) R 9+ 0 2 ~
R O O ~ (Eq.
10)
The solubility and the rate of decomposition both depend on the structure of R [100]. 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) is a water-soluble radical generator, whereas 2,2'-azobis(2,4dimethylvaleronitrile) (AMVN) is lipid-soluble. The oxygen radical absorbing capacity (ORAC) assay is based on the generation of ROO ~ by AAPH and the detection of the chemical damage to the indicator protein ~l-phycoerythrin (]3-PE), which is monitored by a decrease in its fluorescence emission [101-102]. The inhibition of the fluorescence loss, which is expressed in ORAC units, is related to the antioxidant ability to protect ~-PE against free radical damage. One ORAC unit is equivalent to the protection provided by 1 IxM Trolox, a watersoluble vitamin E analog [101 ]. Flavones with a single OH group had a lower activity than Trolox, whereas kaempferol, quercetin, and myricetin had respectively ORAC values of 2.7, 3.3, and 4.3 [29]. Methylation of all the OH groups of luteolin resulted in an undetectable ORAC value [29]. Cao et al. concluded that a catechol moiety on ring B is essential for a high ROO 9 scavenging activity and that this activity is proportional to the number of OH groups [29].
318
VANDEN B E R G H E et al.
Wang and Zheng [103] studied the ROO 9scavenging activity of several flavonoids by AAPH-initiated auto-oxidation of linoleic acid in cetyl trimethylammonium bromide micelles. The activity decreased in the following order: {x-tocopherol (a well-known chain-breaking antioxidant) > morin > rutin > quercetin. Naringin (naringenin 7-O-neohesperidoside) and hesperidin (hesperetin 7-O-rutinoside) had no significant activity. According to Wang and Zheng, this was due to the blockade of the 7-OH group - which is most likely the ROO 9target site - with a glycoside [103]. Terao et al. [104] and Ioku et al. [105] investigated the ROO ~ scavenging activity of flavonoids by measuring the inhibition of an azo compound-induced peroxidation of methyl linoleate in a solution of nhexane, isopropanol, and ethanol [106]. According to Terao et al., the rate constants for the ROO ~ scavenging activity decreased in the following order: o~-tocopherol > quercetin > epicatechin and epicatechin gallate [104]. loku et al. demonstrated the effect of glycosylation on the ROO 9 scavenging activity [105]. All quercetin monoglucosides had a lower ROO ~ scavenging activity than the aglycone. However, it appeared that the position of the glucosyl moiety on the flavonoid was important for both the ROO 9scavenging activity and the antioxidant mechanism. The 7-OH position appeared to affect the stability of trapped radicals, resulting in a decrease of chain-breaking activity, whereas an ortho-dihydroxy structure in the B-ring and to a lesser degree the 3-OH group were essential for the ROO 9scavenging activity of flavonoids [ 105]. Terao et al. [104] and Ioku et al. [105] also examined the hydroperoxide formation from liposomal phosphatidylcholine, which contained the chelator diethylenetriaminepenta-acetic acid (DETAPAC) for preventing the pro-oxidant effect of traces of iron, by exposure to the water-soluble radical initiator AAPH. Terao et al. demonstrated that - in contrast to the rate constants for the ROO 9scavenging activity - the effectiveness of epicatechin and epicatechin gallate in membrane phospholipids was comparable to that of quercetin, but much higher than observed for {xtocopherol, o~-Tocopherol is located within the membranes, whereas flavonoid aglycones interact with the polar head of phospholipid bilayers, being located near the surface of membranes [107]. This location may be favorable for scavenging of ROO" originating from the aqueous phase [104105].
DPPH Scavenging A ctivity Several studies described the interaction of flavonoids with the 1,1diphenyl-2-picrylhydrazyl (DPPH) free radical [76, 79, 107-108] (Fig. (3)). DPPH is a "stable" free radical and is frequently used in ESR studies [109]. The DPPH assay provides information on the reactivity of flavonoids with a stable free radical [79]. Because of its odd electron, DPPH gives a strong absorption band in ethanol at 517 nm. As this
STRUCTURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS
319
electron becomes paired off, the absorption vanishes and the degree of resulting decolorization is stoichiometric with respect to the number of N{C6Hs}2 I *N O2N " ~y NO2
NO2 Fig. (3). Structure
of the I,l-diphenyl-2-picrylhydrazyl (DPPH) free radical.
electrons taken up [109]. Cotelle et al. demonstrated that a catechol or a pyrogallol type moiety on the B-ring was essential for the DPPH radical scavenging activity [79]. Ratty et al. studied the flavonoid-DPPH interaction in Tris/NaCl buffer (pH=7.4) and in liposomal suspension [107]. The DPPH scavenging activity decreased in the following orders: OH
OH
OH
OH HO
~
O
HO
i"i~
/ OH
",s OH
OH OH I C~-O
(-)-Epicatechln
Ho.2 OH
OH
OH Gallic acid
X
HO
OH ,,,pOH
OH OH
OH
(-)-Eplcatechin gallate
H O i_i i i ~l ( ~_0.
~
OH OH
o
--
"*e
OH
OH (-}-Eplgallocatechln Fig. (4). Structures
OH
{-}-EplgaIlocatechin gallate of some flavanols and flavanol-gallate esters.
-\
OH
320
VANDEN BERGHE et at
quercitrin = quercetin > morin > rutin = myricetin > taxifolin > catechin for the buffer, and morin > quercetin = rutin > myricetin > quercitrin > catechin > taxifolin for the liposomal suspension [107]. Rekka and Kourounakis examined the interaction of five hydroxyethyl rutosides and quercetin with the DPPH free radical and found quercetin and 7-monohydroxyethyl rutoside to be more active than di-, tri- and tetrahydroxyethylated rutosides [108]. According to Chen and Ho, the DPPH radical scavenging activity of some flavanols (see Fig. (4) for structures) decreased in the following order: epigallocatechin gallate > epicatechin gallate > epigallocatechin > epicatechin [76]. Free Radicals and Pulse Radiolysis
Pulse radiolysis is a unique technique to generate specific radicals and to study the kinetic parameters for the primary radical attack, the stability of the secondary formed radicals, and the redox potentials of these secondary formed radicals [110]. In pulse radiolysis a short pulse of ionizing radiation will generate excited states, ions, and radicals in a sample solution [111]. In aqueous solutions a short electron pulse will result within 1 nanosecond (10 -9 sec) in the formation of the following primary radicals (Eq. 11) [112]:
H20 - - - / ~ - - - -
e-aq + H" + OH* (Eq. 11)
The radicals e-aq (hydrated electron) and H. (hydrogen atom or hydrogen radical) are reducing species, whereas the OH* is an oxidizing agent I112]. It is possible to 'select out' a particular radical by alterations of pH and addition of various compounds [35]. One method of generating almost exclusively OH* is irradiation of an aqueous solution, saturated with nitrous oxide gas (N20), converting e-aq into extra OH* (Eq. 12-13) [112]. e-aq + N 2 0 --4 N2 + O -
(Eq. 12)
O-+ H + - ) OH-(Eq. 13) In an oxygenated formate (HCOO-) solution the following reactions occur to produce 02" (Eq. 14-17) [35, 112]. e-aq + 02 --) 02"- (Eq. 14) H" + 02 --) 02"- + H + (Eq. 15) OH" + HCOO- --) CO2"- + H20 (Eq. 16) CO2 ~ + 02 ---) C O 2 + 02 ~
(Eq. 17)
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
321
Azide radicals (N3") are produced by reaction of radiolytieally generated OH" with azide anions (N3-) (Eq. 18) [ 113]. N3- + OH" --r N3~+ OH- (Eq. 18) The application of N3~ in pulse radiolysis studies includes several advantages: (1) the low absorption of N3 ~ facilitates optical pulse radiolysis experiments, (2) the non-ionic character of N3" excludes effects of charge and dependence on ionic strength, and (3) in contrast to OH', N3~ react primarily via electron transfer, thereby simplifying the secondary chemistry [113]. Bors et al. used these electrophilic N3" to produce flavonoid aroxyl radicals, whose generation rates and stability were determined [114-117]. The stability of the flavonoid aroxyl radical prevents radical chain reaction and defines the extent of flavonoid activity as antioxidant [116]. It was shown that quercetin and kaempferol have good radical scavenging activities, but only the quercetin aroxyl radical decays slowly enough (more than 40 times slower compared to kaempferol) to act as a potential antioxidant [ 116]. According to Bors et al., three structural elements are required for optimal radical scavenging activity [ 115-117]: (1)
(2) (3)
An ortho-dihydroxy structure (catechol moiety) in the B-ring, which is the radical target site for flavonoids with a saturated C-2 and C-3 double bond, confers a high stability to the flavonoid aroxyl radical and participates in electron delocalization. A 2,3-double bond in conjugation with a 4-oxo function is responsible for electron delocalization from the B-ring. The 3- and 5-OH groups are, together with the 4-oxo function, essential for maximal radical scavenging activity. In the absence of a C2-C3 double bond, the presence of an aliphatic 3-OH group does not contribute to the radical scavenging activity [ 117].
Bors et al. also investigated the reaction rate constants of flavonoids with different radical species and found for quercetin the following values: 43xl 08 M -1 sec-I with OH', 0.9x105 M -! see-1 with 02"- and ranging from 107 until 108 M -! sec-l with different ROO- [ 116, 118]. Jovanovic et al. studied the acid-base and redox properties of the flavonoid aroxyl radicals by pulse radiolysis of aqueous solutions [119]. The flavonoid aroxyl radicals were generated by bromide radical ion (Br2"-) induced oxidation of flavonoids (F-OH) (Eq. 19), followed by a rapid loss of a proton to form the neutral flavonoid aroxyl radical (F-O') (Eq. 20). F-OH + Br2"- --->F-OH "+ + 2Br-(Eq. 19) F-OH .+ --->F-O. + H + (Eq. 20)
322
V A N D E N B E R G H E et a2
The absorption spectra of the flavonoid aroxyl radicals resembled those produced by the action of N3 9[114-117]. The dissociation constants (pKa) of the 3',4'-dihydroxyflavonoid aroxyl radicals were similar to those of the 3,4-dihydroxybenzoate and the 3,4-dihydroxycinnamate radicals, namely ranging from 4 to 5. Consequently, at physiological pH the 3',4'dihydroxyflavonoid aroxyl radicals are negatively charged which can hinder their passage through the cell membranes. The reduction potentials of flavonoids depend strongly on the electron-donating properties of the substituents of the B-ring. Hesperidin aroxyl radical had a reduction potential of 0.72V, whereas the aroxyl radicals of catechin and rutin had reduction potentials of respectively 0.57V and 0.60V. According to the low reduction potentials of the flavonoid radicals, it is expected that they can efficiently scavenge ROO 9and 02 ~ [119]. Their rate constants with 02 ~ were determined by kinetic conductivity at pH=10 and by optical pulse radiolysis at pH=7. At pH=10, the flavonoids were ionized, in either the A- or B-ring, and their reaction with 02 ~ resulted in the reduction of 02" to 022- and the formation of the flavonoid aroxyl radical (Eq. 21). O2"- 4- F-O- --> 022- + F-O ~ (Eq. 21)
The rate constants were in the following increasing order: 8.8xl 02, and 5.1• M -1 sec -l for respectively galangin, kaempferol, quercetin, and rutin [119]. These values confirm the importance of a catechol moiety for O2o- scavenging activity (see also section O2~ Scavenging Activity). According to Jovanovic et al., the reactivity of flavonoids with O2~ depends on their charge. Uncharged catechin reacted at pH=7 four times faster than negatively charged catechin at pH-10. Nevertheless, flavonoids can scavenge O2"- at pH values ranging from 7 to l0 [119]. Trichloromethylperoxyl radicals (CC1302~ are frequently used to study the ability of compounds to scavenge ROO ~ [ 120]. CC1302 ~ were generated by radiolysis of an aqueous mixture of propan-2-ol and CCI4. Flavonoids showed variable rate constants: 108 M -l see -~ for fisetin and morin, 107 M -l sec -! for myricetin and quercetin, and 106 M -l see -l for catechin and epicateehin [ 121 ].
2.4x103, 4.7•
Free Radicals and Electron Spin Resonance (ESR) ESR is a spectroscopic technique for studying paramagnetic molecules or molecules with unpaired electrons, which include free radicals and many transition metals [ 122]. An unpaired electron can align itself in an external magnetic field either parallel or antiparallel to that field, yielding two possible energy levels. Application of electromagnetic radiation of the proper energy will induce transitions and an ESR signal will appear. The
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
323
basic ESR line of an electron can "split" into two or more "hyperfine" lines as a consequence of the interaction of the unpaired electron with nuclei having magnetic moments (e.g. IH, 2H and 13C). The distance between two lines in the ESR spectrum is called the hyperfine coupling constant. Hyperfine splitting can greatly enhance the identification of free radicals through analysis of their ESR spectrum [122]. Van Acker et al. investigated the antioxidant activity of flavonoids by quantum chemical calculations together with experimental parameters, such as half peak oxidation potentials (Ep/2) and ESR data [123]. Calculations of the geometry of several flavonoids indicated that the structure of a flavonol aglycone was planar, i.e., complete conjugation, in contrast to the flavones and the flavonol derivative rutin. The lack of a 3OH group in a flavone and the resulting loss of the corresponding hydrogen bond caused a slight twist (+ 20%) between the B-ring and the rest of the molecule. The introduction of a sugar moiety on the 3-OH group of a flavonol caused the loss of eoplanarity of the B-ring with the rest of the molecule and could therefore explain the lower scavenging activity of rutin compared to quercetin [123]. Consequently, the planarity of a flavonoid is related to its scavenging activity and a free 3-OH group is required for high activity. Actually, a free 3-OH group interacts with the B-ring through a hydrogen bond, holding the B-ring in the same plane as the rest of the molecule. The difference in heat of formation (AAHf) between the flavonoid molecule and its corresponding radical is a suitable parameter to describe the abstraction of a H 9from an O-H bond and is well correlated for the flavonols with their Ep/2. Studies of the spin distributions of quercetin and taxifolin showed that most spins remained in the B-ring. More precisely, 84% of the spins were located on the 4'-oxygen. However, spin densities and ESR hyperfine coupling constants both measured and calculated indicated that the delocalization was larger in the flavonol quercetin than in the dihydroflavonol taxifolin [ 123]. Cotelle et al. [77, 79] and Kuhnle et al. [124] investigated by ESR the ability of flavonoids to form stable radicals. The flavonoid radicals were generated by aerial, alkaline oxidation of the corresponding flavonoids. It was shown that flavonoids with a pyrogallol or catechol moiety in the Bring gave rise to semiquinone or pyrogallol-type anion radicals which were stable enough to be detected by ESR.
Trolox Equivalent Antioxidant Capacity (TEAC) Miller and Rice-Evans have recently developed a method to measure the total antioxidant capacity of solutions of pure compounds, as well as plasma, serum and other body fluids, plant extracts, beverages, etc. [125126]. This method is based on the inhibition of the absorbance of the 2,2'azinobis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS o§ by antioxidants. Although the chromophore ABTS o§ has
324
VANDEN BERGHE et as
characteristic absorption maxima at 417, 645, 734, and 815 nm, the suppression of the absorbance is quantified at 734 nm, because at this wavelength myoglobin and many other compounds do not interfere [ 127]. The relatively long-lived radical cation ABTS ~ is formed through the interaction of ABTS with the ferrylmyoglobin radical ('X-Few=O) (Eq. 23) [ 126], which is derived from the oxidation of metmyoglobin (HX-Fe m) by H202 (Eq. 22) [128]. HX-Fe m + H202 ~ "X-Fdv=o + H20 (Eq. 22) 9X-Few=O + ABTS ~ HX-Fd n + ABTS "+ (Eq. 23) The Trolox Equivalent Antioxidant Capacity (TEAC) assay reflects the relative ability of antioxidants ( h y d r o g e n - o r electron-donating compounds) to scavenge the ABTS o§ generated in the aqueous phase, compared to the antioxidant standard Trolox [127]. The TEAC is defined as the millimolar concentration of Trolox having an antioxidant capacity equivalent to 1 mM of the test compound [127]. However, according to Strube et al. the decrease in absorbance of ABTS o+measured at a fixed time could be originated from a scavenging effect and/or a decrease in the rate of ABTS -+ formation [129]. It was shown that Trolox scavenges ABTS "+, whereas quercetin acts by both mechanisms [129]. By using the TEAC assay, Rice-Evans et al. established a structureantioxidant activity relationship of flavonoids [28, 130-134]. The antioxidant activity of a flavonoid is determined by the following structural elements [28, 130-134]:
(1)
(2)
(3)
A catechol moiety in the B-ring is essential for a high antioxidant activity, whereas the presence of a pyrogallol moiety or a single OH group in the B-ring reduces the activity. The TEAC values were 4.72, 3.12, 2.55, and 1.34 mM for respectively quercetin, myricetin, morin, and kaempferol. The C2-C3 double bond enhances the activity by stabilization of the flavonoid aroxyl radical through electron delocalization across the molecule. Taxifolin (dihydroquercetin) had a TEAC value of 1.9 mM compared to 4.72 mM for quercetin. In the absence of a catechol moiety, the C2-C3 double bond has no significant influence on the antioxidant activity. The TEAC values of kaempferol (1.34 mM) and dihydrokaempferol (1.39 mM) were quite similar, because of the low hydrogen-donating capacity of a monophenolic B-ring. A free 3-OH group is required to get the maximal antioxidant activity out of a flavonoid with a catechol moiety in the B-ring and a C2-C3 double bond, such as quercetin. Blockade of the 3-OH group by introducing a glycoside reduces the antioxidant activity, which was showed by a TEAC value of 2.4 mM for rutin compared to 4.72
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
(4)
(5)
325
mM for quercetin. Removing the 3-OH group from quercetin led to a lower TEAC value of 2.1 mM for luteolin. The 3-OH group has no influence on the activity when there is no ortho-dihydroxy structure in the B-ring present. The TEAC values were quite similar for apigenin (1.45 mM) and kaempferol (1.34 mM). The 5,7-dihydroxyphenolic A-ring contributes to the antioxidant activity with a TEAC value ranging from 1.35 to 1.5 mM. Glycosylation of the 7-OH group has a negative influence on the TEAC values, like naringenin (1.5 mM) and hesperetin (1.37 mM) compared to respectively their glycosides narirutin (naringenin 7-Orutinoside) (0.76 mM) and hesperidin (1.08 mM). The flavanol catechin, which lacks the C2-C3 double bond together with the 4-oxo function in the C-ring, had a TEAC value of 2.4 mM, whereas epigallocatechin had a TEAC value of 3.82 mM. It appears that when there is no conjugation between the A- and B-ring via the C-ring, the insertion of a third adjacent OH group in the B-ring enhances the antioxidant activity. Another possibility to increase the antioxidant activity is the introduction of a gallic acid moiety by ester linkage via the 3-OH group (epicatechin gallate) giving a TEAC value of 4.93 mM.
These results are in agreement with the structure-activity relationship proposed by Bors et al., who used pulse radiolysis to study the antioxidant activity of flavonoids [116-118].
Quenching of Singlet Oxygen Singlet oxygen ( I O 2 ) is an excited state of 0 2 where spin restriction is removed. There are two types of singlet oxygen, namely the very shortlived sigma singlet oxygen (lEg§ and the, because of its longer life-time, biologically more important delta singlet oxygen (IAgO2) [35]. In IAgO2 the two outer electrons occupy the same orbital, whereas in leg+ they occupy separate orbitals [42]. IO2 is formed in vivo via photosensitized reactions or by chemical excitation reactions which do not require light excitation [35]. Flavonoids can interact with IO2 in two different ways: they can combine chemically with ~O2 or they can transfer the excitation energy of tOE to the flavonoid molecule, which then enters an excited state. The latter phenomenon is called quenching [35]. Flavonoids have been reported to quench singlet oxygen [ 17, 135-137], but only Toumaire et al. tried to establish a structure-activity relationship [17]. Sorata et al. studied the suppression of photosensitized hemolysis of human erythrocytes by the flavonols quercetin and rutin [135]. At lower concentrations, the effect of quercetin was more important than that of rutin [135]. According to Takahama et al., quercetin suppressed JOE-dependent photobleaching of
326
VANDEN B E R G H E et al
crocin in the presence of rose bengal as photosensitizer [136]. This was accompanied by the transformation of quercetin into a compound with an absorption maximum at approximately 330 nm. Devasagayam et al. studied the protective role of flavonoids in inhibiting single-strand breaks in plasmid pBR322 DNA induced by IO2, which was generated by thermal dissociation of the endoperoxide of 3,3'-(1,4naphtylene)dipropionate [137]. In this assay, all flavonoids tested protected the DNA mainly by chemical reaction or quenching of IO2, whereas quercetin also might react with DNA. The protective ability of the flavonoids decreased in the following order: myricetin > catechin > rutin > fisetin > luteolin > apigenin > quercetin (which showed no protection) [137]. At equimolar concentration (100 ~tM) myricetin showed a better protective effect than ct-tocopherol. However, increasing the concentration of myricetin did not increase its protective effect [137]. Toumaire et al. produced ~O2 by photosensitization with rose bengal [17]. The rate constants of the chemical reaction of flavonoids with IO2 and their rate constants of ~O2 quenching were determined by kinetic measurements and near-IR luminescence of IO2 [ 17]. Flavones were much more reactive than the corresponding flavonols, whereas flavanones, dihydroflavonols, and flavanols were chemically inert towards IO2 [17]. These results were explained by the dioxetane formation of IO2 with the C2-C3 double bond of the flavonoid molecule [17]. However, it is important to consider that a five-membered cyclic peroxide intermediate was proposed for the chemical reaction of 3-hydroxyflavone with IO2 [138]. The dioxetane formation of a flavonoid is activated by an electrondonating substituent in position 3 [35]. Dioxetanes are unstable and decompose into two carbonylated fragments. In conclusion, the efficiency of the chemical reactivity between flavonoids and IO2 is greatly affected by the structure of ring C" the presence of a OH group in the 3-position increases the reactivity of the C2-C3 double bond towards IO2. This was confirmed by the fact that glycosylation of the 3-OH (e.g. rutin compared to quercetin) decreased the chemical reactivity [17]. The degree of quenching of ~O2 decreased in the following order: catechin > fisetin > quercetin = rutin > luteolin > taxifolin [ 17]. Catechin was the most efficient quencher with a rate constant of 5.8xl 06 M -l see-~, being 3 to 5 times higher than the other flavonoids measured. This might be due to the absence of a carbonyl group in ring C of catechin, resulting in a less planar structure [17]. The most important structural element for efficient IO2 quenching by flavonoids is the presence of a catechol moiety on ring B [17]. Chelation of Transition Metals
The transition metals iron and copper are essential cofactors of several enzymes which are involved in oxygen metabolism. Approximately two-
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
327
third of the human body's iron is found in hemoglobin, with smaller amounts in myoglobin, various enzymes (e.g. catalase, cytochrome P450, ...), the iron carder proteins transferrin and lactoferrin, and the iron storage proteins ferritin and hemosiderin [39]. Copper is present in several enzymes, such as Cu, Zn-SOD and dopamine-I]-hydroxylase. In plasma it is mainly attached to ceruloplasmin, a large protein containing six or seven copper ions per molecule, and to a minor extent to the plasma protein albumin [139]. When these transition metals are present in free state in biological systems, they can catalyze free radical reactions. For example, iron and copper act as catalysts in the generation of OH" through the Fenton reaction (see section OH" Scavenging Activity) and the HaberWeiss reaction. The Haber-Weiss reaction or superoxide-driven Fenton reaction is an interaction between H202 and 02 ~ in the presence of traces of iron or copper, and results in the formation of OH" (Eq. 24-26) [35]. Fe 3+ + O2"- ~ Fe 2+ +
02
(Eq. 24)
Fe 2§ + H202 ~ Fe 3§ + OH- +OH" (Fenton reaction) (Eq. 25)
O2"- + H202 ---) 02 § OH- + OH ~ (Haber-Weiss reaction) (Eq. 26) Traces of transition metals accelerate also auto-oxidation reactions and the decomposition of LOOH to LOO o, LO o, and cytotoxic aldehydes [139]. Agents that complex these transition metals decrease their biological effects dramatically [36]. Flavonoids possess the ability to form a complex with Cu 2§ ions [140141]. Hudson and Lewis investigated this Cu2§ activity by studying their UV spectra [140]. The 4-carbonyl group in cooperation with either the 3-OH group or the 5-OH group was responsible for the complexation of Cu 2§ with flavones and flavanones. This was confirmed by the formation of a weak complex with the flavanol catechin [140]. According to Thompson et al., flavonoids chelated Cu 2§ ions mainly at neutral and high pH values, and to a minor extent at low pH values [ 141 ]. Actually, the degree of Cu 2§ complexation - but not the stability - is related to the basicity of the flavonoid molecule. Quercetin was found to form the most stable complex with Cu 2§ 5-Hydroxyflavone formed a slightly more stable complex than 3-hydroxyflavone. In fact, the sixmembered chelate ring of the former had a higher stability compared to the five-membered ring of the latter [141 ]. The relative chelating capacity of a series of flavonoids was studied spectrophotometrically by measuring the ability to release Fe 2§ ions from a Fe2§ complex and to chelate Fe 2§ ions [27]. The flavonoids were classified into four groups" (1) flavonoids, such as apigenin, that could release Fe 2§ from this complex were ranked as good chelators, (2) flavonoids, such as quercetin, that could not remove Fe 2§ from the
328
V A N D E N B E R G H E et al.
complex but chelated Fe 2§ (3) flavonoids, such as galangin, that could not remove Fe 2§ from the complex and chelated Fe2§ to a minor extent, and (4) flavonoids, such as naringin, that did not chelate Fe 2§ [27]. In conclusion, a 3-OH group and a catechol moiety are more important for iron chelation than a 5-OH group [27]. Afanas'ev et al. demonstrated the ability of rutin to form a stable complex with Fe 2§ at physiological pH [142]. The absorption spectrum of the Fe2§ complex did not change during 8 hours [142]. Morel et al. investigated radiochemically the capacity of three flavonoids (catechin, quercetin and diosmetin) and desferrioxamine - a powerful chelator of Fe 3§ - to remove Fe 3§ from iron-loaded hepatocytes [18, 143]. Nitrilotriacetic acid - a low affinity iron-chelator- was used to maintain Fe 3§ in a soluble state. The iron-chelating ability decreased in the following order: desferrioxamine > catechin > quercetin > diosmetin (which had a very low activity) [18, 143]. PRO-OXIDANT ACTIVITY OF FLAVONOIDS Nowadays, there is a growing interest in the pro-oxidant effects of flavonoids. It is of great importance to understand that a compound cannot just be classified as an antioxidant on the basis of one antioxidant experiment, because it can act as a pro-oxidant in another system. For example, diethylstilboestrol is an inhibitor of lipid peroxidation in vitro [144], but can accelerate oxidative DNA damage in vivo [145]. It is therefore recommended to use a battery of test systems involving DNA, lipids, proteins, and carbohydrates to determine both the antioxidant and pro-oxidant properties [ 146]. Some of the pro-oxidant effects of flavonoids have been attributed to the fact that they can undergo auto-oxidation reactions when they are dissolved in aqueous buffers. The rate of auto-oxidation can be determined by measuring the oxygen consumption. Hodnick et al. studied the inhibition of succinoxidase in isolated mitochondria and found that in a series of flavonols with various B-ring OH substitution patterns the strongest enzyme inhibitors were those with a pyrogallol (myricetin) or catechol moiety (quercetin) [ 147-148]. The flavonols myricetin, quercetin, and quercetagetin (6-hydroxyquercetin) were found to undergo autooxidation, resulting in the production of 0 2 " - a n d H202 during mitochondrial respiratory bursts. According to Hodnick et al., 02"-was involved in the auto-oxidation of myricetin, since addition of SOD inhibited this process, in contrast to the auto-oxidation of quercetagetin [147]. Myricetin, quercetin, and quercetagetin had oxidation potentials significantly lower than those that did not undergo auto-oxidation. Consequently, inhibition of succinoxidase by the three flavonols appears to be associated with their ability to participate in redox reactions [ 148].
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
329
In agreement with the results from Hodnick et al. [ 147-148], Canada et al. [149] found myricetin underwent auto-oxidation more rapidly than quercetin, but this process was prevented by glycosylation of the 3-OH group (rutin). Virtually no auto-oxidation was detected for quercetin at physiological pH, whereas the rate of auto-oxidation increased considerably with increasing pH. Both the addition of iron for the flavonols quercetin and myricetin, and the addition of iron followed by SOD for quercetin resulted in an increase of the auto-oxidation rate, whereas the addition of SOD to the two flavonols reduced the autooxidation rate [149]. Consequently, 02 ~ might be involved in the autooxidation of myricetin [ 149] - a flavonol with a pyrogallol moiety in the Bring - as indicated for the auto-oxidation of pyrogallol [150]. The ROS produced by auto-oxidation were identified as OH" and H202 by ESR [149]. The deoxyribose assay is a simple test tube assay to determine the antioxidant or pro-oxidant properties of test compounds against carbohydrates [ 151-152]. In the deoxyribose assay, OH" are produced from the reaction of H202 with Fe 2+ (Fenton reaction), where the latter is formed by the ability of ascorbic acid to reduce ferric iron (Fe 3§ to ferrous iron (Fe 2+) (Eq. 27). Fe 3§ + ascorbate ---) Fe 2+ + semidehydroascorbate (Eq. 27) Deoxyribose attacked by OH- produces malondialdehyde (MDA); following heating in acidic conditions, MDA forms with thiobarbituric acid (TBA) a pink chromogen that can be quantified spectrophotometrically at 532 nm (Fig. (5)). H
~,~N~
OH +
~HO
T~ ~.~
~H2
H+
"~
"~
OH
OH
SH
CHO OH
TBA
MDA
|TBA)2-MDA adduct (pink chromogen)
Fig. (5). Detection of malondialdehyde (MDA) by the thiobarbituric acid (TBA) test.
Two different antioxidant actions can be measured using this method. Firstly, the addition of Fe 3§ as Fe3+-EDTA to the assay results in free OH- in solution and therefore a compound can inhibit the deoxyribose damage by scavenging these OH*. Secondly, the addition of free Fe 3§ instead of Fe3+-EDTA, will result in the binding of some of these Fe 3+ to deoxyribose, followed by a reduction to Fe 2§ which then stimulates OH" generation at the site of damage [153]. The ability of a compound to inhibit the site-specific deoxyribose degradation is due to its iron chelating
9
330
V A N D E N B E R G H E et as
activity, thereby preventing the iron ion to catalyze the Fenton reaction. Potential pro-oxidant properties of test compounds can be determined when ascorbic acid is omitted from the test system. A potential prooxidant compound will reduce Fe 3§ to Fe2§ resulting in the generation of OH" [33]. A disadvantage of this easy-to-use assay is the restriction to water-soluble compounds, because organic solvents, like ethanol and DMSO are powerful OH" scavengers. Laughton et al. [32] and Puppo [93] investigated the pro-oxidant activity of flavonoids with the deoxyribose assay. According to Laughton et al., the flavonols quercetin and myricetin accelerated at 100 IxM the generation of OH" from a mixture of H202 and Fe3+-EDTA [32]. This specific pro-oxidant activity was higher for myricetin than for quercetin. Interestingly, the addition of SOD prevented their pro-oxidant effect, which excludes a simple reduction of Fe 3+ to Fe 2§ by the flavonols. Consequently, Laughton et al. suggest therefore that Fe3+-EDTA induces an oxidation of the flavonols, yielding O2o-, which is responsible for the reduction of Fe3§ to Fe2§ These pro-oxidant effects were not demonstrated with Fe 3§ alone or with the physiologically more relevant Fe3§ diphosphate (ADP) or Fe3§ complexes. The pro-oxidant results were explained by the action of EDTA altering the redox potential and iron solubility [154]. Puppo found that the OH" generating effect of flavonoids decreased in the following order: myricetin > quercetin > catechin > morin > kaempferol; flavone had no effect [93]. Changes in UV/VIS absorbance, which were due to the oxidation of the flavonoid molecule, were related to their pro-oxidant activity and were faster with myricetin than with quercetin. These results, together with the observation that the addition of SOD inhibited the pro-oxidant activity, support the pro-oxidant hypothesis of Laughton et al. [32]. In the presence of the biologically more relevant chelators ATP and citrate, the flavonoids did not act as pro-oxidants. Consequently, iron chelators influence the pro-oxidant or antioxidant capacities of flavonoids. Laughton et al. investigated also the possible pro-oxidant effects of quercetin and myricetin on DNA with the bleomycin assay [32]. Bleomycin is an antitumor antibiotic that binds both iron ions and DNA. A bleomycin-Fe 3§ complex will degrade DNA in the presence of 02 and a reducing agent (e.g. ascorbic acid) or H202 [33, 155]. In addition, deoxyribose - a sugar originating from DNA - degrades to form MDA, which can be measured with TBA (see also deoxyribose assay) [155]. Laughton et al. found that quercetin and myricetin accelerated DNA damage, most likely by reducing the Fe3+-eomplex to a Fe2+-complex [32]. Hanasaki et al. [ 16] investigated the antioxidant and pro-oxidant activities of flavonoids with a method [156] based on the oxidation of DMSO by OH" generated by the Fenton reaction - to form the stable compound methanesulfinic acid (MSA) as shown in Eq. 28. -
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
331
OHH3C---S----CH
3 +
OH 9
~
C H 3" +
DMSO
H3C---S
OH
(Eq. 2 8 )
MSA
MSA is derivatized with a diazonium salt to produce a diazosulfone, which is quantified by HPLC [157]. The flavonoids catechin, epicatechin, 7,8-dihydroxyflavone, and rutin showed a OH" scavenging activity 100 to 300 times higher than mannitol, whereas baicalein (5,6,7trihydroxyflavone), quercetin, myricetin, and morin enhanced the OHproduction [16]. The pro-oxidant activity of morin increased with an increasing concentration of morin, whereas an increasing concentration of the other three flavonoids did not enhance their pro-oxidant activity. Hanasaki et al. suggested that these pro-oxidant flavonoids produce H202 during their auto-oxidation, which then stimulates the Fenton reaction [16]. Cao et al. investigated the possible pro-oxidant activity of flavonoids by the ORAC assay [29]. Depending on their concentration, flavonoids scavenged OH- in a CuE+-n202 system. However, at higher flavonoid concentrations the activity declined with increasing concentration. In the presence of Cu 2+ without H202, the flavonoids acted as pro-oxidants rather than as antioxidants [29]. The pro-oxidant activity decreased in the following order: myricetin > quercetin > kaempferol > taxifolin [29]. Flavone and 6-hydroxyflavone showed no significant pro-oxidant activity. Clearly, these results demonstrate the importance of a C2-C3 double bond and the number of OH substitutions regarding the Cu2+-initiated prooxidant activity of flavonoids [29]. Several flavonoids were investigated in one assay for both the inhibition of XO and the O2"- scavenging activity, measured spectrophotometrically S u m m a r y of the Classification of Flavonoids into Six Categories According to their Inhibition of Xanthine Oxidase (XO) and Superoxide
Table 3.
Radical (02*-) Scavenging Activity [78] Category
Inhibition of XO
02 ~ Scavenging Activity i
Example i
A
o
+
(-)-epigallocatechin
B
+
o
baicalein
C
+
+
myricetin
D
+
-
galangin
E
o
-
7-hydroxyflavanone
F
o i
o: No effect; +: Effect; -: Pro-oxidant effect
O
naringenin
332
FANDEN BERGHE et aL
and with the nitrite method, respectively [78]. The relative concentrations of uric acid (as a measure of the inhibitory activity on XO) and 02"- were displayed as a function of the concentration of the flavonoid tested (Fig. (6)). According to these two activities, the flavonoids were classified into six categories (Table 3). Since a reduction in uric acid production and consequently an inhibition of XO results automatically in an equivalent reduction in 02"-- which is the result of the XO activity - a flavonoid with a complementary pro-oxidant activity will not show an equivalent reduction of 02"- due to complementary 02"- production. The 02"- curve will be situated above the uric acid curve. Galangin, chrysin, apigenin, and luteolin were classified as XO inhibitors with an additional pro-oxidant effect on the production of 02"- (category D) (Fig. (6)), whereas 7hydroxyflavanone had a marginal effect on XO, but a pro-oxidant effect on the production of 02"- (category E) (Fig. (6)) [78]. 02"- Scavengers without inhibitory activity on XO and XO inhibitors with an additional 02"- scavenging activity were classified into categories A and C, respectively (Fig. (6)) [78]. category A
(%)120' 110
~.
superoxld'e.=.~ m,,
90.
8o~ 70 ~ 60 ~ 50: 40: 3o-
2O ~
~o~ 0
. . . . . . . .
.I
i
. . . . . . . .
,
120(%) " uric acid 110 100 90 .80 "70 "60 "50 "4O -30 "20 .10 0 -
-
- J ~ - - -
I I0 lO0 concentration of taxffolin Qd~ ~tegory C
(%)12G
120{%)
Superoxlde~
uric acld [ llO 100
90 ~
90 ~' 80 p
80~ 7O~
60~ 30 ~
~ 60 "50 p ~ 4o 30
20 ~
,2o
5o ~ 4o ~
:~o
IO: 0
,
.1
.
.
.
.
.
.
.
.
~JL,
.-.
. . . .
concentration of fl~e~in (/dUO
0 100
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
{%} 120
11o;r
(Fig. 6). contd .....
category D
~
superoxld~
333
120{%}
uric acid llO I00
9o; 8o ~ 7o ~
'80
6o.
"60
50' 40" 30 ~ 2O ~ ]0~
o!1
(%} 120~_~
140 "30 "2O
I I0 I00 concentration of galangin {glD category E 0--- superoxide
II0' O0 =
90 ~
soi
70" 6O; 5O ~ 4O ~ 30 ~ 20"
120(%} uric acld
II0 I00 9O 8O 70 6O 5O 4O 3O 2O I0
0
0
.I I I0 I00 concentration of 7-hydroxTfiavanone Fig. (6). The concentration of flavonoid vs. the relative superoxide amount detected and the uric acid production. The results are expressed in % = (Ain the presence of flavonoid / Ain the
absence of flavonoid) x 100 [78].
CONCLUDING REMARKS According to the studies discussed above, it can be concluded that flavonoids act as antioxidants through different mechanisms. Most studies propose three structural elements as essential for optimal antioxidant activity: (1) a catechol or pyrogallol moiety in the B-ring, (2) a free 3-OH group, and (3) a C2-C3 double bond. Flavanols are exceptions to the rule, since they do not possess a C2-C3 double bond. The flavonols quercetin and myricetin meet these conditions and are therefore good candidates for antioxidant use. However, similar flavonoids can act as pro-oxidants,
334
V A N D E N B E R G H E et aL
possibly promoting oxidative damage to biological molecules. Consequently, there is a need for more detailed studies to elucidate the mechanism of the pro-oxidant effect and to determine its relevancy in vivo. There is also a lack of studies concerning the in vivo antioxidant actvity of flavonoids. Some studies demonstrate that humans absorb considerable amounts of flavonoids, but the absorption and metabolism of most flavonoids are not known. Many techniques currently used in the antioxidant research are highly specialized and the value of the obtained results depends often on the applied techniques. Consequently, there is a need for collaborative studies to standardize these methods. First, in most studies the purity of flavonoids is not mentioned and this can mask their activity. Second, most flavonoids are not readily water-soluble and are therefore dissolved in organic solvents. But some organic solvents, like DMSO and ethanol, are powerful OH" scavengers. Third, measuring flavonoids at millimolar concentrations is not relevant, because such plasma levels are never obtained. Some flavonoids show interesting activity at micr0molar or lower concentrations. Research should be focused on the pharmacology of such molecules. A lot of these flavonoids exhibit not only antioxidant activity but also other biological activities (anti-inflammatory, antiviral, ...) so that these molecules could be very useful in the future for the treatment of some diseases due to their combined activities. ABBREVIATIONS AAPH ABTS ABTS o+
= = =
AMVN CC1302 ~
= = =
2,2'-azobis(2-amidinopropane) dihydrochloride 2,2'-azinobis(3-ethylbenz-thiazoline-6-sulfonic acid) 2,2'-azinobis(3-ethylbenz-thiazoline-6-sulfonie acid) radical cation 2,2'-azobis(2,4-dimethylvaleronitrile) bromide radical ion trichloromethylperoxyl radical
Cu,Zn-SOD
=
copper- and zinc-containing superoxide dismutase
DETAPAC
=
diethylenetriaminepenta-acetic acid
DMPO
=
5,5-dimethyl- 1-pyrroline N-oxide
DPPH
=
1,1-diphenyl-2-picrylhydrazyl (free radical)
e-aq
=
hydrated electron
Eva EDTA
=
half peak oxidation potential
=
ethylenediaminetetra-acetic acid
Br2"-
STRUCTURE-ACTIVITY RELATIONSHIP OF FLAVONOIDS
ESR
=
electron spin resonance
GSH
=
reduced glutathione
GSHPx
=
glutathione peroxidase
GSSG
=
oxidized glutathione
H-
=
hydrogen atom or hydrogen radical
HCOO-
=
formate
HO2 ~
=
hydroperoxyl radical
H202
=
hydrogen peroxide
HOC1
=
hypochlorous acid
HX-FeIII
=
metmyoglobin
MDA
=
malondialdehyde
Mn-SOD
=
manganese-containing superoxide dismutase
N3"
=
azide radical
N3-
=
azide anion
NBT
=
nitro-blue tetrazolium
IO 2
=
singlet oxygen
IAgO2 leg+
=
delta singlet oxygen
=
sigma singlet oxygen
02"-"
=
superoxide radical
03
=
ozone
OH"
=
hydroxyl radical
ORAC
=
oxygen radical absorbing capacity
13-PE
=
13-phycoerythrin
R 9
=
carbon radical
RO-
=
alkoxyl radical
ROO 9
=
peroxyl radical
ROS
=
reactive oxygen species
SOD
=
superoxide dismutase
TBA
=
thiobarbituric acid
TEAC
=
trolox equivalent antioxidant capacity
oX.Few=O
=
ferrylmyoglobin radical
XO
=
xanthine oxidase
335
336
VANDEN BERGHE et as
REFERENCES
[I] [2] [3]
[4] [s] [6] [7]
[8] [9] [101 [11]
[12] [13]
[14] [15] [16] [17]
[is] [19]
[20] [21] [22]
[23] [24]
De Meyer, N.; Haemers, A.; Mishra, L.; Pandey, H.-K.; Pieters, L.A.C.; Vanden Berghe D.A.; Vlietinck, A.J.J. Med. Chem., 1991, 34, 736-746. Vanden Berghe, D.A.R.; Haemers, A.; Vlietinck, A.J. In Bioactive Natural Products: detection, isolation and structural determination; Colegate, S.M., Molyneux, R.J., Eds.; CRC Press: London, 1993; Chapter 17, pp. 405-440. Edenharder, R.; von Petersdorff, I.; Rauscher, R. Murat. Res., 1993, 287, 261274. Calomme, M.; Pieters, L.; Vlietinck, A.; Vanden Berghe, D. Planta Med., 1996, 62, 222-226. Mora, A.; Paya, M.; Rios, J.L.; Alcaraz, M.J. Biochem. Pharmacol., 1990, 40, 793-797. Sanz, M.J.; Ferrandiz, M.L.; Cejudo, M.; Terencio, M.C.; Gil, B.; Bustos, G.; Ubeda, A.; Gunasegaran, R.; Alcaraz, M.J. Xenobiotica, 1994, 24, 689-699. Shimoi, K.; Masuda, S.; Shen, B.; Furugori, M.; Kinae, N. Murat. Res., 1996, 350, 153-161. Shimoi, K.; Masuda, S.; Furugori, M.; Esaki, S.; Kinae, N. Carcinogenesis, 1994, 15, 2669-2672. Lasure, A.; Van Poel, B.; Cimanga, K.; Pieters, L.; Vanden Berghe, D.; Vlietinck, A.J. Pharm. Pharmacol. Lett., 1994, 4, 32-35. De Bruyne, T.; Van Driessen, G.; Van Poel, B.; Laekeman, G.; Vlietinck, A.J. Phytochemistry (Life Sci. Adv.), 1992, 11, 63-66. Middleton, E.; Kandaswami, C., Biochem. Pharmacol., 1992, 43, 1167-1179. Panthong, A.; Kanjanapothi, D.; Tuntiwachwuttikul, P.; Pancharoen, O.; Reutrakul, V. Phytomedicine, 1994, 1, 141-144. Hertog, M.G.; Feskens, E.J.; Hollman, P.C.; Katan, M.B., Kromhout, D. Lancet, 1993, 342, 1007-1011. Knekt, P.; Jarvinen, R.; Reunanen, A.; Maatela, J. Brit. Meal J., 1996, 312, 478481. Hertog, M.G. et al. Arch. Intern. Med., 1995, 155, 381-386. Hanasaki, Y.; Ogawa, S.; Fukui, S. Free Radic. Biol. Meal., 1994, 16, 845- 850. Tournaire, C.; Croux, S.; Maurette, M.-T.; Beck, I.; Hocquaux, M.; Braun, A.M.; Oliveros, E. J. Photochem. Photobiol. B: Biol., 1993, 19, 205-215. Morel, I.; Lescoat, G.; Cillard, P.; Cillard, J. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 437443. Laughton, M.J.; Evans, P.J.; Moroney, M.A.; Hoult, J.R.C.; Halliwell, B. Biochem. Pharmacol., 1991, 42, 1673-1681. Hoult, J.R.S.; Moroney, M.A.; Paya, M. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 443- 455. Siess, M.H.; Leclerc, J.; Canivenc-Lavier, M.C.; Rat, P.; Suschetet, M. Toxicol. Appl. Pharmacol., 1995, 130, 73-78. Chang, W.S.; Lee, Y.J.; Lu, F.J.; Chiang, H.C. Anticancer Res., 1993, 13, 2165-2170. Lindahl, M.; Tagesson, C. Inflammation, 1993, 17, 573-582. Gil, B.; Sanz, M.J.; Terencio, M.C.; Ferrandiz, M.L.; Bustos, G.; Paya, M.; Gunasegaran, R.; Alcaraz, M.J. Life Sci., 1994, 54, 333-338.
STRUCTURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS
[25] [26] [27] [28] [29] [301 [31] [32] [33] [34] [35] [36] [37]
[38] [39] [40] [41]
[42] [43] [44] [45]
[46]
[47] [48] [49]
[50] [51] [52] [53] [541
[551 [561 [571
[58] [59]
[60]
337
Middleton, E.; Kandaswami, C. In The Flavonoids: Advances in research since 1986; Harbome, J.B., Ed.; Chapman & Hall: London, 1993; Chapter 15, pp. 619-652. Middleton, E. Trends Pharmacol. Sci., 1984, 5, 335-338. Van Acker, S.A.B.E.; van den Berg, D.-J.; Tromp, M.N.J.L.; Griffioen, D.H.; van Bennekom, W.P.; van der Vijgh, W.J.F.; Bast, A. Free Radic. Biol. Med., 1996, 20, 331-342. Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Free Radic. Biol. Med., 1996, 20, 933-956. Cao, G.; Sofic, E.; Prior, R.L. Free Radic. Biol. Med., 1997, 22, 749-760. Hollman, P.C.H.; van Trijp, J.M.P.; Mengelers, M.J.B.; de Vries, J.H.M.; Katan, M.B. Cancer Lett., 1997, 114, 139-140. Hollman, P.C.H.; de Vries, J.H.M.; van Leeuwen, S.D.; Mengr M.J.B.; Katan, M.B. Am. J. Clin. Nutr., 1995, 62, 1276-1282. Laughton, M.J.; Halliwell, B.; Evans, P.J.; Hoult, J.R.S. Biochem. Pharmacol., 1989, 38, 2859-2865. Aruoma, O.I. JAOCS, 1996, 73, 1617-1625. Halliwell, B. Free Rad. Res., 1996, 25, 57-74. Halliwell, B.; Gutteridge, J.M.C. Free Radicals in Biology and Medicine; Clarendon Press: Oxford, 1989. Ahmad, S. Oxidative Stress and Antioxidant Defenses in Biology; Chapman & Hall: New York, 1995. Halliwell, B. Biochem. Pharmacol., 1995, 49, 1341-1348. Halliwell, B. Free Rad. Res. Comms., 1990, 9, 1-32. Gutteridge, J.M.C.; Halliwell, B. ,4ntioxidants in Nutrition, Health, and Disease; Oxford University Press: New York, 1994. Halliwell, B. Annu Rev. Nutr., 1996, 16, 33-50. Krinsky, N.I. Proc. Soc. Exp. Biol. Med., 1992, 200, 248-254. Sies, H. Oxidative Stress; Academic Press: London, 1985. Demple, B.; Harrison, L. Annu Rev. Biochem., 1994, 63, 915-948. Antunes, F.; Salvador, A.; Pinto, R.E. Free Radic. Biol. Med, 1995, 19, 669677. Grune, T.; Reinheckel, T.; Joshi, M.; Davies, K.J.A.J. Biol. Chem., 1995, 270, 2344-2351. Harris, E.D. Faseb J., 1992, 6, 2675-2683. Cerutti, P.A. Lancet, 1994, 344, 862-863. Wiseman, H.; Halliwell, B. Biochem. J., 1996, 313, 17-29. Jenner, P. Lancet, 1994, 344, 796-798. Witztum, J.L. Lancet, 1994, 344, 793-795. Halliwell, B. Haemostasis, 1993, 23, 118-126. Halliwell, B.; Gutteridge, J.M.C.; Cross, C.E.J. Lab. Clin. Med., 1992, 119, 598-620. Taylor, A. Ann. N.Y. Acad. Sci., 1992, 669, 111-123. Wiseman, H. Nutritional Biochemistry, 1996, 7, 2-15. Gutteridge, J.M.C. Free Rad. Res. Comms., 1993, 19, 141-158. Gaziano, J.M. Nutrition, 1996, 12, 583-588. Gordon, M.H. Nat. Prod. Rep., 1996, 13, 265-273. Willett, W.C. Science, 1994, 264, 532-537. Block, G.; Patterson, B.; Subar, A. Nutr. Cancer., 1992, 18, 1-29. Byers, T.; Perry, G. dnnu. Rev. Nutr., 1992, 12, 139-159.
338
[61] [621 [63]
[64] [65] [66] [67] [68]
[69] [70] [71]
[72] [73] [74] [75]
[76] [77] [78] [79]
[80] [81]
[82] [83] [84]
[85] [86]
[87]
[88]
[89] [90] [911
V A N D E N B E R G H E et as
Diplock, A.T. MoL Aspects Med., 1994, 15, 293-376. Havsteen, B. Biochem. PharmacoL, 1983, 32, 1141-1148. Harborne, J.B. The Flavonoids: Advances in Research since 1986, Chapman & Hall: London, 1993. Kandaswami, C.; Middleton, E. Adv. Exp. Med. BioL, 1994, 366,351-376. Pierpoint, W.S. Prog. Clin. BioL Res., 1986, 213, 125-140. Hollman, P.C.H. Eur. d. Clin. Nutr., 1997, 51, $66-$69. Ktlhnai, J. Wld. Rev. Nutr. Diet., 1976, 24, 117-191. Hertog, M.G.L.; Hollman, P.C.H.; Katan, M.B.; Kromhout, D. Nutr. Cancer, 1993, 20, 21-29. Das, N.P. Biochem. Pharmacol., 1971, 20, 3435-3445. Pryor, W.A. Ann. Rev. PhysioL, 1986, 48, 657-667. Shahidi, F.; Wanasundara, P.K.J.P.D. Crit. Rev. Food Sci. Nutr., 1992, 32, 67103. Baumann, J.; Wurm, G.; von Bruchhausen, F. Arch Pharm., 1980, 313, 330337. Robak, J.; Gryglewski, R.J. Biochem. PharmacoL, 1988, 37, 837-841. Huguet, A.I.; Manez, S.; Alcaraz, M.J.Z. Naturforsch., 1990, 45c, 19-24. Yuting, C.; Rongliang, Z.; Zhongjian, J.; Yong, J. Free Radic. Biol. Med., 1990, 9, 19-21. Chen, C.-W.; Ho, C.-T. d. Food Lipids, 1995, 2, 35-46. Cotelle, N.; Bernier, J.L.; Henichart, J.P.; Catteau, J.P.; Gaydou, E.; Wallet, J.C. Free Radic. BioL Med., 1992, 13, 211-219. Cos, P.; Ying, L.; Calomme, M.; Hu, J.P.; Cimanga, K.; Van Poel, B.; Pieters, L.; Vlietinck, A.J.; Vanden Berghe, D. d. Nat. Prod., 1998, 61, 71-76. Cotelle, N.; Bernier, J.-L.; Catteau, J.-P.; Pommery, J.; Wallet, J.-C.; Gaydou, E.M. Free Radic. BioL Med., 1996, 20, 35-43. Nishikimi, M.; Appaji Rao, N.; Yagi, K. Biochem. Bioph. Res. Co., 1972, 46, 849-854. Richter, H.W.; Fetrow, M.A.; Lewis, R.E.; Waddell, W.H.d. Am. Chem. Soc., 1982, 104, 1666-1671. Terada, L.S.; Left, J.A.; Repine, J.E. In Methods in Enzymology; Packer, L., Glazer,A.N., Eds.; Academic Press: New York, 1990, Vol. 186, pp. 651-656. Hayashi, T.; Sawa, K.; Kawasaki, M.; Arisawa, M.; Shimizu, M.; Morita, N. jr. Nat. Prod, 1988, 51, 345-348. Rastelli, G.; Constantino, L.; Albasini, A. Eur. d; Med. Chem., 1995, 30, 141146. Rastelli, G.; Constantino, L.; Albasini, A. Eur. d. Med. Chem., 1996, 31, 693699. Chang, W.S.; Lee, Y.J.; Lu, F.J.; Chiang, H.C. Anticancer Res., 1993, 13, 2165-2170. Ponti, V.; Dianzani, M.U.; Cheeseman, K.; Slater, T.F. Chem.-Biol. Interact., 1978, 23, 281-291. Sichel, G.; Corsaro, C.; Sealia, M.; Di Bilio, A.J.; Bonomo, R.P. Free Radic. BioL Med., 1991, 11, 1-8. Koppenol, W.H. Free Radic. Biol. Med., 1993, 15, 645-651. Sutton, H.C.; Winterbourn, C.C. Free Radic. Biol. Med., 1989, 6, 53-60. Lloyd, R.V.; Hanna, P.M.; Mason, R.P. Free Radic. BioL Med., 1997, 22, 885888.
STRUCFURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS [92] [93] [94] [95] [96] [97] [98]
[99]
[loo] [1ol] [102]
[103] [104] [1o5] [1061
[107] [lOS] [109] [llO] [Ill] [1~2] [113] Ill4] [~15] [116] [117]
[118] [119] [12o]
339
Yoshiki, Y.; Okubo, K.; Onuma, M.; Igarashi, K. Phytochemistry, 1995, 39, 225-229. Puppo, A. Phytochemistry, 1992, 31, 85-88. Husain, S.R.; Cillard, J.; Cillard, P. phytochemistry, 1987, 26, 2489-2491. Floyd, R.A.; Lewis, C.A.; Wong, P.K. In Methods in Enzymology; Packer, L., Ed.; Academic Press: New York, 1984, Vol. 105, pp. 231-237. Finkelstein, E.; Rosen, G.M.; Rauckman, E.J. Arch. Biochem. Biophys., 1980, 200, 1-16. Aruoma, O.I. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 233, pp. 67-82. Torel, J.; Cillard, J.; Cillard, P. Phytochemistry, 1986, 25, 383-385. Porter, N.A.; Weber, B.A.; Weenen, H.; Khan, J.A.J. Am. Chem. Soc., 1980, 102, 5597-5601. Niki, E. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1990, Vol. 186, 100-108. Cao, G.; Alessio, H.M.; Cutler, R.G. Free Radic. Biol. Med., 1993, 14, 303311. Cao, G.; Verdon, C.P.; Wu, A.H.B.; Wang, H.; Prior, R.L. Clin. Chem., 1995, 41/42, 1738-1744. Wang, P.-F.; Zheng, R.-L. Chem. Phys. Lipids, 1992, 63, 37-40. Terao, J.; Piskula, M.; Yao, Q. Arch. Biochem. Biophys., 1994, 308, 278-284. Ioku, K.; Tsushida, T.; Takei, Y.; Nakatani, N.; Terao, J. Biochim. Biophys. Acta, 1995, 1234, 99-104. Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. J. Biol. Chem., 1984, 259, 41774182. Ratty, A.K.; Sunamoto, J.; Das, N.P. Biochem. Pharmacol., 1988, 37, 989- 995. Rekka, E.; Kourounakis, P.N.J. Pharm. Pharmacol., 1991, 43, 486-491. Blois, M.S. Nature, 1958, 181, 1199-1200. Fomi, L.G.; Willson, R.L. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press" New York, 1990, Vol. 186, pp. 179-188. Adams, G.E.; Wardman, P. In Free Radicals in Biology: Volume III; Pryor, W.A., Ed.; Academic Press: New York, 1977; Chapter 2, pp. 53-95. Butler, J.; Land, E.J. In Free Radicals: A practical approach; Punchard, N.A., Kelly, F.J., Eds.; IRL Press: Oxford, 1996; Chapter 4, pp. 47-61. Alfassi, Z.B.; Schuler, R.H.J. Phys. Chem., 1985, 89, 3359-3363. Erben-Russ, M.; Bors, W.; Saran, M. Int. J. Radiat. Biol., 1987, 52, 393-412. Bors, W.; Saran, M. Free Radical Res. Com., 1987, 2, 289-294. Bors, W.; Heller, W.; Michel, C.; Saran, M. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1990, Vol. 186, pp. 343355. Bors, W.; Heller, W.; Michel, C.; Saran, M. In Antioxidants in Therapy and Preventive Medicine; Emerit, I., Ed.; Plenum Press: New York, 1990; pp. 165170. Bors, W.; Heller, W.; Michel, C.; Saran, M. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 420429. Jovanovir S.V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M.G.J. Am. Chem. Soc., 1994, 116, 4846-4851. Aruoma, O.I. Food Chem. Toxicol., 1994, 32, 671-685.
340
VANDEN B E R G H E et al.
[121] Aruoma, O.I.; Spencer, J.P.E.; Butler, J.; Halliwell, B. Free Rad. Res., 1995, 22, 187-190. [122] Swartz, H.M.; Swartz, S.M. In Methods of Biochemical Analysis, Glick, D., Ed.; John Wiley & Sons: New York, 1983; Volume 29, pp. 207-323. [123] van Acker, S.A.B.E.; de Groot, M.J.; van den Berg, D.-J.; Tromp, M.N.J.L.; Donn6-Op den Kelder, G.; van der Vijgh, W.J.F.; Bast, A. Chem. Res. Toxicol., 1996, 9, 1305-1312. [124] Kuhnle, J.A.; Windlr J.J.; Waiss, A.C.J. Chem. Soc. (B), 1969, 613-616. [125] Miller, N.J.; Rice-Evans, C.; Davies, M.J.; Gopinathan, V.; Milner, A. Clin. Sci., 1993, 84, 407-412. [1261 Rice-Evans, C.; Miller, N.J. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 279-293. [127] Miller, N.J.; Rice-Evans, C.A. Redox Rep., 1996, 2, 161-171. [128] Giulivi, C.; Cadenas, E. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 234, pp. 189-202. [1291 Strube, M.; Haenen, G.R.M.M.; van den Berg, H.; Bast, A. Free Radical Res., 1997, 26, 515-521. [130] Rice-Evans, C.A.; Miller, N.J.; Bolwell, P.G.; Bramley, P.M.; Pridham, J.B. Free Radical Res., 1995, 22, 375-383. [131] Salah, N.; Miller, N.J.; Paganga, G.; Tijburg, L.; Bolwell, G.P.; Rice-Evans, C. Arch. Biochem. Biophys., 1995, 322, 339-346. [1321 Rice-Evans, C.A.; Miller, N.J. Biochem. Soc. T., 1996, 24, 790-795. [133] Paganga, G.; AI-Hashim, H.; Khodr, H.; Scott, B.C.; Aruoma, O.I.; Hider, R.C.; Halliwell, B.; Rice-Evans, C.A. Redox Rep., 1996, 2, 359-364. [134] Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Trends Plant Sci., 1997, 2, 152159. [135] Sorata, Y.; Takahama, U.; Kimura, M. Biochim. Biophys. Acta, 1984, 799, 313317. [136] Takahama, U.; Youngman, R.J.; Elstner, E.F. Photobiochem. Photobiophys., 1984, 7, 175-181. [137] Devasagayam, T.P.A.; Subramanian, M.; Singh, B.B.; Ramanathan, R.; Das, N.P.J. Photochem. Photobiol. B: Biol., 1995, 30, 97-103. [138] Studer, S.L.; Brewer, W.E.; Martinez, M.L.; Chou, P.-T. J. Am. Chem. Soc., 1989, 111, 7643-7644. [139] Halliwell, B.; Gutteridge, J.M.C. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1990, Vol. 186, pp. 1-85. [140] Hudson, B.J.F.; Lewis, J.l. Food Chem., 1983, 10, 47-55. [141] Thompson, M.; Williams, C.R.; Elliot, G.E.P. Anal. Chim. Acta, 1976, 85, 375-381. [142] Afanas'ev, I.B.; Dorozhko, A.I.; Brodskii, A.V.; Kostyuk, V.A.; Potapovitch, A.I. Biochem. Pharmacol., 1989, 38, 1763-1769. [1431 Morel, I.; Lescoat, G.; Cogrel, P.; Sergent, O.; Pasdeloup, N.; Brissot, P.; Cillard, P.; Cillard, J. Biochem. Pharmacol., 1993, 45, 13-19. [144] Wiseman, H.; Halliwell, B. FEBS Lett., 1993, 322, 159-163. [145] Roy, D.; Liehr, J.G. Cancer Res., 1991, 51, 3882-3885. [146] Aruoma, O.I. Free Radic. Biol. Med., 1996, 20, 675-705. [147] Hodnick, W.F.; Kung, F.S.; Roettger, W.J.; Bohmont, C.W.; Pardini, R.S. Biochem. Pharmacol., 1986, 35, 2345-2357. [148] Hodnick, W.F.; Milosavljevic, E.B.; Nelson, J.H.; Pardini, R.S. Biochem. Pharmacol., 1988, 37, 2607-2611.
STRUCTURE-ACTIVITYRELATIONSHIPOF FLAVONOIDS
341
[149] Canada, A.T.; Giannella, E.; Nguyen, T.D.; Mason, R.P. Free Radic. Biol. Med., 1990, 9, 441-449. [150] Marklund, S.; Marklund, G. Eur. J. Biochem., 1974, 47, 469-474. [151] Halliwell, B.; Gutteridge, J.M.C.; Aruoma, O.I. Anal Biochem., 1987, 165, 215-219. [152] Aruoma, O.I. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 233, pp. 57-66. [153] Aruoma, O.I.; Chaudhary, S.S.; Grootveld, M.; Halliwell, B. J. Inorg. Biochem., 1989, 35, 149-155. [154] Grootveld, M.; Halliwell, B. Free Radical Res. Com., 1986, 1, 243-250. [155] Evans, P.J.; Halliwell, B. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press: New York, 1994, Vol. 233, pp. 82-92. [156] Babbs, C.F.; Steiner, M.G. In Methods in Enzymology; Packer, L., Glazer, A.N., Eds.; Academic Press" New York, 1990, Vol. 186, pp. 137-147. [157] Fukui, S.; Hanasaki, Y.; Ogawa, S. d. Chromatogr., 1993, 630, 187-193.
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natw'al Products Chemistry, Vol. 22
343
9 2000 Elsevier Science B.V. All rights reserved
R E C E N T A D V A N C E S IN T H E S E A R C H FOR A N T I O X I D A N T A C T I V I T Y IN S O U T H A M E R I C A N PLANTS C. DESMARCHELIER I, G. CICCIA l, and J. COUSSIO 2 t Cdtedra de Microbiologia Industrialy Biotecnologia, 2 Cdtedra de Farmacognosia, IQUIMEFA-CONICET; Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 1113 Buenos Aires, Argentina.
Abstract: Over the past two decades, researchers have turned to many of the traditional
folk medicines used in the Far East, Europe and North America in order to uncover the presence of new natural occurring compounds with antioxidant activity. However, in recent years, this interest has also awoken for plants in South America. The existence of geographic areas with highly diverse flora, the unique ecological and physiological conditions under which many of these plants grow and the presence of human societies with a strong tradition in the use of plant resources as medicinal agents, makes this region extremely interesting for this purpose. The present paper reviews the advances in the search for antioxidant activity in plants used as medicinal agents in different areas of South America.
FREE RADICALS, NATURAL ANTIOXIDANTS AND THEIR ROLE IN HUMAN HEALTH It has been determined that active oxygen molecules such as superoxide (O2, OOH.), hydroxyl (OH.) and peroxyl (ROOH o) radicals play an important role in oxidative stress related to the pathogenesis of different diseases such as Alzheimer, Parkinson and Hodgkin's disease, cataracts, acute liver toxicity, inflammation processes and DNA damage that leads to carcinogenesis. These free radicals and other related compounds are generated in (a) mitochondria, which produce the superoxide radical and hydrogen peroxide; (b) phagocytes, generators of nitric oxide and hydrogen peroxide during the "respiratory burst" that takes place in activated phagocytic cells in order to kill bacteria after phagoeytosis; (c) peroxisomes (microbodies), which degrade fatty acids and other substances yielding hydrogen peroxide; and (d) citocrome P-450 enzymes, responsible for many oxidation reactions of endogenous substrates [1 ]. An elaborate cellular defence system against oxygen-free radical toxicity exists, which includes, in a first line, factors that prohibit the fomlation of, or scavenge primary initiators of the lipid peroxidation process, such as metal-ion-binding proteins or the enzymes superoxide dismutase (SOD),
344
DESMARCHELIER et aL
catalase and selenium-dependent glutathione (GSH) peroxidase [2]. Vitamins E and C as well as 13-carotene may also scavenge oxygen radicals. These antioxidants are natural occurring compounds which also include flavonoids, phenolic acids and nitrogen compounds such as alkaloids and chlorophyll derivatives [1,3,4]. Recent epidemiological data suggest that the consumption of polyphenol rich items in the diet is associated to an increase in plasma antioxidant concentrations, thus reducing the exposure to cellular oxidative stress [5,6,7,8,9]. Moreover, it has been determined that the intake of essential antioxidants such as vitamin C, vitamin E and various carotenoids is inversely related to the risk of coronary heart disease and certain forms of cancer [ 10]. Plants, therefore, have become a rich source of potential antioxidant agents that can be useful to mankind. The aim of the present review is to describe the advances in the search for antioxidant compounds and antioxidant activity in medicinal plants used for different purposes in South America. E C O L O G Y AND E T H N O B O T A N Y IN THE S E A R C H FOR NATURAL ANTIOXIDANTS
The search for new active natural compounds such as antioxidants is most productive when plant-surveying takes place in areas where biological diversity is high [ 11 ], such as tropical and subtropical ecosystems, due to the unique ecological characteristics of these regions. For example, it has been stated that some flavonoids, with their strong absorption in the 300400 nm UV region, act as internal light filters for the protection of chloroplasts and other organelles from UV damage. Leaves of nearly all species in high-UV environments, such as arctic, alpine and tropical latitudes, have very low epidermal UV transmittance. The light-filtering ability of these compounds may reinforce their antioxidant effects to provide a high level of protection against damaging oxidants generated either thermally or by light [ 12]. There is evidence that alkaloids also occur with more frequency in species of tropical origin, where UV and UV-B intensities are much higher than in temperate regions [ 13]. Although light intensities also vary with altitude, there is little data on alkaloids concentration as a function of altitude, except for the case of Lycopersicon species growing at high elevations in Peru, which contain higher concentrations of tomatine, a steroidal alkaloid, than related species from lower elevations [14]. Coevolutionary biochemical interactions of plants with their natural predators could contribute to uncover medicinally useful chemicals such as antioxidants [15]. For example, many flavonoids possess allelochemical properties by warding off microbial or animal pathogens as phytoalexins or as antifeedants, and the phytoalexin response is frequently accompanied by an oxidative burst in the cell [ 16]. Moreover, it has been demonstrated that root flavonoids, mainly flavones, isoflavones and some
SEARCH FOR ANTIOXIDANT ACTIVITY
345
chalcones, participate in the process of the induction of nodulation genes during the Rhizobium-legume symbiosis that occurs in root nodulation in Leguminoseae [ 17] a highly represented family in tropical and subtropical areas. Gathering information on the traditional knowledge of the use of medicinal plants by local inhabitants in these regions may also increase the chances of success, since this information could suggest the presence of biologically active principles in the plant, thus serving as a guide that may help in discovering active compounds [ 18]. Although medicinal plants will be rarely used as "antioxidants" in traditional medicine, the therapeutic properties of many active principles present in them could be due, in part, to their capacity of scavenging oxygen free radicals which may be involved in many diseases. For example, plants used to treat inflammatory diseases could act by reducing the oxidative stress that takes place in cells undergoing this process [19]. Likewise, several plants used for hepatoprotective purposes have shown to be active as antioxidants [20,21,22]. Thus, the search for antioxidant activity should centre its attention in plants used traditionally in oxidative stress-related pathologies. "CHACO" AND THE SOUTHERN LOWLANDS This region occupies large areas of south Brazil, north-east Argentina, south-east Bolivia and the territories of Uruguay and Paraguay. The main ecological systems found in the region are the subtropical rainforests, gallery forests, xerofitie deciduous forests ("Chaco"), pyrogenir savannahs, halophylous shrubby steppes, palm forests ("palmares") and herbaceous prairies formed by different Gramineae ("pampas"). The presence of indigenous human groups with an ethnopharmacologieal legacy is only important in the areas of Argentina, Bolivia and Paraguay. Different flavonoids and phenolic compounds have been isolated from medicinal plants of this region, many of which have shown antioxidant properties. A variety of experiments indicate that selected flavonoids and polyphenols possess antialergie, anti-inflammatory, antiviral, anticarcinogenie and antioxidant activities [23]. This latter activity has been attributed to the presence of phenolic groups in their chemical structure. It has been shown that lipid peroxidation can be inhibited by flavonoids, which act as strong 02- scavengers [24] and singlet oxygen (IO2) quenchers [25], and that this activity is closely associated with their chemical structure, especially the number of hydroxyl groups linked to the basic skeleton and also to their configuration [26]. It has also been proposed that these compounds act as H-atom donors to the peroxyl radical, thus inhibiting the autoxidation of fatty acids by means of chain radical termination [27].
346
DESMARCilELIER et aL
OH H HO
OH
O
Fig. (1). Quercetin from Achyrocline satureioides and Pterocaulon polystachium.
Achyrocline satureioides (Lam.) DC. (Compositae), known as "marcela" or "yatei carl", is a medicinal plant widely used in this region by its choleretic, antispasmodic and hepatoprotective properties. Phytochemical analysis of this species has confirmed the presence of flavonoids quercetin, Fig. (1), and its derivatives, and caffeic, chlorogenic and isochlorogenic acids, Fig. (2), in the aerial parts [28]. The high content of polyphenolic compounds in A. satureioides has led us to study its antioxidant properties, in order to determine the free radical scavenging activity and capacity to reduce lipid peroxidation and iron (II)-dependent DNA damage [29]. The inhibition of luminol-enhanced chemiluminescence induced by an azo-bis initiator (a compound that decays spontaneously to generate peroxyl radicals), allowed the determination of the TRAP (total reactive antioxidant potential) and TAR (total antioxidant reactivity) indices [30] for the aqueous and methanolic extracts, using Trolox, a vitamin E hydrosoluble synthetic analog, as a standard. A reduction in iron (II)-dependent DNA damage was also observed, indicating that the extracts studied are capable of reducing the in vitro oxidation of DNA deoxyribose. Lipid peroxidation, on the other hand, was assessed using two different methods: hydroperoxide-initiated chemiluminescence (CL) and the production of thiobarbituric acid-reactive substances (TBARS) in rat liver homogenates. As expected, the extracts of A. satureioides were effective in reducing lipid peroxidation, and the results obtained are summarised in Table 1.
of~~~ HO0 0H
ogr..o o.
oH
OH O E ~ OH OH Cafeic acid
Chlorogenic acid
Isochlorogenic acid
Fig. (2). Caffeoylquinic acids in Achyrocline satureioides and Pterocaulon polystachium.
SEARCH FOR ANTIOXIDANT ACTIVITY
T a b l e 1.
A n t i o x i d a n t Activity in Different Extracts of
TRAP
Extract
TAR
Achyrocline satureioides
CL
TBARS
DNA damage
(~tg/mi)
(~tg/ml)
225 (404-139)
> 1000
NA
ND
> 1000
ND
0tg/ml) | ',,,"
Aqueous Methanolic
Ill
,
,,
J
,
91-F 15
1537 ~ 148
128 4" 20
1910 4- 171
347
II ill
IIIl~
ill
i
Total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) are expressed in (M Trolox equivalents. CL and TBARS are expressed as IC50 ((g/mi), and include 95% confidence intervals. NA: not active; b/D: not determined due to lack of dose dependence.
Another related species, Achyroclineflaccida (Weinm) DC., has also shown to contain different polyphenolic compounds, the most interesting being the flavanone 7,4" dihydroxy 5-methoxy flavanone, and the corresponding chalcone, Fig (3), [31 ]. The latter was highly effective in reducing hydroperoxide-initiated chemiluminescence in rat liver homogenates [32]. Recent studies have also demonstrated that different extracts of this plant exhibited chain-breaking antioxidant activity in FeE+/ascorbate-induced lipid peroxidation in rat liver microsomal fractions and scavenged peroxyl radicals in an aqueous assay system [33]. These activities, together with an inhibition in neutrophil functions, were also observed in Pterocaulon polystachium DC. (Compositae), another medicinal plant widely used in the region. The antioxidant properties 0
H HO
HO
OMe
O
Fig. (3). 7,4" Dihydroxy 5-methoxy flavanone and 4,2',4'-trihydroxy 6'-methoxychalcone from
dchyrocline flaccida. OH
I
O Fig. (4). Rhamnetin from
Pterocaulon polystachium.
H
348
DESMARCtlELIER et
observed were attributed to the presence of quercetin, rhamnetin, Fig. (4), caffeic, chlorogenic and isochlorogenic acids, all of these previously identified in this species [34,35]. Baccharis coridifolia DC. (Compositae) is a sympatric shrub of ,4. satureioides, commonly known as "mio-mio" or "romerillo". The infusion of aerial parts such as leaves is used as an hepatoprotective, and topical use of these parts is also extended in order to treat inflammation. Toxicity of the plant to cattle and other domestic animals has been claimed in this region, probably due to the presence of macrocyclic tricothecens, which show high toxicity towards eucariotic organisms [36]. The involvement of free radical-mediated cell damage in major hepatic diseases has led us to study the in vitro antioxidant activity of the aqueous extract of B. coridifolia [37]. As expected, the extract significantly reduced CL and the production of TBARS in rat liver homogenates, due to the presence of different flavonoids and hydrosoluble polyphenolic compounds in the plant l, thus confirming its antioxidant properties. Other species which were active in these assays are included in Table 2 [38]. Although the active principles in ,4canthospermum australe (Loefl.) have not been identified to the moment, chlorogenic and isochlorogenic acids have been isolated from B. crispa Spr Ok. and P. purpurascens DC. [34]. Table 2.
Antioxidant Activity of Argentine Aqueous Plant Extracts: IC50 and 95% C o n f i d e n c e Interval for Inhibition of H y d r o p e r o x i d e - i n i t i a t e d Chemiluminescence (CL) and the Production of Thiobarbituric Acidreactive Substances (TBARS) in Rat Liver Homogenates i
ii
i
.
ii
IC50 and 95% confidence interval (pg/ml) Botanical name CL ',
",,
,,"
L, ,
i
TBARS i
i
i
i,
Acanthospermum australe (Locfl.)Ok.
123 (183-84)
767 (2953-352)
Baccharis coridifolia DC.
141 (251-84)
556 (1213-320)
B. crispa Spr.
544 ( 1018-340)
554 (2286-247)
Pterocaulon polystachium Malme
462 (776-306)
> 1000
P. purpurascens DC.
376 634-247)
> I000
CL: chemiluminescence. TBARS" thiobarbituric acid reactive substances.
1Bianchi, N.; Sebold, D. Ensayo de toxicidade excessiva e screening fitoquimico de algumas esp~cies do g~nero Baccharis L. (Asteraceae). In: XIV Simp6sio de Plantas Medicinais do Brasil. Florian6polis, September, 1996 (abstract M 004).
SEARCH FOR ANTIOXIDANT ACTIVITY
349
The methoxylated flavonols 5,7,3',4'-tetrahydroxy-3,6,8-trimethoxy flavone, 5,7,3'-trihydroxy-3,6,4'-trimethoxyflavone and 5,3',4'trihydroxy-3,6,7-trimethoxyflavone have been isolated from aerial parts of Plucchea sagittalis (Lam.) Cabrera (Compositae), Fig. (5), locally known as "cuatrocantos" or "lucero" [39]. Although the biological activity of these compounds has not been determined, these could be responsible for the peroxyl radical scavenging activity observed in different extracts of this plant. Recent studies have also demonstrated that taraxasteryl acetate, also present in this species, is active against topical inflammation, as well as on reactive oxygen species and stress protein production 2. Other studies indicated that 5,6,3'-trihydroxy-7,4'-dimethoxyflavone and pedalitin, Fig. (6), identified in another medicinal plant of the region, namely Eupatorium inulaefolium H.B.K. Hier. (Compositae) [40], were capable of reducing lipid peroxidation in mouse liver homogenates, measured as a reduction in rat liver CL [32]. OH OC H3
H3CO"
~
"~ OH
H
CH3 O
5,7,3',4'-tetrahydroxy-3,6,8-trimethoxy flavone
OH
OH
O
R I -- H; R2 = CH3; 5,7,3'-trihydroxy-3,6,4'-trimethoxyflavone R 1 -" CH3; R2 - H; 5,3",4"-trihydroxy-3,6,7-trimethoxyflavone Fig. (5). Flavonols from Plucchea sagittalis.
2p~rez-Garc|a, F.; Matin, E." Adzet, T.; Cafligueral, S. Taraxasteryl acetate, an active principle from Pluchea sagittalis: effects on inflammation, free radicals and stress protein synthesis. In: II World Congress on Medicinal and Aromatic Plants for Human Welfare (WOCMAP II). Mendoza, Argentina, November,
1997. (abstract O 02 I).
350
DESMARCHELIER eta/.
OH
R MeO,
O
HO
(1) R = Me; 5,6,3"-trihydroxy-7,4"-dimethoxyflavone (2) R = H; pedalitin Fig. (6). Flavonoids from Eupatorium inulaefolium. Table 3.
Effects of Polyphenols Isolated from Argentine Medicinal Plants on tertbutyl H y d r o p e r o x i d e - i n i t i a t e d Chemiluminescence of Mouse Liver Homogenates* ,
i
i
ii
Polyphenol ,
IC50 (gM) ,,
i
Catechin
3
Eriodictyol
Myricetin 4,2',4'-Trihydroxy-6'-methoxychalcone 3,4-Dicafeoylquinic acid
20
Isochlorogenic acid
30
Cafeic acid
50
5,6,3'-Trihydroxy-7,4'-dimethoxyflavone
5O
Cynarin
50
Chlorogenic acid
150
Apigenin
150
Quercetin
200
Pedalitin
200
Sylimarin
200
Quercetin-3-methyl ether
200
7,4'-Dihhydroxy-5-methoxyflavonone
500
Kaempferol-3,7-dirhamnoside
500
Quercitrin i
*Taken from Fraga et al., 1987, [32].
900 ii,
1
SEARCtl FOR ANTIOXIDANT ACTIVITY
351
Different flavonoids and polyphenols isolated from Argentine medicinal plants were assayed in vivo as liver chemiluminescence inhibitors to determine their protection against oxidative stress produced by CC14, a potent stimulator of lipid peroxidation [32]. Previously, a large number of polyphenols was screened in vitro for their abilities to inhibit CL in liver homogenates (Table 3). The highest activities were observed in catechin, Fig. (7), and eriodyctiol, Fig. (8), suggesting that these compounds are highly effective as water-soluble protectors against lipid peroxidation and other free radical-mediated cell injury. H HO
H
Fig. (7). Catechin from Heisteria pallida. OH H HO
OH
O
Fig. (8). Eriodyctiol from Argentine medicinal plants.
ANTIOXIDANT ACTIVITY IN MEDICINAL PLANTS FROM THE ANDES The Andes is a mountain range system extended along the Pacific coast of South America, from northern Venezuela down to the far south of Chile and Argentina. The average altitude of the system is 3,500 m, and with an extension of 8,500 km, also occupies large areas of Colombia, Ecuador, Peru and Bolivia. The remarkable variations in altitude that occur along the Andes are the source of unique biological communities to particular and small areas, sometimes harbouring many endemic plant species [41 ]. As in other regions of the Continent, the traditional use of plants in healing practices is very extended among the inhabitants throughout this region [e.g. 42]. The antioxidant capacity of closely related lignans isolated from Chilean medicinal plants was assessed by their effects upon the rate of rat
352
DESMARCHELIER et aL
Moo/
XoMo
RI = H; R2 = H; dihydroguayaretic acid RI = H; R2 = OMe; isopregomisin R I = OMe; R 2 = OMe; guayacasin Fig. (9). Lignans from Porlieria chilensis.
brain homogenate autoxidation [43]. The structurally similar lignans dihydroguayaretic acid, guayacasin and isopregomisin, Fig. (9), isolated from Porlieria chilensis Johnst. (Zygophyllaceae), commonly known as "guayacfin" and traditionally used as an antirheumatic, proved to be powerful antioxidants with activities similar to that of propyl gallate. The results obtained (Table 4) suggest that the free radical scavenging activity in these compounds can be explained in terms of their highly substituted phenolic structures and to the number of methoxyl groups present in their structure. The flavonoids 3"-methoxycalicoptedn and 7"-methylsudachitin, Fig. (10), from Baecharis incarum Wedd. (Compositae), also tested under this method, were considerably less active than the studied lignans or quercetin, isolated from Aristotelia chilensis (Mol.) Stuntz. (Elaeocarpaceae), another medicinal plant of the region commonly known as "maqui". Table 4.
Antioxidant Capacities of Lignans and Flavonoids from Chilean Medicinal Plants* ii
Compound
(IC50)TBAR 0tM)
Dihydroguayaretic acid
2.8
Guayacasin
1.1
Isopregomisin
0.7
Propyl gallate
1.0
Quercetin
!.] (0.8) !
3~
64.0 (48.0) 1
7"-Methyisudachitin
60.0 (51.0) 1 ,
I IC50 determined through luminescence measurements. *Taken from Faur~ et al., 1990, [43].
i
lll l
L
SEARCH FOR ANTIOXIDANT ACTIVITY
353
MeO o.e
Meo- .I .fL OH
O
R -- OMe; 3"-methoxycalieopterin R = H; 7"-methylsudanehitin Fig. (10). Flavonoids from Baccharis incarum.
The leaf decoctions of Tessaria integrifolia R. et P. and Mikania cordifolia Willd. (Compositae) are used in traditional medicine of the Peruvian Andes as anti-inflammatory agents. Three caffeoylquinic acids isolated from these species were tested for their activities on monoeyte migration and superoxide anion production [44]. 3,5-Di-O-caffeoylquinic and 4,5-Di-O-caffeoylquinic acids exhibited an appreciable antiinflammatory activity in vitro, while the tricaffeoyl derivative was inactive. The compounds in study also decreased the zymosan induced liberation of O2-by macrophages, which was dependent on the concentration of the inhibitory molecules. These results indicate that the caffeoylquinic acids isolated from the leaves of T. integrifolia and M. cordifolia could affect migration and 02-secretion in activated human macrophages. The aerial parts of Eupatorium articulatum L. (Compositae) are widely used in the Andes region of Ecuador for the treatment of inflammatory diseases. The methanolic extract of this species exhibited a significant in vivo anti-inflammatory activity, when tested for its ability to reduce carrageenan-induced inflammation in rat paw oedema, thus supporting the traditional use claimed for this plant. Since the inflammation process is associated to the generation of reactive oxygen species and the induction of lipid peroxidation, the antioxidant properties of the extract were also studied [45]. Significant activity was observed when tested for its ability Table 5.
Percentage Inhibition in C a r r a g e e n a n Induced I n f l a m m a t i o n , M i c r o s o m a i Lipid Peroxidation, Superoxide Generation and Xanthine Oxidase Production in the Presence of the Ethanolic Extract of E. artlculatum*
Lipid peroxidation
inflammation
i
I00 (g/ml I
3hi
592 i
0 2 9generation
xanthine oxidase
70.6 (I.I
81.2 (2.1
i
5h
I
7h
....
42.3 I 53.6 I I
*Taken from de las Heras et al, 1997, [45].
.
I
61.7 (I.1 .
.
.
DESMARCHELIER eta/.
354
to inhibit non-enzymatic lipid peroxidation in rat liver microsomes induced by FeCl3-ascorbate and in scavenging superoxide anions by the inhibition xanthine oxidase activity (Table 5). Alkaloids and other nitrogen compounds of higher plants have also shown to exert antioxidant effects in various systems. These effects include inhibition of peroxidation in rat liver homogenates and physical quenching of superoxide radicals [3]. Boldine, Fig. (11), is an alkaloid present in the leaves of Peumus boldus Mol. (Monimiaceae), a widely distributed evergreen tree native to Chile. Boldine-containing boldo leaf extracts and infusions are used in traditional medicine in the treatment of a variety of conditions, among which liver complaints and dysfunctions are generally mentioned [46]. Anti-inflammatory and antipyretic effects of boldine have been previously described [47], and recent studies have shown that boldine behaves as a very potent antioxidant in biological systems undergoing lipid peroxidation, thus providing a possible rationale for the hepatoprotective properties attributed to boldo .[48,49,50,51,52]. HO CH30
CH30"
y OH
Fig. (11). Boldine from Peumus boldus.
Leaf extracts of another Chilean medicinal plant, Psoralea glandulosa L. (Fabaceae), have shown to possess anti-pyretic and anti-inflammatory activities [53]. A major component of this plant is the meroterpenoid bakuchiol, Fig. (12), which has been claimed as the active principle, responsible for the therapeutic properties described [54]. Although bakuchiol was demonstrated to be a natural anti-inflammatory agent able to control leukocyte functions such as eicosanoid production, migration and degranulation in the inflammatory site, it only showed a weak inhibition in the generation of superoxide. Future studies will be necessary in order to determine if this compound is capable of reducing lipid peroxidation and other free radical induced cell damage.
HO Fig. (12). Bakuchiol from Psoralea glandulosa.
SEARCH FOR ANTIOXIDANT ACTIVITY
355
BRAZILIAN AMAZON The region of the Amazon forest in Brazil is roughly delimited by latitudes 0 ~ and 12~ and longitudes 48 ~ and 74~ and has an average elevation of 100 m and an area of 3.4 million km 2 [55]. Besides high indices of botanical diversity and endemism, this large and heterogeneous territory contains a large number of new undescribed species [56], and is inhabited by different native groups which have extensive experience with medicinal plants. Palm trees are extensively distributed in the Amazon and in other tropical ecosystems of South America. It has been determined that oil palm (Euterpe spp., Arecaceae) is the best reported source of tocotrienols, Fig. (13), which have been regarded as better than ct-tocopherol and vitamin E for their antioxidant activity [57]. Tocotrienols are natural Vitamin E analogues which differ from tocopherols in the number of methyl groups besides having an unsaturated chain [58]. Another Brazilian palm, Elaeis guineensis Jacq. (Arecaceae), has also been stated as an excellent source of 13-carotene and tocotrienols [59]. Carotenoids such as 13-carotene, "t-carotene and lycopene have shown to act in vitro as antioxidants at low oxygen concentrations by forming a stabilised radical after addition of peroxyl radicals [60], and by reducing the in vivo production of pentane, an oxidation product of fatty acids during lipid peroxidation [61 ]. HO
Fig. (13). y-tocotrienol from Euterpe, Elaeis, lryanthera and Virola spp.
Different species of the rainforest trees Virola and lryanthera (Myristicaceae) contain a large number of chemically different substances with antioxidant activity, including tocotrienols [62]. For example, two tocotrienols isolated from the ethanolic fruit extract of Iryanthera grandis Duke. have demonstrated in vitro antioxidant activity. Tocotrienol-8 antioxidant capacity measured as the inhibition of spontaneous brain autoxidation was 10 times higher than Vitamin E in the MDA assay and 8 times higher in the chemiluminescence assay 3. Bioassay guided fractionation also showed that two flavonoids, 3-0-rhamnosil-kaempferol and 3-0-rhamnosil-quercetin, together with a novel dimmeric dihydrochalcone, Fig. (14), are responsible for the inhibition of the spon-
3Barros, S. Higher plants as sources of antioxidants for the treatment of oxidative stress mediated disease. In: International Symposium: Oxygen Radicals in Biochemistry, Biophysics and Medicine. Buenos Aires, March, 1994 (abstract $8).
356
DESMARCIIELIER et aL
OH HO
OH
O
R = H; 3-0-rhamnosil-kaempferol
R = OH; 3-0-rhamnosil-quercetin
O
HO,
OH
Me<) Fig. (14). Two flavonoids and a novel dimmeric dihydrochalcone from Iryanthera sagotiana.
taneous oxidation in rat brain homogenates, measured as a reduction in the production MDA observed in the presence of leaf extracts of Iryanthera sagotiana ~. Tocotrienols, dihydrochalcones and flavonolignoids have also been identified in Iryanthera lancifolia Ducke s, popularly known as "cumala blanca". Finally, both in vitro and in vivo antioxidant activity were also detected in Virola sebifera Aubl. and K elongata (Benth.) Warb. fruit extracts 6. Although the active compounds have not yet been identified, different lignans have been previously isolated from the pericarp, aril and seeds of Ksebifera, suggesting that these structures could be responsible for the activity described 7. Leaves and roots of the tropical shrubs Pothomorphe umbellata (L.) Miq. and P. peltata (L.) Miq. (Piperaceae), locally known as "pariparoba" and "hoja santamaria" respectively, are widely used in the treatment of liver diseases and other inflammatory disorders not only by the local 4Silva, D.; Davino, S.; Barros, S." Kato, M.; Yoshida, M. Atividade antixidante de flavonbides de Iryanthera sagotiana (Myristicaceae) In: )(IV Simp6sio de Plantas Medicinais do Brasil. Florian6polis, September, 1996 (abstract Q 053). 5Diqueira Silva, D." Yoshida, M. Chemical constituents from inflorescences of lyanthera lancifolia (Myristicaceae) In: II World Congress on Medicinal and Aromatic Plants for Human Welfare (WOCMAP II). Mendoza, Argentina, November, 1997. (abstract P 221). 6Davino, S.; Tamasiro, V.; Santos, E.; Almeida, M." Kato, M." Barros, S. Atividade antixidante de produtos naturais de esp~cies de Virola (Familia Myristicaceae) In: )(IV Simp6sio de Plantas Medicinais do Brasil. FIorian6polis, September, 1996 (abstract M 004). 7Danelutte, A.; Cavalheiro, A.; Kato, M. Determination of lignoids in seedlings of Virola sebifera Aubl. by HPLC. In: II Worm Congress on Medicinal and Aromatic Plants for Human Welfare (WOCMAP II). Mendoza, Argentina, November, 1997. (abstract P 152).
357
SEARCH FOR ANTIOXIDANT ACTIVITY
Table 6.
Inhibition of Lipid Peroxidation (Chemiluminescence and TBARS) and Free Radical-mediated DNA-sugar Damage Induced by the Presence of Fe (II) Salts ( I C 5 0 ' s ) in M e t h a n o l i c Extracts and Isolated Compounds of
Pothomorphe spp. , , ,
,
,
Tested sample ,i
CL (Bg/mi) ,
ii
,
,,,
,,..,
TBARS (Bg/ml)
DNA damage (Bg/ml)
,.,
P. umbellata
ND
35*
21
P. peltata
4
> 1000
5
4-nerolydilcatechol ((M)
0.8
I
25
ND: not determined. *measured in autoxidation of brain homogenates
inhabitants of Amazonia, but also in other regions of tropical America. Different extracts of these plants have shown to reduce oxidative stress when tested in in vitro lipid peroxidation models (Table 6) [63,64]. Catechol derivative 4-nerolidylcatechol, Fig. (15), present in both species studied [65,66], also showed high antioxidant activity when compared to o~-tocopherol, indicating that the activity observed in the extracts could be attributed to the presence of this compound [63]. OH HO
Fig. (15). 4-Nerolidylcatechol from Pothomorphe spp.
Recent comparative studies have demonstrated that extracts of P.
peltata and P. umbellata, as well as isolated 4-nerolidylcatechol, reduce hydroxyl radical-mediated degradation of DNA (Table 7) [64,67]. The TRAP and TAR indices were also determined by monitoring the intensity of luminol enhanced chemiluminescence (Table 7). The results obtained suggested that 4-nerolidylcatechol and the methanolic extracts of P. umbellata and P. peltata that contain this compound can scavenge peroxyl and hydroxyl radicals, thus reducing oxidative stress that can lead to lipid peroxidation and DNA damage. It has been stated that the antioxidant properties of phenols are due to the phenolic groups present in their chemical structure [68]. However, part of the higher activity observed for 4-nerolidylcatechol in protecting DNA from hydroxyl radical-mediated oxidation could also be due to the presence of two hydroxyl groups and an aliphatic unsaturated chain, which can contribute to an increased antioxidant activity when compared to tocopherols, such as in the case of tocotrienols [63]. o~-Tocopherol, used as a standard, has only one
358
DESMARCHELIER et aL
hydroxyl group in the aromatic ring and a saturated side chain. On the other hand, the extract of at least one plant studied shower higher activity than 4-nerolidylcatechol in each assay, suggesting that the isolation of an active compound may not always account for all the qualitative or quantitative activity of the total extract. There is increasing experimental support for the synergistic effects of compounds administered together, such as the increased protection against oxidation of low density lipoproteins and depletion of o~-tocopherol when quercetin and catechin, both common plant constituents, are coadministered [69]. Table 7.
Total Reactive Antioxidant Potential (TRAP) and Total Antioxidant Reactivity (TAR) in (M Trolox Equivalents; Number of Radicals (n) Trapped Per Molecule of Additive, IC50's and Percentage of Efficiency, O b t a i n e d From the Quenching of Luminol Chemiluminescence in Methanolic Extracts and Isolated Compounds of Pothomorphe spp.
Tested sample
TRAP ((M)
IC50 ((g/(i)
efficiency (%)
0.6 ( 0.1
13.3
26
5.0 ( 1.9
21.7
TAR ((M)
i
ii
,i
i
P. umbellata
97.2 ( 10.8
....
P. peltata
4-nerolidylcatechol
16.4 ( 7.1
-
33.6 ( 23.0
0.71
190 ,,,
4.9 ( 0.2
4.9
164
ND: not determined.
In vitro antioxidant activity was also evaluated in Piper regnellii, another plant from the Piperaceae family commonly used as it were P. umbellata. The activity of the extracts was evaluated in spontaneous lipid peroxidation of brain homogenates, and both TBARS and chemiluminescence were employed as parameters for lipid peroxidation evaluation. Although P. regnellii showed some antioxidant capacity, this was below that of P. umbellata, probably due to the absence of 4nerolidylcatechol in the former. Leaves of Coleus barbatus (Andr.) Benth. (Labiateae), known as "falso boldo", are used in the region as an effective hepatoprotective agent. A comparative study of the hydroalcoholic extract of leaves of C. barbatus, P. boldus ("Chilean boldo") and boldine indicated the presence of antioxidant activity for all the samples, measured by means of an inhibition in lipid peroxidation (V. Tamasiro, personal communication). However, C. barbatus was prooxidant when tested in vivo, thus indicating that this species should not be used as it were P. boldus. THE WESTERN AMAZON BASIN About 16% of the total plants that exist in the world today, at least 35,000 species, are found in the tropical rain forests of the Amazon, and
SEARCH FOR ANTIOXIDANT ACTIVITY
359
many of them occur in the westernmost part of that region, which is the least studied [70]. This region, which includes areas of Bolivia, Peru, Ecuador and Colombia, presents high indices of biological diversity [41 ], relatively lower rates of deforestation and is inhabited by different groups of Amerindians which possess a wide pharmacopoeia and a strong tradition in which healers transmit their medicinal plant knowledge from generation to generation [e.g. 71 ] The edible fruits of Myrciaria dubia (HBK) McVaugh. (Myrtaceae) and A verrhoa carambola (Sw.) Beauv. (Oxalidaceae) have become known for their high content of vitamin C, Fig. (16), the former being the highest known source of ascorbic acid, with 2-3 g/kg [57]. Water-soluble vitamin C has many physiological roles; antioxidant activity, including the recycling of vitamin E in membranes and lipoproteins [72,73], is only one of them. Ascorbate appears to be the most important non-protein antioxidant in plasma, and in vitro studies suggest a strong inhibition of the oxidation of lipoproteins in the presence of this compound [74]. However, prooxidant activity should also be mentioned in the case of vitamin C, since it has long been known that the combination of ascorbate and ferrous ions generates hydroxyl radicals which can induce lipid peroxidation [4].
X_.o. Fig. (16). Vitamin C (aseorbir acid) from Myreiaria dubia and Averrhoa earambola.
Portulaca oleracea L. (Portulacaceae), known locally as "verdolaga", is widely used in the region for the treatment of different diseases. Several poli-unsaturated fatty acids with potential cholesterolemic properties have been identified in this species [75], and pharmacological studies have shown that the aqueous extract of the leaves is hypoglycaemic [76]. Phytochemical analysis of P. oleracea has shown that this plant is a good source of antioxidants such as o~-tocopherol (vitamin E), Fig. (17), ascorbic acid (vitamin C) and 13-carotene, Fig. (18), [77], making it a promising chemopreventive herb. o~-Tocopherol is a lipid-soluble compound found in lipoproteins and membranes that acts to block the chain reaction of lipid OH
Fig. (17). Vitamin E (a-tocopherol) from Portulaca oleracea.
360
DESMARCHELIERel as
peroxidation by scavenging the intermediate peroxyl radicals which are generated in the hydrophilic domains of membranes during this process [4], probably due to the long lipophilic chain present in its structure [78]. H3C~CH3
~
~H3
~
qH3
u
I H3
Fig. (18). [3-carotene from Portulaca oleracea.
The bark and latex of Chlorophora tinctoria (L.) Gaud. (Moraceae) are used as an anti-inflammatory and pain reliever by some indigenous groups in Peruvian Amazon. The wood of this tree, locally known as "turcash", has shown to contain morin, Fig. (19), a highly hydroxylated flavonoid [79]. Morine, like other flavonoids, significantly reduces the autoxidation rate of fatty acids and inhibits the formation of hydroperoxide isomers of linoleic acid, thus exhibiting a great H-atom donating ability to peroxyl radicals that terminate the chain radical reaction [27]. HO
HO~OH
OH
0
Fig. (19). Morin from Chlorophora tinctoria.
The charge of antioxidants in different extracts of medicinal plants used in south-west Amazonia regions of Beni (Bolivia) and Madre de Dios (Peru) was determined employing luminol-enhanced chemiluminescence [80]. TRAP and TAR values were determined by using this method, and the highest activity was recorded in the aqueous and methanolic bark extracts of Copaifera reticulata (Caesalpinaceae) and Heisteria pallida (Olacaceae). Both species are widely used as anti-inflammatory agents throughout the region, the former locally known as "copaibo" and the later as "chichiguazo" or "chuchuhuasi". Further studies also showed that these extracts protected DNA against in vitro oxidative damage in terms of deoxyribose oxidation [81 ] and reduced lipid peroxidation in vitro and in vivo (Table 8). The compounds responsible for the antioxidant activity in C. reticulata have been identified as glieosylated procyanidins, and it has been determined that the bark of H. pallida contains high concentrations of catechin and catechin-based tannins (H. Constant, personal communication). Flavonoids such as catechin have shown to exert antiinflammatory effects by inhibiting arachidonic acid metabolism as one of
SEARCH FOR ANTIOXIDANT ACTIVITY
361
their mechanisms [82], and this could therefore explain the antiinflammatory properties claimed for H. pallida. T a b l e 8. i
i
A n t i o x i d a n t A c t i v i t y in E x t r a c t s o f S o u t h w e s t A m a z o n i a n M e d i c i n a l Plants
,
Botanical name i
TRAP
TAR
(pM) (pM)
CL (pg/ml)
TBARS
(pg/ml)
DNA damage (pg/ml)
(pg/ml)
in vivo
1
C. reticulata i
4200
22
NA
> 1000
3 (! 7-0.01)
561 (1239-337)
C. lechleri 2
935
NA
NA
161 (912-48)
ND
200 (950-154)
H. pallida 1
3850
12
NA
636 (1201-397)
ND
NA
U. tomentosa I
84
56 (36-83)
889 (2415.469)
17 (50-I)
NA
I methanolic extract, 21iophylized latex. Total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) are expressed in (M Trolox equivalents. CL and TBARS are expressed as IC50 ((g/ml), and include 95% confidence intervals. NA: not active; ND: not determined due to lack of dose dependence.
"Sangre de drago" is a red viscous latex obtained from the bark of Croton lechleri Muell. Arg. (Euphorbiaceae) and other species of Croton. Cicatrizant, anti-inflammatory, antiviral and antitumour properties have been claimed for "sangre de drago" by indigenous populations in many parts of the westernmost part of the Amazon valley, including Colombia, Ecuador, Peru and Bolivia (83). In addition to the alkaloid taspine [84] and the lignan 3",4-O-dimethylcedrusin [85], responsible for the antiinflammatory, antiviral, antitumour and wound healing properties, the latex of C. lechleri was found to contain several proanthocyanidins as major constituents, which account for up to 90 % of the dried weight. These include catechin, epicatechin, gallocatechin, epigalloeatechin, Fig. (20), and five novel dimmers and trimmers of these compounds [86], Fig. (20). The implication of antioxidation in the wound healing process, together with the participation of oxygen free radicals in inflammation and carcinogenesis, has lead us to study the free radical trapping capacity of "sangre de drago" [87]. The capacity of the lyophilised latex from C. lechleri to scavenge peroxyl radicals derived from thermolysis of ABAP was determined by monitoring the intensity of luminol enhanced chemiluminescence. Although "sangre de drago" was highly effective in scavenging free radicals at high concentrations, the additive incorporation of lower concentrations of the latex yielded an instantaneous increase in chemiluminescence, suggesting prooxidant activity. DNA sugar damage induced by Fe (II) salts was also used to determine the capacity of the latex to suppress hydroxyl radical-mediated degradation of DNA. As in the case of luminol enhanced chemiluminescence, "sangre de drago" was
362
DESMARCHELIER et aL
Epieatechin OH
HO
~H
,,,( H Galloeateehin
OH
HO
,,,( ~H '~'t3H Epigallocateehin
Fig. (20). Proanthocyanidins from Croton lechleri.
highly effective in reducing oxidation of DNA at higher concentrations, but showed an increase in the production of TBARS at lower doses, compared to the control. Lipid peroxidation was also inhibited in the presence of the latex, as determined by a reduction in the production of TBARS in rat liver homogenates, both in vitro and in vivo. The results obtained are summarised in Table 8. Uncaria t o m e n t o s a (Willd.) DC. is a vine belonging to the family Rubiaceae that grows in the Amazon forests of Bolivia and Peru, and is locally known as "ufia de gato". The aqueous extract and decoction of the bark of this plant are widely used in traditional Peruvian medicine for the treatment of cancer and as a potent anti-inflammatory agent. Antioxidant activity in extracts of U. tomentosa was determined using different bioassays, and the free radical scavenging capacity was observed both against lipid-peroxidation and DNA damage in methanolic extracts of bark
SEARCH FOR ANTIOXIDANT ACTIVITY
363
and roots (Table 8) [88]. Since U. tomentosa extracts and fractions previously showed significant anti-inflammatory activity in rat paw oedema [89] and antimutagenic effects against induced photomutagenesis [90], it may be proposed that their efficacy could be partially due to their free radical scavenging activity. Although the compounds responsible for the antioxidant activity observed in U. tomentosa have not yet been identified, the presence of flavonoids kaempferol and dihydrokaempferol, Fig. (21), has been previously reported in the bark of the related species U. guianensis, also known as "ufia de gato" [91 ].
HO
Kaempferol
OH HO
Dihydrokaempferol Fig. (21). Flavonoids from Uncaria guianensis.
FUTURE The search for new active natural products such as antioxidants in South American plants has, in many aspects, only just begun. The poorly known flora of the drylands of"Patagonia", located in the far south of Argentina; the xerofitic vegetation of the "Caatinga" region, which covers almost 1 million km 2 in north-eastern Brazil and is extensively used in local popular medicinal practice, particularly among the African derived populations which inhabit the region; the flora of the "Pantanal", a swamp ecosystem in west centre Brazil, which is highly preserved and with scarce registered ethnobotanical data; and the Chocoan rainforests in north-west Colombia, all enclose, with no doubt, pharmacological secrets that are waiting to be unveiled in the future.
364
DESMARCHELIER eta/.
ACKNOWLEDGEMENTS Studies on antioxidant activity in South American medicinal plants are funded by the International Foundation for Science (IFS), in Stockholm, Sweden (Grant agreement No. F/2628-1), BID-CONICET (grant PMTSID0370) and the University of Buenos Aires (grant UBACYT FAO12/J). REFERENCES [~] [21 [31 [4] [51 [6] [71 [8] [91 [10] [ll] [121 [131 [14]
[151 [161 [171 [18]
[19] [201
[21] [22] [23] [24]
Gordon, M. Nat. Prod. Rep., 1996, 265. Bast, A.; Goris, R. Pharm. Weekbl. [Sci.], 1989, 11, 199. Larson, R. Phytochemistry, 1988, 27, 969. Haslam, E., J. Nat. Prod., 1996, 59, 205. Kinsella, J.; Frankel, E.; German, J.; Kanner, J. J. Food Technol., 1993, 85. Kinsella, J.; Frankel, E.; German, J.; Parks, E., Kanner, J. Lancet, 1993, 341, 454. Maxwell, S.; Cruickshank, A; Thorpe, G. Lancet, 1994, 344, 193. Serafini, M.; Ghiselli, A.; Ferro-Luzzi, A. Lancet, 1994, 344, 626. Kondo, K.; Matsumoto, A.; Kurata, H.; Tanahashi, H.; Koda, H., Amachi, T.; Itakura, H. Lancet, 1994, 344, 1152. Rice-Evans, C. Biochemist, 1996, 17, 8. Cox, P; Balick, M. Sci. Am.,1994, June, 82. Caldwell, M.; Robberecht, R.; Flint, S. Physiol. Plant., 1983, 58, 445. Levin, D. Am. Nat., 1976, 110, 261. Larson, R.; Marley, K. Phytochemistry, 1984, 23, 2351. Gentry, A. In Human Medicinal Agents from Plants, Kinghorn, D.; Balandrin, M., Eds.; ACS Symposium Series, Maple Press, York, P.A., 1993; pp. 13-24. Bors, In Handbook ofAntioxidants; Cadenas, E.; Packer, L., Eds.; Marcel Dekker Inc., N.Y., 1996; pp. 409-466. Phillips, D. In Recent Advances in Phytochemistry. Vol. 26. Phenolic Metabolism in Plants, Stafford, H., Ibrahim, R., Eds.; Plenum Press, N.Y., 1992; pp. 201231. Soejarto, D.; Gyllenhaal, C.; Ashton, P.; Sohmer, S. In Medicinal Resources of the Tropical Forest. Biodiversity and its Importance to Human Health; M.J. Balick, E. Elisabetsky, S.A. Laird, Eds.; Columbia University Press, N.Y, 1996; pp. 284-310. Cross, C.; Halliwell, B.; Borish, E.; Pryor, W.; Ames, B.; Saul, R.; McCord, or. Ann. Intern. Med., 1987, 107, 526. Lin, J.; Lin, C.; Chen, M.; Ujiie, T.; Takada, A. J. Ethnopharmacol., 1995, 47, 33. Seivam, R.; Subramanian, R.; Gayathri, R.; Angayarkanni, N. J. Ethnopharmacol., 1995, 47, 59. Sultana, S.; Perwaiz, S.; Iqbal, M.; Athar, M. J. Ethnopharmacol., 1995, 45, 189. Middleton, E. Int. J. Pharmacognosy, 1996, 34, 344. Baumann, J.; Wurm, G.; Bruchhausen, F. Arch. Pharm., 1980, 313, 330.
SEARCH FOR ANTIOXIDANT ACTIVITY
365
[25] Sorata, Y.; Takahama, U.; Kimura, M. Biochem. Biophys. Acta, 1984, 799, 313. [26] Yokozawa, T.; Dong, E.; Wu Liu, Z.; Shimizu, M. Phytother. Res., 1997, 11, 446.
[27] Torel, J.; Cillard, J.; Cillard, P. Phytochemistry, 1986, 25,383. [281 Broussalis, A.; Ferraro, G.; Gurni, A.; Coussio, J. Acta Farm. Bonaerense, [29]
[30] [31] [32] [33] [34]
[35] [36] [37]
[38] [39]
[40] [41] M2] [43]
[44] [45] [461 [471
[48] [49] [50] [51] [52]
1989, 8, 11. Desmarchelier, C.; Coussio, J.; Ciccia, G.; Braz. J. Med. Biol. Res. 1998, 31, 1163. Lissi, E.; Pascual, C.; del Castillo, M. Free Rad. Res. Com. 1992, 17, 299. Norbedo, C.; Ferraro, G.; Coussio, J. or. Nat. Prod., 1982, 45, 635. Fraga, C.; Martino, V.; Ferraro, G.; Coussio, J.; Boveris, A. Biochem. Pharmacol., 1987, 36, 717. PayS, M.; Silla, M.; Vay~, E.; Alcaraz, M.J.; Coussio, J.; Ferraro, G.; Martino, V.; Hnatyszyn, O.; Debenedetti, S.; Broussalis, A.; Muschietti, L. Phytother. Res., 1996, 10, 228. Debenedetti, S.; Palacios, P.; Wilson, E.; Coussio, J. Acta Hort., 1993, 333, 191. Debenedetti, S.; Palacios, P.; Wilson, E.; Coussio, J. Fitoterapia, 1994, LXV, 188. Jarvis, B.; Midiwo, J.; Bean, G.; AbouI-Nasr, B.; Barros, C. (1988) J. Nat. Prod., 1988, 51,736. Mongeili, E.; Desmarchelier, C.; Rodriguez Talou, J.; Coussio, J.; Ciceia, G. J. Ethnopharmacol. 1997, 58, 157. Desmarchelier, C.; Novoa Bermudez, M.J.; Coussio, J.; Ciccia, G.; Boveris, A. Int. d. Pharmacognosy 1997, 35, 116. Martino, V.; Ferraro, G.; Debenedetti, S.; Coussio, J. ,4cta Farm. Bonaerense, 1984, 3, 141. Ferraro, G.; Martino, V.; Coussio, J. Phytochemistry, 1977, 16, 1618. Gentry, A. In Conservation Biology; M.E. Soule, Ed., Sinauer Associates, Sunderland, Mass., 1986, p. 78. Girault, L. Kallawaya: curanderos itinerantes de los Andes. UNICEF-OPSOMS" Paris, 1987. Faur6, M.; Lissi, E.; Torres, R.; Videla, L. Phytochemistry, 1990, 29, 3773. Peluso, G.; de Feo, V.; de Simone, F.; Bresciano, E.; Vuotto, M.L.J. Nat. Prod. 1995, 58, 639. de las Heras, B.; Slowing. K.; Bened, J.; Carretero, E.; Ortega, T.; Toledo, C.; Bermejo, P.; Iglesias, I.; Abad, M.J.; Serranillos, P.G.; Liso, P.A.; Villar, A.; Chiriboga, X. J. Ethnopharmacol. 1998, 61, 161. Speisky, H.; Cassels, B.; Nieto, S.; Valenzuela, A.; Nuflez-Vergara, L. J. Chromatogr., 1993, 612, 315. Backhouse, N.; Delporte, M.; Guivemau, M.; Cassels, B.; Speisky, H. Rev. Med. Chile, 1994, 31, 114. Bannach, R.; Valenzuela, A.; Cassels, L.; Nuflez-Vergara; Speisky, H. Cell Biol. Toxicol., 1996, 12, 89. Campos, A.; Lissi, E. Bol. Soc. Chil. Quire., 1995, 40, 375. Speisky, H.; Cassels, B.; Lissi, A.; Videla, L. Biochem. Pharmacol., 1991, 41, 1575. Speisky, H.; Cassels, B. Pharmacol. Res. 1994, 29, 1. Valenzuela, A.; Nieto, S.; Cassels, B.; Speisky, H. J. Am. Oil Chem. Soc., 1991, 68, 935.
366
[53] [54]
[551
[56] [571
[58] [59]
[60] [611 [62] [63]
[64] [651 [66] [67] [68]
[69] [70] [71] [72] [73]
[74] [75] [76] [77]
[78] [79]
[80] [81]
DESMARCHELIER et aL
Backhouse, N.; Delporte, C.; Negrete, R.; Salinas, P.; Pinto, A.; Aravena, S.; Cassels, B. Int. d. Pharmacognosy, 1996, 34, 53. Ferr~ndiz, M.L.; Gil, B.; Sanz, M.; Ubeda, A.; Erazo, S.; Gonzfilez, E.; Negrete, R.; Pacheco, S.; Pay~t, M.; AIcaraz, M.J.J. Pharm. Pharmacol., 1996, 48, 975. Souza Brito, A. R. M.; Souza Brito, A. In Medicinal Resources of the Tropical Forest. Biodiversity and its Importance to Human Health; M.J. Balick, E. Elisabetsky, S.A. Laird, Ed.; Columbia University Press, N.Y, 1996; pp. 386401. Gentry, A. In Biological Diversification in the Tropics; Prance, G., Ed.; Columbia University Press, N.Y. 1982; p., 714. Duke, J.A.; Vasquez, R. Amazonian Ethnobotanical Dictionary, CRC Press: Boca Rat6n, 1994. Traber, M. In Handbook ofAntioxidants; Cadenas, E.; Packer, L., Eds.; Marcel Dekker Inc., N.Y., 1996; p. 43-61. Melo, M.; Mancini, J. Rev. Farm. Bioquim. Univ. Sao Paulo, 1989, 25, 147. Burton, G.; Ingold, K., Science, 1984, 224, 569. Allard, J.; Royall, D.; Kurian, R.; Muggli, R.; Jreejeebhoy, K. Am. J. Cli. Nutr., 1994, 59, 884. Dugall, S.; Kartha, A. Indian J. Agric. Sci., 1956, 26, 391. Barros, S.; Teixeira, D.; Aznar, A.; Moreira Jr, J.; lshii, I.; Freitas, C. Cienc. Cult. Sao Paulo, 1996, 48, 114. Desmarchelier, C.; Mongelli, E.; Coussio, J.; Ciccia, G. Braz. d. Med. Biol. Res. 1997, 30, 85. Kijjoa, A.; Giesbrecht, A.; Akisue, M.; Gottlieb, O.; Gottlieb, H. Planta Med., 1980, 39, 85. Gustafson, K.; Cardellina, H.; McMahon, J.; Pannell, L.; Cragg, G.; Boyd, M. J. Org. Chem. 1992, 57, 2809. Desmarchelier, C.; Repetto, M.; Coussio, J.; Ribeiro Latorre, L.; Kato, M.; Barros, S.; Ciccia, G., Planta Meal, 1997, 63, 561. Van Acker, S.; Koymans, L.; Blast, A. d. Free Radic. Biol. Med., 1991, 15, 311. Houghton, P. J. Alt. Compl. Med., 1995, I, 131. Tyler, V. In Medicinal Resources of the Tropical Forest. Biodiversity and its Importance to Human Health; M.J. Balick, E. Elisabetsky, S.A. Laird, Eds.; Columbia University Press, N.Y, 1996; pp. 3-10. Desmarchelier, C.; Gurni, A.; Ciccia, G.; Giulietti, A. d. Ethnopharmacol., 1996, 52, 45. Lambelet, F.; Saucy, F.; Loliger, J. Experientia, 1985, 41, 1384. Serbinova, E.; Kagan, V.; Han, D.; Packer, L. Free Radical Biol. Med., 1991, 10, 263. Rifici, V.; Khachadurian, K. J. Am. Coll. Nutr., 1993, 12, 631. Koch, H. Deut. Apoth. Ztg., 1988, 128, 2493. Akhtar, M.; Khan, Q.; Khaliq, T. J. Pak. Med. Ass., 1985, 35, 207. Simopoulos, A.; Norman, H.; Gillaspy, J.; Duke, J. Journal of the American College of Nutrition, 1992, 11,374. Burton, G.; Ingold, K., J. Am. Chem. Soc., 1981, 103, 6472. Gupta, M. (Ed.) 270 plantas medicinales iberoamericanas. CYTED, Santaf6 de Bogota, 1995. Desmarchelier, C.; Repetto, M.; Coussio, J.; Llesuy, S.; Ciceia, G. Int. d. Pharmacognosy 1997, 35, 288. Desmarchelier, C.; Coussio, J.; Ciccia, G.; Phytother. Res. 1997, 11, 460.
SEARCHFOR ANTIOXIDANTACTIVITY
[82] [83] [84]
[85] [86] [87]
[88] [89] [90] [91]
367
Ferrfindiz, M.; Alcaraz, M. Agents and Actions, 1991, 32, 283 Anonymous. Plantas medicinales amaz6nicas: realidad y perspectivas. Tratado de Cooperaci6n Amaz6nica, Secrctaria Pro-Tempore, Lima, 1995. Vaisberg, A..; Milla, M.; Planas, M.; Cordova, J. de Agusti, E.; Ferreyra, R.; Mustiga, M.; Carlin, L.; Hammond, G. Planta Med., 1988, 55, 140. Picters, L.; de Bruyne, T. Claeys, M.; Vlietinck, J. J. Nat. Prod., 1993, 56, 899. Cai, Y.; Evans, F.; Roberts, M.; Phillipson, J.D.; Zenk, M.; Gleba, Y.; Phytochemistry, 1991, 30, 2033. Desmarchelier, C.; Witting Schaus, F.; Coussio, J.; Ciccia, G.; J. Ethnopharmacol. 1997, 58, 103. Desmarchelier, C.; Mongelli, E.; Coussio, J.; Ciccia, G. Phytother. Res. 1997, 11,254. Aquino, R.; De Feo, V.; De Simonr F.; Pizza, C.; Cirino, G. J. Nat. Prod. 1991, 54, 453. Rizzi, R.; Re, F.; Bianchi, A.; de Feo, V.; de Simone, F.; Bianchi, L.; Stivala, L.A.J. Ethnopharmacol. 1993, 38, 63. Lock de Ugaz, O.; Callo, N. Revista de Quimica, 1991, 5, 47.
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22
369
9 2000 Elsevier Science B.V. All rights reserved
INSECT JUVENH E HORMONES IN PLANTS JACQUELINE C. BEDE and STEPHEN S. TOBE*
Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, M5S 3G5, Canada. A s Gregor Samsa a,:.,oke one morning from uneasy dreams he found hanaelf transformed ln hls bed lnto a glgantie lnsect. -
en "The
ea.n xpho a" E t )
ABSTRACT: In the defence against insect herbivory, plants may produce compounds which interfere with the insect endocrine system [2, 3, 4]. The focus of this review is to provide an overview of plant secondary metabolites which act either as insect juvenile hormone (JH) mimics or interfere with JH biosynthesis. Juvenile hormones are
sesquiterpenoids which are involved in the regulation of developmental processes such as metamorphosis and reproduction in most insect species [5]. The first section of this review provides an overview of insect JHs, including their physiological action and biosynthesis in hemimetabolous insects such as the cockroach, Diplopterapunctata, and their general chemistry. In the next part, examples of phytochemicals which act as JH mimics (juvenoids) or which inhibit JH biosynthesis will be presented, illustrating the diversity of compounds which are able to interfere with the endocrine system of the insect. Finally, a unique example of the production of insect juvenile hormone III (JH III), methyl-1OR,11-epoxy-3,7,1l-trimethyl 2E,6E-dodeeadienoate, in the sedge Cyperus iria L. will be presented and the possible biological role(s) of this compound discussed. INTRODUCTION One defensive strategy of plants against insect herbivory is the production of secondary metabolites which interfere with the physiology of the insect [6, 7]. It is thought that targeting the insect endocrine system makes it difficult for the insect to evolve counteradaptive strategies [3]. Insect juvenile hormones (JHs) are sesquiterpenoids which are involved in the regulation of insect metamorphosis and reproduction [5, 8]. In some insect species, these hormones are also involved in the regulation of other physiological processes including diapause and behaviour [8]. Plants have been shown to contain compounds which can either mimic JH activity or act as antagonists by inhibiting JH biosynthesis. The purpose of this review is to highlight some of these phytochemicals which are known to
370
BEDE and TOBE
interfere with the endocrine system of insects, specifically with the function of insect JH. Although there are many examples of plant secondary compounds which interfere with the physiology of insects [6, 7], there is debate as to the nature of the selective forces responsible for the evolution of these compounds [9, 10]. The argument has been made that although it appears that these secondary metabolites serve to protect the plant against insect herbivory today, it is likely that they were initially selected as a response to vertebrate herbivory [ 11 ]. However, phytochemicals which specifically interfere with the insect endocrine system possibly represent compounds which have evolved as protection against insect attack and therefore represent plant adaptations to insect herbivory. INSECT JUVENILE HORMONES To date, six JHs have been identified and their structures elucidated (Fig. (1)). Structurally, they share a sesquiterpenoid skeleton, a methyl ester O OMe
JH O
0 OMe
4-methyl J H I
O JH I
OMe O J H II OMe O J H Ill OMe
O J H III blsepoxide
OMe Fig. (1). Structures of insect juvenile hormones (JHs).
JUVENILE HORMONES
371
moiety on C-1 and an epoxide function. Juvenile hormone III (JH llI), methyl- 1OR, 11-epoxy-3,7,11-trimethyl 2E,6E-dodecadienoate, is the most common of these hormones and has been isolated from Lepidoptera (moths and butterflies), Coleoptera (beetles), Hymenoptera (sawflies, wasps, ants and bees), Orthoptera (grasshoppers, locusts, katydids, crickets), Dictyoptera (cockroaches) and Isoptera (termites) [ 12, 13]. In most of these insect orders, JH III is the only JH present. In the Lepidoptera, four other homologs have been isolated in addition to JH III. The ethyl branched homologs, juvenile hormone I (JH I), methyl- 1OR, 11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate, and juvenile hormone II, methyl-10R, 1 1-epoxy-3,7,11-trimethyl-2,6tridecadienoate, and their corresponding acids predominate in this order [ 14, 15, 16]. Two additional homologs, juvenile hormone 0 (JH 0), 1OR, 11-epoxy-3,7-diethyl-11-methyl-2,6-tridecadienoate, and 4-methyl JH I have been isolated from embryos of the tobacco homworm, Manduca sexta; JH 0 has also been isolated from males of the silkworm, Hylophora cecropia [ 17, 18, 19]. In higher Diptera (flies), a unique JH bisepoxide, methyl-6,7,10,11-bisepoxy-3,7,11-trimethyl 2E-dodecenoate, has been isolated and appears to be the principle JH of some species in this order [20]. CHEMISTRY OF JUVENILE HORMONE HI Chemical data for JH III is summarized in Appendix I, Table 1. In the infrared spectrum, the band at 1720 cm -~ is characteristic of an ester carbonyl group (C=O) and the band at 1650 cm -I is reflective of the alkene nature of the molecule (C=C) [ 14, 21 ].
Mass Spectroscopy Electron impact mass spectroscopy routinely uses a high energy beam of 70 eV; however, in the fragmentation pattern of JH III (Appendix I, Table 2), important high mass ions are also observed in the low energy resolution spectrum (15 eV) [22, 23, 24]. The observed peak at m/z 248 results from the migration of two hydrogen atoms to the epoxide oxygen and the subsequent loss of water. Mass ions at m/z 234 and 206 characterize the methyl ester moiety; m/z 234 represents the loss of the methyl ester group (CH3OH) and m/z 206 represents the loss of the methyl ester and the C=O from the acid. Fragmentation cleavage patterns give rise to mass ions at m/z 195, 114, 81 and 71 [22]. Low mass ion fragments (m/z 135, 114, 81, 71 and 43), which are detected in both the high and low energy resolution spectra, arise from hydrogen migration patterns suggestive of McLafferty-type rearrangements [25]. Scheme I illustrates the hydrogen
372
BEDE and TOBE
m/e 266
1 §
o~
§ o
o§
m / e 43
mle 71
Scheme I. Mass spectroscopy fragmentation pattern of juvenile hormone III [23] (reprinted with kind permission from the American Chemical Society).
rearrangement which generates an ion of m/z 71 and demonstrates the relationship between fragments produced at m/z 71 and 43 [23]. The base peak ion (m/z 81) results from two carbon-carbon bond cleavages and the transfer of one hydrogen atom [23] and can be generated through mass ions of m/z 195, as shown by Trost [22], or m/z 153 [23]. The mass ion m/z 135 is generated through the fragmentation of ions at m/z 153 and 195 and also m/z 163 [23] (Lietke). Lastly, the intense ion peak at m/z 114, which is present in the spectrum of JH III and its biosynthetic precursor methyl famesoate (MF), may occur by hydrogen transfer through a McLaffertytype rearrangement or by the migration of a hydrogen from C-4 to the carbonyl oxygen followed by transfer of another hydrogen from C-8 or C8' to C-4 [23-26].
JUVENILE HORMONES
373
§
~
9
m/e 266
H
m/e 195
OH * -H
CloHI5+
Of"
~
+ m/e 81
CH3OH
* - {CO+CH3OH) ~
CIoH15 +
~
* -
m / e 135
~
m/e 135
CIIHI5O+
CO
m/e 163
II. Mass spectroscopy fragmentation pattern of juvenile hormone III [23] (reprinted with kind permission from the American Chemical Society). Scheme
Nuclear Magnetic Resonance Proton Nuclear Magnetic Resonance Spectrum of Juvenile Hormone III Table 3 compares the assignment of chemical shifts based on three reports in the literature (Appendix I) [21, 22, 27]. The singlet at 8 3.72 represents the three protons on the methyl ester. The vinyl proton on C-2 is denoted by a signal at 8 5.75. The sharp doublet at 8 2.18 represents a vinyl methyl group which is cis and 13 to the carbonyl group. The vinyl proton on C-6 is indicated by ~55.22. The methyl on C-7 is shown by a singlet at ~i 1.65 The unusual chemical shift at 8 2.74 represents the epoxide proton on c - J 0 and the singlets at 8 1.27 and ~i 1.32 are due to the two methyl groups on C-11. The remaining protons on C-4, C-5, C-8 and C-9 are represented by bands at ~i 1.7 and ~i 2.1.
374
BEDE and TOBE
13CNuclear Magnetic Resonance Spectrum of Juvenile Hormone III The 13C nuclear magnetic spectrum assignment for JH III is listed in Table 4 (Appendix I) [28].
Chirality of Juvenile Hormone III
The absolute configuration of the epoxide group of insect JHs was determined by spectroscopy and circular dichroism [29]. In the first method, perchloric acid was used to catalyze the hydrolysis of the epoxide in the presence of H2180. The resulting vic-diol was analyzed by mass spectroscopy and definitively demonstrated the 10R,11S chirality of the epoxide [30]. The chirality was confirmed by circular dichroism. The epoxide was hydrolyzed by sulfuric acid and tetrahydrofuran and the circular dichroism cotton effect of the resulting acyclic glycol was measured in the presence of tris(dipivaloylmethanato)praseodymium [31]. General Chemistry of Juvenile Hormone III
Juvenile hormones are lipophilic molecules and relatively insoluble in aqueous solutions. The solubility of JH I is approximately 3 x 10.2 mM in 0.2 M Tris-HC1 buffer, pH 7.5 [32]; changes in pH, buffer and ionic strength had no effect on solubility. The addition of proteins such as bovine serum albumin (BSA) and immunoglobulin G increases the solubility of JH I; for example, in 5% BSA, a 1 mM solution of JH I can be made. Sonication also increases the solublity of the hormone by producing stable, finely dispersed micelles. Juvenile hormone III is more hydrophilic than JH I; in 5 mM Tris-HCl, pH 8.3, the solubility of JH III is greater than 2 x 10-1 mM [33]. However, its limited solubility must be recognized when preparing solutions. The epoxide group on insect JHs is particularly susceptible to hydrolysis through a SN2-type mechanism, generating the transdihydrodiol [27, 34, 35]. Therefore, protic acids, in aqueous or methanolic solutions readily convert the JH to its diol or 11-methoxy-10-hydroxy derivative, respectively [36, 37]. This reaction can be used to quantify JHs in insect haemolymph by generating derivatives which can either be analyzed directly or derivatized further and analyzed by gas chromatography-mass spectroscopy (GC-MS) (See Appendix I, Table 5 for references). Oxidation of the epoxide with neutral alumina or silica or heating to temperatures above 150oC results in an allylic alcohol which will undergo cyclization at higher temperatures (180oC)to a tetrahydrofuran derivative [27, 38]. Therefore, in the past, the direct analysis of JH by GC was contraindicated by the tendency for thermolysis at the temperatures required for volatization [38, 39].
JUVENILE HORMONES
375
However, this can now be overcome through the use of a cool-on-line injector and programming of the oven after sample injection (P. Teal, personal comm.). Cyclization of JH III to mono- and bicyclic products also occurs readily in the presence of boron trifluoride or phosphoric acid (H3PO4) [34]. Transition metals may catalyze similar reactions in aqueous solutions [12]. Inorganic salts, such as ferric chloride and zinc and magnesium sulfate, react with JH I, producing undetermined products [35]. The methyl ester function on C-1 of JH III is resistant to saponification by strong base [14]. One must also be aware of the binding affinity of JHs to different substrates. Juvenile hormones strongly adsorb to many commonly used plastic materials such as polystyrene, Millipore filters PSAC 02510, polyvinylchloride, polyethylene and plexiglass and to a lesser extent, glass and teflon [32]. Therefore, if plastics are used, it is recommended that they should be tested to determine the degree of JH affinity. Glassware should be treated with siliconizing agents or polyethylene glycol (PEG) to block JH binding sites [32]. Prior to this treatment, glassware should be washed with a nonionic detergent, rinsed in distilled water and heated to at least 200oC for 5 hours [27]. The use of an acid, such as chromic acid, in the washing of glassware is contraindicated because of the susceptible nature of the epoxide group to acid hydrolysis [27, 35]. Then the glassware can be treated with PEG or a siliconizing agent. Traces of acid catalyst in the PEG (Carbowax) may need to be removed before coating of the glassware, again because of the susceptibility of the epoxide ring to hydrolysis in the presence of acid. The solvents used should be of the highest quality and glass distillation is recommended in some cases [27]. Pure JHs are oils which can be stored for a number of years at -20oC or lower [ 12]. Solutions of JH, which are made up in aprotic, relatively nonvolatile solvents such as hexane or iso-octane, can also be stored at this temperature [12, 40]. In both these situations, the solution should be stored under an inert gas such as N2 to prevent the oxidation of the double bonds. Extraction and Quantification of the Juvenile Hormones
Methods for the extraction and quantification of physiological levels of insect JHs by chromatographic techniques (thin-layer chromatography, high performance liquid chromatography, gas chromatography-mass spectroscopy (GC-MS), immunological techniques (radioimmunoassay)) and the radiochemical assay will not be reviewed here but key references are listed in Appendix I, Table 5. Analysis by electron impact GC-MS requires an initial derivatization(s) due to the susceptibility of the epoxide group to thermolysis, followed by detection through electron capture or single ion monitoring. This treatment is not necessary with chemical ionization GC-MS.
376
BEDE and TOBE
Chemical Synthesis of Juvenile Hormone III The chemical synthesis of JH IIl will not discussed in this chapter; references on the synthesis are included in Appendix I, Table 5 and reviewed in the following [29, 41, 42]. ~
IOLOGICALROIEOF INSl ~ I ' ~ H O R M O N I ~
Metamorphosis Insect growth is discontinuous and comprised of discrete periods of metamorphosis, the change of form, which includes redifferentiation of body tissue and the shedding of the old cuticle. Pterygote (winged) insects undergo either complete metamorphosis (holometabolous, such as Lepidoptera which show larval (caterpillar), pupal and adult stages) or incomplete (partial) metamorphosis (hemimetabolous, such as the cockroach in which the larval or juvenile form resembles the adult form). The regulation of this metamorphosis by insect hormones, the ecdysones and JHs, differs in holometabolous and hemimetabolous insects. This chapter will focus on the latter. In the events leading up to metamorphosis, neurosecretory cells secrete prothoracicotropic hormone which acts on the prothoracic gland to stimulate the synthesis and release of the moulting hormone, 20hydroxyecdysone [5]. Ecdysone and its metabolites are responsible for the induction of the moulting process whereas the titre of JH in the insect haemolymph in the interval prior to the moult determines the characteristics. In hemimetabolous insects such as the Pacific beetle cockroach, Diploptera punctata, the presence of JH III is necessary for the maintenance of juvenile characteristics: JH III biosynthesis by the corpora allata (CA) is relatively high throughout immature stages and there is a drop in the rate of production during the penultimate and final nymphal stages prior to the moult to an adult (Fig. (2)) [43].
Gonadotrophic Cycle In adult females of most insect species, JHs are involved in the regulation of reproduction and oocyte maturation. Juvenile hormone-dependent processes include the synthesis of vitellogenin, the precursor of the yolk protein, by the fat body and the release of vitellogenin and its subsequent uptake and incorporation into the oocyte [44, 45]. Most insects are oviparous, laying eggs externally which undergo subsequent embryogenesis and development. However, there are also examples of viviparous insects, in which the fertilized egg undergoes early development within the female and derives nourishment from her, and
JUVENILE HORMONES
Penultimate instar 3.5 -mm
=3
377
Last instar 3.5-
Ecdysis
2.5
3.02.5-
l
2.0
2.0-
1.5
1.5
1.0
1.0
is
3.0
", S 0.5 0.0
~
0.5o
g
1'o
2'o
0.0
0
5
1'0
..... 1'5 270
Age (days) Fig. (2). Rates of juvenile hormone III (JH III) biosynthesis by the corpora ailata (CA) of the penultimate and final instars of the cockroach, Diploptera punctata [43]. The rate of biosynthesis, which is closely correlated to haemolymph titers, was measured by an in vitro JH III radiochemical assay [45, 46, 47]. Each point represents the mean of 3-12 determinations. Arrows indicate approximate times of ecdysis (reprinted with kind permission from Academic Press).
ovoviviparous insects, in which the eggs are incubated within the reproductive tract of the female. In these last two situations, there is a period of gestation within the insect. These three categories are represented within the order Dietyoptera (cockroaches), allowing comparison of the relationship between reproductive strategies and JH III titres in these insects [51, 52]. In the oviparous brown-banded cockroach, Supella longipalpa, females produce successive oothecae (eggs and protective easing) at five to seven day intervals. Each cycle of maturing oocytes is closely associated with a cycle of JH III biosynthesis by the corpora allata (CA) (Fig. (3)) [48]. Interestingly, mating is not necessary for the production and release of JH III and a similar trend is observed in virgin females [48]. This can be compared to JH III titres in another oviparous insect, the American cockroach, Periplaneta americana. In this species, mated adult females produce batches of eggs in rapid succession and the vitellogenir cycle encompasses two asynchronous egg-laying periods. In this situation, there is continuous JH III biosynthesis and no time when the hormone is completely absent [53, 54].
378
BEDE and TOBE
30 t 0
o,,,,4
m
Mating
25
20
=i "=1
& 0'
-
0
| . . . . . . .
5
1'0
1'5
2'0
Age (days) Fig. (3). Juvenile hormone III (JH III) release from the corpora allata (CA) of the oviparous cockroach, Supella longipalpa [48]. Juvenile hormone release was measured by the in vitro radiochemical assay [49, 50] and values represent the means of 5-12 determinations. Approximate times of mating and oviposition are indicated by arrows (reprinted with kind permission from Elsevier Scientific).
In both ovoviviparous and viviparous species of cockroaches, JH III biosynthesis is associated with vitellogenic growth of oocytes followed by periods of ovarian arrest and reduction of JH III biosynthesis during gestation [55, 56]. In mated females of the viviparous cockroach, Diploptera punctata, JH III titre is closely correlated to oocyte development (Fig. (4)) [57]; high rates of biosynthesis correspond to the rapid growth of oocytes as compared with the low synthesis observed during pre- and post-vitellogenic periods [52]. In adult virgin females, only basal JH III biosynthesis is observed. Similar results are seen in the ovoviviparous cockroach, Nauphoeta cinerea [58.]. BIOSYNTHESIS OF INSECT JUVENILE HORMONE III In D. punctata, JH III is biosynthesized in retrocerebral endocrine organs, the corpora allata (CA) [45]. The sesquiterpenoid skeleton of this compound is formed through the terpenoid biosynthetic pathway from acetyl-CoA. The early steps of this pathway involve the sequential condensation of three acetyl-CoA molecules (3 x 2C) to form the biosynthetic intermediate mevalonate (MVA, 6C). 3-Hydroxy-3methylglutaryl CoA reductase, which catalyzes the formation of MVA, is thought to be the rate-limiting enzyme in this pathway [59], although the
JUVENILE HORMONES
379
1800 1600 1400 1200 Q; *I,U
Oviposition
1000 800 600 400 200 0
-
o
....
....... 5"
--.,.,,~~
6'....... 7'
8"
lO
Age (days) Fig. (4). Juvenile hormone III (JH III) haemolymph titres in mated adult females of the viviparous cockroach, Diploptera punctata [57]. Haemolymph titre was measured by derivitizing JH III and subjecting the resultant methoxyhydrin to gas chromatography-mass spectroscopy [37]. Arrows indicate approximate time of oviposition (reprinted with kind permission from Birkhaeuser Pubi.).
regulatory importance of this enzyme has recently been questioned [60]. In the next series of steps, MVA undergoes three phosphorylations (net two) and a decarboxylation to generate the important isoprene intermediate, isopentenyl diphosphate (IDP, 5C); isomerization of this compound forms dimethyl allyl diphosphate (DMADP, 5C). Through head-to-tail covalent linkages of DMADP and IDP, prenyl diphosphate intermediates are formed which give rise to the terpenoid classes, such as the monoterpenes (10C), sesquiterpenes (15C), diterpenes (20C) and sterols. However, insects lack the enzymes to synthesize higher terpenoids [61] and JH III is synthesized from the fifteen-carbon farnesyl diphosphate (FDP). In vitro labelling of JH III in the CA of D. punctata has confirmed that the C-15 skeleton is derived from 3 moles of MVA [62]. In these studies, 6 moles of [2-14C]acetate were incorporated per molecule of JH III which reflects the expected incorporation of three acetate molecules, followed by the loss of a labelled carbon dioxide (14CO2) for every isoprene synthesized. From FDP, a phosphatase or pyrophosphatase catalyzes the removal of the pyrophosphate group, generating farnesol (Fig. (5)). The next two steps, oxidation of this alcohol to an aldehyde (farnesal), then a carboxylic acid (farnesoic acid), are catalyzed by one or two NAD+-dependent dehydrogenase(s) [63]. In the cockroach, D. punctata, methylation of
380
BEDE and TOBE
famesoic acid forms methyl famesoate (MF), followed by epoxidation at C 10,C 11 producing JH III [64, 65].
Farnesyl diphosphate
Phosphataseor pyrophosphataseI
Famesol
Famesoldehydrogenase,NAD+ 1
Farnesal
Farnesaldehydrogenase,NAD+ I
Farnesoic acid
JUVENILE HORMONES
381
(Fig. (5) contd .....
Methyl transferase S-adenosyl-methlonlne
!
Methyl farnesoate
Epoxidase 0 2, NADPH + H +
I
0 J u v e n i l e H o r m o n e HI Fig. (5). Biosynthetic pathway of juvenile hormone III in the cockroach, Diploptera punctata [65, 71].
The enzyme which catalyzes this methylation reaction, S-adenosylmethionine: famesoic acid o-methyltransferase (EC 2.1.1.-), is a cytosolic enzyme in the CA of adult female locusts, Locusta migratoria, and cockroaches, D. punctata [66, 67] and adult female tobacco homworms, M. sexta [68]. In a developmental profile, o-methyltransferase activity paralleled JH III biosynthesis in the CA of final larval instars and adult females of D. punctata [43, 57, 67]. The subsequent epoxidation reaction is catalyzed by a methyl farnesoate reduced flavoprotein: oxygen oxidoreductase (EC I. 14.14.-), which is associated with microsomal fractions in CA homogenates from the cockroach, Blaberus gigantus, and the locust, L. migratoria [66, 69, 70]. In Locusta, this enzyme was further shown to be a NADPH-dependent cytochrome P450 monooxygenase with an apparent Km of 7.7 x 10 -6 M, although the solubility of the substrate (methyl famesoate) limited the rate of the reaction. This pathway or variations of it occur in other insect species. The ethyl branches of the higher JH homologs found in the Lepidoptera (Fig. (1)) are derived from isoleucine and valine which are first metabolized to propionate and then incorporated, instead of acetate, in the early steps of biosynthesis [72, 73]. The sequence of the final steps of JH biosynthesis may also be different in these insect species. In the adult female CA of some Lepidoptera, epoxidation of farnesoic acid to the JH acid occurs before the final methylation step [64, 65, 71]. Interestingly, in larval stages of the tobacco hornworm, M. sexta, and adult males of the silkworm, Hyalophora cecropia, the CA synthesize and release JH acids into the
382
BEDE and TOBE
haemolymph which undergo the final methylation step in the imaginal disks or the accessory sex glands, respectively [74, 75]. In D. punctata, the haemolymph titre of JH is determined by the rate of biosynthesis and release from the CA and its degradation and clearance from the haemolymph by tissue uptake and excretion [ 13, 76]. The two primary routes of metabolism are through the cleavage of the methyl ester group by haemolymph esterases or the hydration of the epoxide by tissueassociated epoxide hydrolases. In the CA of final instars and in adult females of D. punctata, the low JH III titre in the haemolymph corresponds to low rates of biosynthesis by the CA and high esterase activity [43, 57, 77]. PLANT JUVENILE HORMONE MIMICS From the previous section, it is evident that JH III titers in insect haemolymph are precisely regulated during development. During the final stadium, there is a drop in haemolymph titer [43]. For example, in the penultimate stadium of the cockroach, D. punctata, the rate of JH III biosynthesis by the CA is between 1 to 3 pmol/hour per pair, but falls to undetectable levels later in the final stadium (Fig. (2)). Application of JH at this stage results in the inappropriate retention of juvenile characteristics at the next moult. The morphological effects are dependent on the dose of hormone, the age of the larvae and the sensitivity of the insect. In susceptible insects, application of JH to final stadium larvae may produce "adultoids" at the next moult or supernumerary larvae. In the latter case, the insect moults into a giant immature stage retaining juvenile features, without reaching sexual maturity. Therefore, application of synthetic JH analogues to insects in their final larval stages could be useful in the control of insects which are pests in their adult stages such as mosquitos and fleas. In adult female D. punctata, JH III is involved in the regulation of vitellogenin synthesis during ovarian development [44, 45, 78]. After the final moult and subsequent mating, the total amount of JH III in whole body extracts rises from 32.1 ng to its highest value on day 4 (196 ng), then falls to 12.8 ng over the next three days and remains at this low level [57]. Application of JH or synthetic JH analogues to insect eggs disrupts embryonic development. This can range from immediate ovicidal effects to delayed developmental effects [7]. Application to eggs result in apparently normal larvae which ultimately moult into supernumerary sixth-instars instead of adults [79]. Interestingly, the male linden bug, Pyrrhorocis apterus, treated with JH analogue, can transfer it to the female during mating, resulting in her sterility [80]. Therefore, application of JH or its synthetic analogues to preoviposition adult females or eggs may result in the disruption of embryogenesis.
JUVENILE HORMONES
383
One defensive strategy of plants against insect herbivory is the production of secondary metabolites which mimic JH activity [3]. Many of these compounds have been extracted from diverse plant species and are termed "juvenoids" based on their activity in vitro. However, a number of criteria should be met before these compounds are defined as JH mimics: Application of this compound should not block the insect's development but rather cause retention of juvenile characteristics in at least a single insect species [81 ]. The compound should demonstrate the ability to mimic JH at the three principle stages of insect development: the eggs, the juvenile and the adult (female). Therefore, observation of ovicidal effects is suggestive ofjuvenoid activity but not definitive. In the assessment of efficacy, dose response curves are necessary and allow the comparison of the activity of different compounds. These effects should represent the biological situation. For example, many of the studies described below have been performed using topical application of the putative juvenoid; this may not reflect the insect's natural contact with the chemical. The plant must be considered; for example, the amount of compound in the tissues must be sufficient to produce a biological effect on the test insect. In the evaluation of this, it should be remembered that many plants have specialized tissues which contain high levels of secondary metabolites. For example, many conifer species contain specialized secretory structures, such as resin ducts, which contain high localized concentrations of terpenoids [82]. There may also be dynamic variations in the amounts of these compounds in the plant, resulting from seasonal fluxes or synthesis induced upon stress. Lastly, other possible biological activities of the compound must be considered.
Juvabione, the "Paper Factor" The best known example of a plant-derived JH mimic is juvabione or the "paper factor" which was identified after it was observed that fifth instars of the linden bug, Pyrrhocoris apterus, from Europe, failed to mature into normal adults and instead metamorphosed into sixth instar supernumerary intermediates when reared in the United States [83]. The apparent cause of this was traced to the paper towels used in the rearing jars. Extracts from these paper towels Were only active against the Hemipteran insect, P. apterus, but inactive against pupae of the silkworms, Hylophora cecropia, H. gloverL Antheraea mylitta and Samia cynthia, and two other Hemipteran insects, Rhodnius prolixus and Oncopeltus fasciatus. Therefore, it appeared that only insects in the family Pyrrhocoridae are sensitive to this JH analogue.
384
BEDE a n d T O B E
In the search for the paper factor, extracts from several gymnosperms were assayed on P. apterus fifth instars (Table 1) [83, 84, 85]. Extracts of wood and bark of a number of these conifers were also injected into pupae of the wax moth, Galleria mellonella; a localized scaleless patch was observed in the pupal cuticle at the site of injection with these extracts. However, no abnormal effects on development were observed [86]. The authors speculated that this absence of activity may be attributable to the dilute concentration of active ingredients in the extracts. T a b l e 1.
Juvenoid
"
"
Activity of Gymnosperm
'"
"
'|
Plant
,
Extracts
"'
1
Insect
Stage
[ Balsam fir
i I ,
Ables balsamea (L.) Miller
.
,
Pyrrhorocis apterus
5th instar larvae
_,
,
!
eggs
Galleria mellonella i
,
Fir
..
|
adult females
I
. . . . . . .
t
pupae
i
'~
Ref
Topical
+++
83
Topical
+++
84
Topical
+++
84
.
.
Injection
86 i
P. apterus
5th instar larvae
Topical
+++
85
5th instar larvae
Topical
+++
85
i
i
'"
Response
l
A. nordmanniana (Steven) Spach
Douglas Fir
i
'
Method of application
|
Pseudotsuga men-ziesii (Mirb.) France
P. apterus
Canadian hemlock
Tsuga canadensis (L.) Carri~re i
P. apterus
5th instar larvae
Topical
+++
83
Hemlock
T. heterophu
G. mellonella
pupae
Injection
-
86
Sarg.
'
Yew
Taxus brev/folia Nutt.
P. apterus
5th instar larvae
Topical
+++
83
American larch
Larix laricina (Du Roi) K. Koch
P. apterus
5th instar larvae
Topical
++
83
European larch
L. decidua Mill.
P. apterus
5th instar larvae
Topical
+
83
Southern pine
Pinus echinata Sarg.
P. apterus
5th instal" larvae
Topical
Pine
P. contorta Engelm.
pupae
Injection
G. mellonella I
83 86
i i
Red spruce
Picea rubra Sarg.
P. apterus
5th instar larvae
Topical
Spruce
Pc. sitchensis (Bonn.) Cam
G. mellonella
pupae
Injection
Juniperus procumbens
P. apterus
Juniper
P. apterus
Thuja plicata Donn ex D. ii
i
I
5th instar larvae i
J. virginiana L.
Red cedar
-
86
t
,,.
Juniper
83
..
5th instar larvae
G. mellonella
Topical
_.
l
Topical
pupae
Injection
Don
83 i
-
:
-
! 86
83
i ii
i
i
ii
i
The symbols represent the potency of the extract: +++ (high activity); ++ (moderate activity); + (slight activity); -(inactive).
!
JUVENILE HORMONES
COOCH 3
385
duvablone
Juvoclmene I
O
J u v o c i m e n e II HO
Bakuchiol
Echtnolone
Fig. (6). Structures of phytojuvenoids.
Juvabione, the compound responsible for this activity, was isolated from the balsam fir, Abies balsamea (L.) Miller, and identified as the methyl ester of todomamic acid, (+)-4(R)-[ l'(R)-5'-dimethyl-3'-oxohexyl]1-cyclohexene-1-carboxylic acid [87]. This compound is a sesquiterpenoid (Fig. (6)) with a cyclohexene group and an a,13-unsaturated methyl ester group; the chemical data for this compound are summarized in Appendix If, Table 1. The IR spectrum suggests that there is a carbonyl ester group present in conjunction with a double bond (1722 and 1645 cm -1) and also an isolated carbonyl group (1712 cm -l) [87]. Mass spectroscopy confirms
386
BEDE
and TOBE
the presence of the methyl ester and proton nuclear magnetic resonance identifies an isopropyl unit attatched to a non-asymmetric carbon atom and a methyl group attatched to a disubstituted carbon. Juvabione and juvabione-related compounds have been isolated from a number of gymnosperms (Appendix II, Table 2 and Table 3). There is a debate in the literature as to the proper stereochemical assignment of these compounds. This paper will follow the recommendations proposed by Manville [88]. The possible biosynthetic relationship between these compounds has also been proposed by Manville and coworkers [89, 90]. Application of juvabione to fifth larval instars of P. apterus produced the expected supernumerary sixth instars at the subsequent moult [87]. However, at higher doses, juvabione was also active against the box elder bug, Leptocoris trivittatus, and the mealworm beetle, Tenebrio molitor T a b l e 2.
Juvenoid Effects of Juvabione
!
Insect
Stage
Method of application
t
ii
.,
Dose
|
Ref
k ....
Hemiptera
i
Pyrrhocoris apterus
5th larval instar
P. apterus
[
Topical
5 Ixg
87
5th larval instar
Topical
1.7 lag*
91
Leptocoris trivittatus
5th larval instar
Topical
100 p.g
87
Dysdercus cingulatus
5th larval instar
Topical
2.3 Ixg*
91
Graphosoma italicum
5th larval instar
Topical
- (500 btg)
91
Tenebrio molitor
pupae
Injection
500 p.g
87
T. molitor
pupae
Injection
+
92
T. molitor
pupae
Topical
- (500 I~g)
91
Dendroctonus pseudotsugae
adults
Topical
- (100 lag)
93
Manduca sexta
pupae
Injection
-
92
Antheraea polyphemus
pupae
Injection
-
92
Choristoneurafumiferana
eggs
Topical
Coleoptera
[
I
Lepidoptera
9
,
100 ~tg/egg mass .
94 ,,
.
.
A plus sign (+) represents an observed response where the effective concentration was not reported. A minus sign (-) indicates that the compound was inactive at the highest concentration tested (in brackets). Starred values represent the ED50; the dose at which 50% of the biological activity is observed.
JUVENILE HORMONES
387
(Table 2). Similar results were obtained following treatment of these insects with JH III. These results suggest that the JH-activity ofjuvabione is not specific to insects from the family Pyrrhocoridae, as was previously thought, but that these insects may be more sensitive to treatment. Pure juvabione was less active than the extract from balsam fir in the P. aperatus assay [84, 87]. Possible explanations for this observation are that there were differences in the strain of insect tested or, more likely, that the extract contained other compounds which either functioned as juvenoids or which acted as synergists to juvabione. In subsequent studies, the juvenoid effects ofjuvabione have been confirmed on fitch instars of P. apterus [91 ] and pupae of 7'. molitor [92] and observed on fifth instar nymphs of the red cotton stainer, Dysdercus cingulatus [91]. The variability in the sensitivity of these insects may reflect differences in the rate of penetration across the cuticle, metabolism and excretion etc. (Table 2). In subsequent studies, topical application of juvabione to T. molitor pupae was inactive [91]. This reflects differences in the mode of treatment; activity was observed if the pupae were injected with juvabione but absent if it was topically applied. Juvabione was inactive against pupae of the tobacco hornworm, Manduca sexta, and the polyphemus moth, ,4ntheraea polyphemus [92] and the last instar larvae of the pentamid bug, Graphosoma italicum [91 ]. Bioactivities of structually related juvabiones have also been investigated (Table 3). The isomer of juvabione, epijuvabione, and dehydroepijuvabione inhibited normal development of fifth instars of P. apterus [95]; epijuvabione was approximately ten times more effective than dehydroepijuvabione in the assay [96]. Both these compounds are also active on fifth instar nymphs of five Dysdercus species, which are also in the family Pyrrhocoridae" D. intermedius, D. superstitiosus, D. fluvoniger discolor, D. chaquensis and D. cingulatus, [96, 97]. These compounds are inactive against G. italicum, T. molitor, the Colorado potato beetle, Leptinotarsa decemlineata, the crickets, Gryllus domesticus and Acheta domesticus, the locust, Locusta migratoria, and the wax moth, Galleria mellonella [95, 96]. The juvenoid activity of dihydrojuvabione is similar to juvabione [92]. Bioactivity was observed against P. apterus fifth instars and T. molitor pupae, but this compound was inactive following injection into pupae of the Lepidopterans, M. sexta, and A. polyphemus. As expected, the proposed juvabione biosynthetic intermediates, dehydrojuvabione, juvabiol and its isomer, and dehydrojuvabiol and its isomer, were also active against fifth larval instars of P. apterus, and D. cingulams, but inactive against last instars of G. italicum and the pupae of T. molitor [91 ]. In addition to their effects on metamorphosis, these compounds also disrupt embryonic development. The viability of P. apterus eggs is severely reduced following treatment of adult females or freshly laid eggs with partially purified "paper factor" [98]; this activity is not observed
388
BEDE and TOBE
following application of the extract to eggs of O. fasciatus or R. prolixus. Potent ovicidal activity was observed following application of juvabione to eggs of the spruce budworm, Choristoneurafumiferana, a lepidopteran forest pest (Table 3) [94]. Interestingly, balsam fir, ,4. balsamea, the conifer from which juvabione was first isolated, is a host plant of the spruce budworm. Treatment of C. fumiferana eggs with dihydrojuvabione also reduced hatching [92]. Application of this compound to the adult female of the Douglas-fir beetle, Dendroctonus pseudotsugae, resulted in an increase in fecundity. This is significant because dihydrojuvabione has been isolated from the host plant of this beetle, the Douglas fir, Pseudotsuga menziesii (Beissn.) Franco. This suggests that coadaptation of the beetle and the host plant may have occurred such that dihydrojuvabione is not only non-toxic but increases the fecundity of the insect. T a b l e 3.
J u v e n o i d Activity of Structurally-Related J u v a b i o n e C o m p o u n d s i
m
Compound
Insect
Epijuvabione
Hemiptera
,
i
_
Stage
,
ii
Method of application
Dose
Ref!
i
F i
l
.
,
Pyrrhorocis apterus
5th instar larvae
Topical
10 lag
96
Dysdercus intermedius
5th instar larvae
Topical
I0 lag
96
D. intermedius
5th instar larvae
Topical
5 lag
97
5th instar larvae
Topical
.
.
.
.
.
.
.
D. superstitiosus
. . . . . . . . .
i ; l
I lag .
.
.
97 .
.
.
.
D. discolor
5th instar larvae
Topical
0.5 lag
97
D. chaquensis
5th instar larvae
Topical
I lag
97
D. cingulatus
5th instar larvae
Topical
0.5 pg
97
Graphosoma italicum
5th instar larvae
Topical
- (100 pg)
97
Injection
- (1 mg)
95
Coleopteran
Tenebrio molitor
pupae i
T. molitor
pupae
Leptinotersa decemlineata
pupae
96 -
96
Orthoptera
Gryllus domesticus
last nymph stadium
Injection
- (I mg)
95
Acheta domesticus
last nymph stadium
Topical
- ( 100 lag)
96
! I
JUVENILE HORMONES
389
(Table 3) contd .....
i
Compound
i
Stage
Insect
i
Method of application
l
|
,
. . . . . . .
F
,
,,
,
,
, ....
Dose
,,h
,
Ref ! i
I
last nymph stadium
Locusta migratoria
Lepidoptera
i Dehydroepi- i juvabione
Galleria mellonella
pupae
G. mellonella
pupae
Injection
- (1 mg) L -(100~tg)
95 !
96
Hemiptera
P. apterus
5th instar larvae
Topical
100 Ixg
96
D. intermedius
5th instar larvae
Topical
10 Ixg
96
D. intermedius
5th instar larvae
Topical
3 Ixg
97
D. superstitiosus
5th instar larvae
Topical
0.8 I,tg
97
D. discolor
5th instar larvae
Topical
0.1 Ixg
97
D. chaquensis
5th instar larvae
Topical
0.5 gg
97
D. cingulatus
5th instar larvae
Topical
0.5 l,tg
97
G. italicum
5th instar larvae
Topical
- (100 ~tg)
97
pupae
Injection
- (I mg)
95
Coleoptera T. molitor
,
T. molitor
pupae
- (100 g,g)
95
L. decemlineata
pupae
-
(lO0 p,g)
96
[
I
Orthoptera G. domesticus
last nymph stadium
Injection
- (1 mg)
95
Ac. domesticus
last nymph stadium
Topical
- ( i o o ~tg)
96
L. migratoria
last nymph stadium
Topical
- ( 1 0 o ~tg)
96
G. mellonella
pupae
Injection
- (1 mg)
95
G. mellonella
pupae
- (100 g,g)
96
Lepidoptera
390
BEDE and TOBE
(Table 3) contd.....
Compound
Insect
Stage
Method of application
i
Dihydrojuvabione
Hemiptera P. apterus
Dose
Ref
i
I
i 9,_
5th instar larvae
Topical
T. molitor
pupae
Injection
Dendroctonus pseudotsugae
adult females
Topical
Manduca sexta
pupae
Injection
92
Antheraea polyphemus
pupae
Injection
92
Choristoneura fumiferana
eggs
Topical
reduction in hatching
92
P. apterus
5th larval instar
Topical
5 Ixg*
91
D. cingulatus
5th larval instar
Topical
2 Ixg*
91
G. italicum
5th larval instar
Topical
- (500 ~tg)
91
pupae
Topical
- (500 . g )
91
5th larval instar
Topical
5.6 ~tg*
91
Coleoptera 92 increase in fecundity
92
Lepidoptera
Dehydrojuvabione
Hemiptera
Coleoptera T. molitor
Juvabiol and isomer
Hemiptera J
P. apterus
,,,
D. cingulatus
5th larval instar
Topical
4.7 ~tg*
91
G. italicum
5th larval instar
Topical
- (500 ~ g )
91
Topical
- (500 ~ g )
....
,,
Coleoptera T. molitor
pupae
91
JUVENILE HORMONES
391
(Table 3) eontd ..... :,
Compound
Insect
Stage
Method of application
Dose
Ref
!
b,,, ,,,
Dehydrojuva-biol and isomer
Hemiptera
,,,
|
P. apterus
5th larval instar
Topical
6.0 lag*
D. cingulatus
5th larval instar
Topical
0.7 lag*
G. italicum
5th larval instar
Topical
- (500lag)
91
- (500lag)
91
91
....
Coleoptera i
T. molitor
pupae
Topical i
A plus sign (+) represents an observed response where the effective concentration was not reported. A minus sign (-) indicates that the compound was inactive at the highest concentration tested (in brackets). Starred values represent the
ED50; the dose at which 50% of the biological activity is observed.
In balsam fir, juvabione is found predominantly in the wood; little activity is associated with the foliage or bark [85]. Interestingly, the free acids of juvabione and dehydrojuvabione, todomatuic acid and dehydrotodomatuic acid, were not detected in healthy balsam trees, Abies grandis and A. amabilis [99]. However, significant amounts of these compounds were present in the wood adjacent to sites infected by the balsam wooly aphid, Adelges piceae, suggesting that the synthesis of (+)todomatuic acid and its derivatives or transport of these compounds to the site of infection may be induced upon insect infestation. There are minor discrepancies regarding the biological activity of these compounds. For example, in one assay, juvabione is effective against pupae of T. molitor [87, 92] whereas in subsequent assays, it is inactive [91]. This difference is probably due to the method of delivery (injection vs topical application) which may reflect the ability of juvabione to cross the pupal cuticle or to differences in the metabolism of this compound depending on the route of entry. There also was a difference in the potency of some compounds, for example, epijuvabione and dehydrojuvabione [95, 96]. This could be related to the sensitivity of different strains of insects to the juvenoid or to differences in the physiological state of the insect. For example, in these bioassays, the test compound was applied to fifth larval instars of P. apterus but the age of the insects was not always specified. The activity of the juvenoid could be altered by the physiological stage of the insect and, in particular, the concentration of metabolic enzymes in the haemolymph which change dramatically during this period [57, 76]. This, along with the contradictory reports of the structural stereoisomers ofjuvabione and its derivatives [98, 100, 101, 102], presents a very complicated story.
392
BEDE and TOBE
Other Phytojuvenoids There are many examples of such JH mimics isolated from plants [42, 103]. In the following section, the activities of four of the better characterized JH mimics, juvocimene I and II, bakuchiol and echinolone, are reviewed to illustrate the diversity of the compounds which exhibit JH activity (Fig. (6)) and the range ofbioactivity (Table 4). Juvocimenes I and II are monoterpenoids isolated from the oil of sweet basil, Ocimum basilicum L. (Fig. (6)) (Table 4) [104]. These compounds demonstrate potent JH activity following application to last instar nymphs of the milkweed bug, O. fasciatus. Juvocimene II is approximately 10 times more active than juvocimene I, with biological activities (ED50) in the range of 5 pg and 50 pg, respectively [104]. Therefore, these compounds are far more potent then juvabione when applied to Oncopeltus [92]. The chemical data for these compounds are summarized in Appendix III, Table 1 and 2 and protocols for their chemical synthesis can be found in the references 105 and 106. Table 4.
Comparison of Phytojuvenoids
Compound ,,,
Plant
Plant part
Plant species
,,
Juv~imene
|, !
,
L.
Juvocimene Ii
Sweet basil
O. basilicum
Stage
Method of app.
Dose
Ref
Oncopeltus fasciatus
5th instar larvae
Topical
50 pg*
105
O. fasciatus
5th instar larvae
5 pg*
105
,,
Ocimum basilicum
Sweet basil
lmect
L.
.
b. . . . . . . . . .
seeds
Bakuchiol Echinolone
American coneflower
root
Psoralea cornifolia L .
Dysdercus koenigii
. . . .
,.
Tenebrio
Echinacea augustifolla D e .
molitor
.
Topical .
.
.
.
.
.
.
.
5th instar larvae
Topical
! 0 pg
108
pupae
Injection
0.97 ttg
109
5th instar larvae
Topical
30 ptg
110
......
Juvadecene
Pepper-tree
roots
Macropiper excelsum M i q .
O.
fasclatus
..,
Thujic acid
Red cedar
wood
Thuja plicata
.
.
.
.
T.
monitor
pupae
Injection
51 ! p g
I!I
D.
koenigii
5th instar larvae
Topical
blot reported
112
Donn .....
Tagetone
Marigold
whole plants
Tagetes minuta L. .
Starred values represent the EC50; this is the dose where 50% of the biological response is observed.
Bakuchiol is a phenolic monoterpene (meroterpenoid) isolated from the seeds of Psoralea corylifolia L. (Fig. (6)) [ 107]. Topical application of 10 lag of bakuchiol to fifth instar nymphs of D. koenigii, results in the metamorphosis to nymph-adult intermediates [108]. This juvenoid activity is comparable to juvabione where application of 0.6 lag to fifth instars ofP. apterus or 10 lag to last instar larvae of O. fasciatus, produced morphological abnormalities in 50% of the adults [91, 92]. The chemical data for bakuchiol is compiled in Appendix III, Table 11 and synthesis of the racemate methyl ether from geraniol has been reported [ 113].
JUVENILE HORMONES
393
Root extracts of the American coneflower, Echinacea augustifolia DC., also exhibited juvenoid activity [114]; ether extracts showed high morphogenic activity on T. molitor pupae but none was observed on fifth instar nymphs of O. fasciatus (500 p,g of extract). The active principle in the oil was identified as (E)- 10-hydroxy-4,10-dimethyl-4,11-dodeeadien2-one or eehinolone (Fig. (6)) [109]. However, chemically synthesized eehinolone was not active in the standard T. molitor pupal bioassay [ 115]. The authors of this report acknowledge that this may be due to a failure to synthesize the proper compound. However, the spectral data is consistent with that reported. They were also unable to isolate eehinolone from the roots of E. augustifolia. The chemical data for eehinolone are summarized in Appendix III, Table 12 and the synthesis of raeemie eehinolone has been reported [ 115, 116]. Other phytoehemieals which exhibit JH activity include juvadeeene (1(3,4-methylenedioxyphenyl)-trans-3-decene) isolated from roots of the pepper-tree, Macropiper excelsum Miq. [ 110], thujie acid (5,5-dimethyl1,3,6-eyeloheptatrien-l-carboxylie acid) extracted from the heartwood of western red cedar, Thuja plicata [111] and tagetone ((E)-2,6-dimethyl-5,7oetadien-4-one) from the marigold, Tagetes minuta L. [ 112]. There have been numerous reports of juvenoid activity of plant extracts (Table 5); however, to our knowledge, the compound(s) responsible for this activity have not been isolated and characterized. Plant secondary metabolites which mimic JH activity appear to be active on a narrow range of host species. What account(s) for this effect ? The majority of bioassays used last larval instars of P. apterus, O. faseiatus and pupae of T. molitor to test for activity of the juvenoids. Are these the most sensitive insects ? Six JHs have been identified to date; different homologs have been isolated from specific insect orders. Juvenile hormone III appears to be ubiquitous [ 12, 13] and, in most species, is the only JH present. Juvenile hormone I and II are important in the regulation of metamorphosis and ovarian maturation in Lepidoptera [5] and the bisepoxide appears to be the principle JH in higher Diptera [20]. Therefore, the nature of the JH in the test insect and the role that it plays in development must be considered in the selection of the bioassay; a compound which mimics the action of JH in P. apterus (Hemiptera) is unlikely to be active in a Lepidopteran insect. Originally, it was thought that these compounds could provide the structural basis for the design of pesticides because it is unlikely that insects would develop resistance to their own or closely related hormones [ 120]. However, this has proven not to be the ease. Most insects possess enzymes such as mixed function oxidases and esterases in their alimentary tract and haemolymph which are, among other functions, important for the metabolism of plant toxins [121, 122]. For example, insects which have developed resistance to insecticides may also show resistance to JH
BEDE and TOBE
394
Table 5.
Juvenoid Activity of Plant Extracts
,
Plant
,
Plant pa•
-
i r ~
..,
.
,
.,
,
Method of application
Response
Ref
5th larval instars
Topical
+4+
117
5th larval instars
Topical
++
fasciatus
114
Tenebrio molitor
pupae
Topical
-
!!4
0/ascialas
5th larval instars
Topical
4+
114
pupae . . . 5th larval instars
Topical
Insect ,
,
Stage
Plant species |
,
Roots
Iris
Iris endata Thamb.
Dysdercus koenigii
Roots, stem, fruits
Iris
Iris douglasiana Herb.
Oncopeltus
Stem, leaves, fruits
Sweet pepperbush
Clethra aiternifolia L.
Root, root bark
White sassafras
Sassafras aibidum (Nutt.) Nees
O. fasciatus T. molitor
pupae
Topical
-
! 14
Stem, bark
Murray redgum
Eucalyptus camaldulensis
O. fasciatus
5th larval instars
Topical
4+
! 14
T. molitor
pupae
Topical
Twigs, leaves
Pitch pine
Pinus rigida Mill.
O. fasciatus
5th larval insists
Topical
+
I !4
T. molitor
pupae
Topical
Seeds
Lawson cypress
Chamaecyparis lawso-niana
O. fasctatus
5th larval instars
Topical
T. molitor .
.
.
Dehnh.
(Andr. Murray) Pari
ii i I
Topical
"
.
4+
._
114 i 14
! 14
! 14 -
I 14
7". molitor
pupae
Topical
.H-
I 14
Anthocephalus cadamba
D. cingulatus
5th larval instars
Topical
4-+
I 18
Lantana camara L.
D. cingulatus
5th larval instals
Topical
4+
I 18
Stem
Calophyllum sp. L.
D. cingulatus
5th larval instars i
Topical
~
118
Stem
Phyllanthus emblica
D. cingulatus
5th larval i insists i
Topical
-t-t+
!!8
5th larval instars
Topical
Stem Stem
Yellow sage
.
i
Stem
D. cingulalus
Erythrina indica Lam.
[
119
Stem
i i [
Auracaria excelsa R. Br
D. cingulatus
5th larval instars
Topical
Stem
j
Annona reticulata L.
D. cingulatus
5th larval , instara
Topical
Peltoforum inerme Benth.
D. cingulatus
5th larval instars
Topical
-14+
119
Manihot esculenta Pohl.
D. cingulatus
5th larval instars
Topical
+4-+
119
1 i
5th larval instals
Topical
+4+
119
9
Stem Stem
Cassava
"'
[ [
-4-++
119 119
Stem
Phyllanthas emblica L.
D. cingulalus
Stem
Tabernaemontana dichotoma
D. ctngulatus
5th larval instars
Topical
D. koenigii
5th larval instars
Topical
Aedes aegpti
larvae
Contact in water
112
Aedes aegpti
pupae
Contact in water
112
Roxb. Whole plant
Marigold
Tagetes minuta L.
i Muses domestica ,
pupae ,
I19 +++
Topical ,
.,
,
The symbols represent the potency of the extract: +++ (high activity); ++ (moderate activity); + (slight activity); - (inactive).
,
!i2
L 112
JUVENILE HORMONES
395
analogues [ 123, 124, 125]. This cross-resistance appears to be related to an increase in metabolic enzymes, particularly mixed function oxidases, including cytochrome P450 enzymes. Also, esterases are present at high levels in the insect haemolymph in the final instar and are thought to be partially responsible for the low JH III titers observed at this stage [57, 76]. Differences in such enzyme systems may account for the observed differences in the sensitivities of insect species to juvenoids. Further difficulty with the use of these analogues as a means of control of pest species is that the window of sensitivity to these compounds is short, e.g. to eggs, at metamorphosis or during reproduction. Treatment with these compounds may also result in an arrest of the insect in the feeding period. These compounds also have a relatively broad specificity and would not act exclusively on pest species. However, in certain circumstances, the synthetic JH analogues methoprene and hydroprene have been used successfully in insect control. Much of the interest in phytojuvenoids has focused on the isolation of compounds to use as models for the development of stable, potent pesticides. As a result, there is little information regarding the biological nature of these compounds. In most bioassays, the juvenoid was applied topically, biasing the screen for lipophilic compounds which are able to penetrate the insect cuticle and underlying epidermis. In nature, these compounds can be absorbed, ingested or inhaled depending on the plant and the life history of the insect on the plant. Little information is available regarding juvenoids which elicit these effects by oral administration. Similarly, there have been few reports regarding the amount and distribution of these compounds in the plant. To our knowledge, there is presently no information on whether the levels of these compounds in plant tissues are sufficiently high to affect insect herbivores or whether these compounds play another role(s) in vivo. Consequently, these compounds which have been defined in the literature as insect JH mimics do not meet our criteria: the present evidence suggests that they may function as phytojuvenoids but further studies must be performed. PLANT JUVENILE HORMONE ANTAGONISTS Phytochemicals, such as the precocenes, isolated from A g e r a t u m houstonianum [ 126], interfere with JH biosynthesis. In sensitive insect species, application of these dichromenes to larval instars results in precocious metamorphosis to sterile adults or sterility in adult females following treatment. Pesticides based on these compounds would be useful in the control of insects which are primarily destructive in their immature stages. Other phytochemicals which potentially function as "antijuvenile hormones" include dimethyl sciadinonate, isolated from the leaves of avocado, Persea americana Mill [127, 128]. Ingestion of this
396
BEDEand TOBE
compound results in the direct pupation of fourth instar larvae of the silkworm, Bombyx mori, bypassing the fourth moult. Precocenes
The bioactivities of precocene I, 7-methoxy-2,2-dimethyl chromene, and precocene II, 6,7-dimethoxy-2,2-dimethyl chromene, are well established (Fig. (7)). These two compounds have been isolated from plants throughout the family Asteraceae [ 129-136, 154]. The chemical data for these compounds and references for their synthesis are compiled in Appendix IV, Tables 13 and 14. There is a wealth of information on the effects of these compounds on various insect species [42, 137]. For simplicity, we will focus on the effects of precocene II on the susceptible Hemipteran bug, Oncopeltusfasciatus.
MeO M M e e ~
Precocene I
Precocene II
Fig. (7). Plant insectjuvenile hormone antagonists, the ageratochromenes.
Application of sublethal doses of precocene to the first, second and third instar nymphs of O. fasciatus results in premature metamorphosis to adultoids at the third, fourth and fifth stadium moult, respectively [126]. Interestingly, there is an "in-between" instar following the stage when the chromene is applied and when the effects are observed. These effects can be reversed by the topical application of JH; a decrease in both mortality and the number of insects which undergo precocious metamorphosis is observed following application of JH I to second instars which have been pretreated with precocene II [138]. Fourth instar larvae of O. faseiatus which were treated with precocene developed either into precocious adults or underwent apparently normal development through a fifth instar into an adult, with an increase in the preoviposition period and reduced fecundity observed in females [138, 139]. This stage of Oneopeltus was less sensitive to precocene; a hundred-fold increase in concentration was required to produce these effects [138]. Treatment of these precocious adult females with JH resulted in mating, although oviposition did not
JUVENILE HORMONES
397
ensue [ 126]. Application of precocene to the last nymphal stages has no effect and these insects develop into normal, reproductive adults [ 140]. Treatment of adult female O. fasciatus with precocene results in sterilization [ 126, 138, 139]. Examination of the ovaries revealed a marked difference between control and treated insects [126, 141]. In normal insects, oocyte development begins two to three days after adult moult and continues until day six, at which time oviposition normally begins. Treatment of gravid insects with precocene on day 5 after eclosion resulted in resorption of most oocytes. These eggs hatched and developed normally to third instar juveniles which then moulted into precocious adults [ 126]. Following application of precocene to newly emerged adult females, oocyte development is completely inhibited. Application of JH III to these insects resulted in a rapid increase in oocyte length, demonstrating that precocenes were not acting directly on the ovary. Transplantation experiments in which precocene II was added to CA maintained in vitro from mated adult female O. fasciatus, followed by implantation into fifth instar juveniles, demonstrated that the precocenes directly inactivate the CA and their effects are not the result of signals originating in the brain or other tissues [142]. In complementary experiments on the cockroach, Periplaneta americana, Pratt and Bowers demonstrated that incubation of the CA with precocene II directly inhibits JH III biosynthesis and release, even though in vivo, P. americana is relatively insensitive to these compounds [ 143]. The effects of precocenes on larval and adult O. fasciatus and their reversal by application of JH suggests that these compounds may be affecting JH biosynthesis by the CA. As with the ovary, profound differences are observed in CA volume between normal and precocenetreated animals [141]. After eclosion, the CA of mated adult females normally undergo a five-fold increase in volume over the next nine days. Treatment of these animals with precocene on day 5 results in a reduction in size. If the insect is treated with precocene immediately following the adult moult, the CA size does not change; even if JH III is added on day 5 to these animals, no further development occurs. However, in this species, there is no definitive evidence to suggest that CA volume is correlated with the rescue of JH. Morphological and ultrastructural studies of the CA of precocenetreated adult females of O. fasciatus demonstrate progressive necrotic degeneration, as compared to controls [ 144, 145, 146]. These studies have shown that precocene II not only inhibits the CA but actually stimulates its atrophy. Its specific cytotoxicity is attributed to epoxidation of the precocenes by the epoxidase which catalyzes the final step in JH III biosynthesis, generating extremely reactive 3,4-epoxy intermediates which alkylate cellular proteins [ 147, 148, 149]. This results in the atrophy of the CA and the resultant inhibition of JH biosynthesis [150]. Therefore, the destruction of the CA by precocene is responsible for its biological
398
BEDE and TOBE
activity. This occurs through its epoxidation by an enzyme which is only active at times of JH biosynthesis. Therefore, precocenes have no effect on inactive CA such as in those of last larval instars of O. fasciatus [ 140]. Generally, it is thought that these compounds are only effective in vivo against insects in the orders of Hemiptera, Homoptera, Dictyoptera and Orthoptera, with exceptions [42, 137]. Holometamolous insects are believed to be insensitive; high doses of precocenes may affect development in these and other insect species but it is thought that this action does not occur through inhibition of JH biosynthesis. Rescue experiments with reversal of precocene-induced effects counteracted with JH or JH analogues will allow the differentiation between endocrinespecific and non-specific effects. In vitro inhibition of JH biosynthesis in insect species which are insensitive to precocenes in vivo has also been demonstrated [ 151 ]. It is believed that the difference insusceptibility of different insect species to precocenes may, in part, be related to their metabolism by the insect. The metabolism of precocene II to the diol was demonstrated in nine insect species [ 147] and, presumably, occurs by mixed function oxidases present in the gut and fat body. The wax moth, G. mellonella, metabolized 77% of this compound as compared to 47% by O. fasciatus in the same time period. This difference in metabolic rate may account, at least partially, for differences in the sensitivity of these insect species. Precocene II has also shown antifeedant activity in a non-choice assay with the Mexican bean beetle, Epilachna varivestis [152]. It also has antifungal activity against Pyricuaria oryzae, whereas precocene I was nontoxic in this assay [ 153]. Neither compound demonstrated antibiotic activity against the yeasts, Saccharomyces cerevisiae and Candida albicans, the gram-negative bacteria, Pseudomonas fluorescens and Escherichia coli, and the gram-positive bacteria, Bacillus subtilis and Staphlococcus albus [130]. The distribution of precocene I and II in flowering plants of Ageratum houstonianum has been determined [134]. The highest amounts of precocene II is found in the leaves (1.4 lamol/g fresh weight (FW) to 6.7 ~mol/g FW in older leaves) and the flowerheads (21.5 I.tmol/g FW). It is invalid to compare a dose which would be topically applied versus ingested. However, with information on the amounts in the plant, it would be useful to determine if precocene II levels in the plant would be sufficient to either deter feeding or interfere with development of a susceptible insect species. Considering that application of 0.5 mg of precocene II is ovicidal to O. fasciatus eggs treated by fumigation [ 126], 10 ktg of precocene II resulted in precocious metamorphosis in O. fasciatus [139] and that 0.05 btmol/leaf disc deterred the feeding of E. verivestis [152], it is possible that these compounds are present in sufficient concentrations to play a role in protecting the plant against insect herbivory.
JUVENILE HORMONES
399
INSECT JUVENILE HORMONE III IN PLANTS To date, there has been only one report of the identification of an insect JH in a plant; in 1988, JH III and MF were isolated from the sedges Cyperus iria L. and C. aromaticus (Ridley) Mattf and Kiik [155]. Although JH III has only been detected in these two plant species, structurally similar sesquiterpenoids have been isolated from the roots of C. iria and a number of related Cyperus species (Fig. (8)) [ 156-161 ]. For C iria
Methyl- 1OR, 1 I-epoxy-3,7, I I-trimethyl2E, 6E-dodecadienoate
C monophyllus
C pilosus
C microiria
Methyl-3,7,11 -trimethyl- I I -hydroxy-2E, 6E, 9Z-dodecadienoate
"~-
+
Methyl-3,7, i l-trimethyl- I I-hydroxy-2E, 6E, 9E-dodecadienoate
-!-
+
Methyl-3,7,1 I-trimethyl-2E, 6E, 9Z, I ! Z-dodecatraenoate
"[-
+
Methyl-3,7,1 i-trirnethyl-2E, 6E, 9E, I ! E-dodecatraenoate
~
"]"
Farnesol Methyl farnesoate
C polystachyos
C serotinus
+
+
+
+
+
+
+
+
+
Juvenile Hormone HI: methyl-10R. 11-epoxy-3.7,11-trimethyl-2 E,6Edodecadienoate (CtoH2eOs}
~ methyl 3,7,11-trlmethyl-11-hydroxy trans-2, trans-6, c/s-9-dodeeadfenoate (r methyl 3,7,11-trlrnethyl- 11-hydroxy tmns-2, tmns-6, tr~Ls-9-dodecadlenoate
1
o"
OH methyl 3,7',11-trlmethyl tr~s-2, tn~ls-6, ds-9,l 1-dodeeadlenoate (C16He4021 methyl 3,7',11-trlmethyl trots-2, tr~s-6, trans-9,11-dodeeadlenoate
oi" Methyl F~-nesoate: methyl a,7,11-trlrnethyl trans-2, t r ~ s 6,10-dodeeatrlenoate (e~6H26021
oi" Farnesol: 12N, 6~1-3,7,11-trlmethyl-2,6,10-dodeeatrlen- 1-ol (r 15H2601
Fig. (8). Linear sesquiterpenoids isolated from the roots of Cyperus species [ 155-161 ].
400
BEDE and TOBE
example, MF, the immediate biosynthetic precursor of JH Ill in insects, has been identified in extracts from C iria and three other Cyperus species, C monophyllus, C pilosus and C serotinus [155, 158, 160, 161], as well as from grape skins [ 162] and the bark of Polyalthia viridis Craib [ 163 ]. The linear sesquiterpenoid methyl (E,E)- 10,11 -dihydroxy-3,7,11 trimethyl-2,6-dodecadienoate has been isolated from dried roots of a canopy tree Hortia regia Sandwith., and the root bark of an African rainforest tree, Cleistopholis patens (Benth.) Engl. and Diels, and the stem bark of C1. staudtii (Engl. & Pierre) [ 164, 165, 166]. This compound is structurally identical to JH III except that the epoxide has been hydrated to a vicinal diol, a reaction which readily occurs in the presence of acid or base. These reports suggest that JH III, MF and structurally similar compounds are present in diverse plant species and are more prevalent than previously thought. Distribution of Juvenile Hormone III in Cyperus iria
The distribution of JH III was monitored in C. iria throughout plant development using a radioimmunoassay [ 167]. In seven-month old plants, the amount of JH III in root tissue is 27.2 + 3.3 ~g/g FW [ 168]. This is approximately thirty-five times the whole body concentration of day 5 adult females ofD. punctata [57]. The total level of JH III in the roots was approximately 600 times and 300 times the amount found in either the inflorescence or leaf tissue, respectively, at this stage of development. This profile was maintained over the course of development, with the highest concentrations of JH III observed in the root tissue and substantial quantities also in the leaf tissue. The overall amount of JH III per gram plant tissue was less than that originally reported [ 155]; this discrepancy probably reflects the different cultivars and environmental growth conditions. Possible Biological Role(s) of Juvenile Hormone III
The isolation of JH lII from C. iria and C. aromaticus, the high concentrations found in C iria throughout development and the extraction of structurally similar compounds from closely related species as well as other plant species suggests that this compound may play an important biological function(s) in these plants. At this point, the nature of this role is speculative; however, it is possible that JH III may be involved in plantinsect, plant-plant, plant-nematode or plant-fungal interactions. At present, there are few reports on the ecology of C. iria and associated insects and the information available is vague and contradictory. In the initial report on the isolation of JH III from C. iria, third stadium grasshopper nymphs, Melanoplus sanguinipes, were raised on either
JUVENILE HORMONES
401
wheat seedlings or C. iria; no difference was observed in growth. However, upon moulting to adults, those reared on C. iria displayed deformed wings and colour changes indicative of development under conditions of excess JH [ 155]. Adult female grasshoppers fed on C. iria were infertile and their ovaries contained only immature oocytes [ 155]. In field studies, eggs of the Dipteran leafminer, Hydrellia sp., which were laid on C. iria leaves did not hatch [ 169]. However, in some reports, C. iria had no effect on insect development. For example, nymphs of the planthoppers, Nisia strovenosa and N. nervosa were reared successfully on C. iria and C. rotundus L., although more insects reached maturity on C. rotundus [ 170]. Also, in a feeding preference study, the rice stink bug, Oebalus pugnax, did feed on C. iria, although the mean number of feedings was less than that observed on other plants such as the Vasey grass, Paspalum urvillei Steud. [ 171 ]. It is also possible that these compounds may reduce plant competition by inhibiting the germination and growth of plants in the immediate vicinity. In the search for allelopathic agents in invasive weeds, methyl farnesoate and farnesol were isolated from tubers of C. serotinus Rottb. [ 161 ]. At concentrations of 1 mM, these compounds inhibited the growth of lettuce and rice seedlings. In other studies, treatment of seeds with famesol (83.3 IxM) stimulated root elongation in barley but had a slight inhibitory effect on mustard, tomato, spinach and wheat [ 172]; no effect was observed on lettuce, carrot or cabbage. At higher concentrations (516.6 ~M), famesol inhibited root growth in barley. The high levels of JH III in the plant coupled with the above evidence of allelopathic activity of two of its biosynthetic precursors, farnesol and MF [161], raises the possibility that these compounds may be involved in the inhibition of germination and growth of surrounding plants. Allelochemicals may also be released into the environment as a defence against phytoparasitic nematodes [173]. Juvenile hormones have been shown to affect the development of nematodes [ 174]. Juvenile hormone I and MF inhibited hatching of the eggs ofHaemonchus contortus [175] and application of JH III (3.4 mM) to third-stage larval females of the rodent hookworm, Nippostrongylus brasiliensis, resulted in a 50% reduction in egg production [176]. The biosynthetic precursor of JH III, farnesol, inhibits development of larvae of the nematode Trichinella spiralis [ 177]. However, in the few studies done, these compounds do not appear to have an effect on plant parasitic nematodes [ 175, 178]. CONCLUSION It is apparent from these examples that plants produce compounds which are able to interfere with the endocrine system of insects. It is tempting to speculate that they may be responsible, or at least partially responsible, for the protection of the plant against insect attack. Unfortunately, most
402
BEDE and TOBE
work has focussed on the effects of these compounds on the insect and the development of synthetic pesticides based on their structure. Little is known about the distribution of the compounds in the plant, whether their concentration is sufficient to inhibit insect attack or whether they are induced upon attack to protect the plant tissues. Also, in most studies, the effects of topically applied compounds were observed whereas the dietary effects, which is more likely the mode of entry, on these insects is not known. The mechanisms of insect metabolism of these compounds have also not been extensively studied. There are many examples of plants which produce insect hormones, for example, the insect moulting hormone, 20-hydroxyecdysone [179]. However, to date there had only been one report of the identification of an insect JH from a plant [ 155]. It is obvious from the previous section, that we are in the preliminary stages of determining the role of JH III in C. iria. In an attempt to understand the role of this compound in the plant, we have characterized the distribution of JH III developmentally. At present, we can only speculate as to its possible biological activity(ies). ACKNOWLEDGEMENTS We thank C. Garside and K. Yagi for thoughtful comments on the manuscript and P. Bowser for help with the manuscript. Funding of this research was provided by a National Sciences and Engineering Research Council of Canada Operating Grant (S.S.T).
JUVENILE HORMONES
403
APPENDIX I. JUVENILE HORMONE DATA Table 1.
Chemical Data for Juvenile Hormone II1, Methyl-10R,ll-epoxy-3,7,11trimethyl 2E,6E-dodecadieneoate [21, 38, 180-183]
Beilstein Registry Number: 1316317 MW 266.38 C!6H2603 Boiling point: 125-126~ (0.08 mm3) 30~ (0.02 mm 3) Ultraviolet spectroscopy (ethanol): ~, = 221 rim, ~ = 14 350 Infrared spectroscopy (film): 2950, 1720, 1215, !130 cm"1. 1720, 1650 cm" 1.
Table 2.
Mass Spectral Data for Juvenile Hormone III [23] ,
Mass (m/z) ,
i
,
,
,,
,,
M+ (M-H20)
C16H260 3 C16H2402
234
(M-CH3OH) +
C15H2202
206
(M-CH3OH + CO) +
C14H220
i 95 163 153 135
C 12H 1902 C 11HI5 O C 10H 170 CIOHI5
114 81 71 43 ,
(M-C4H70) + (195-CH3OH) (M-C6HgO2) (153-H20) (] 95-CO + CH3OH ) (M-C I OH 16O) (M-C I OH 1703) (C12H1902) (C13H1903)
C6H 1002 C6H9 C4H70 C3H7 ....
266 248
M+ (M-H20)
C!6H2603 C16H2402
234
(M-CH3OH) +
C 15H2202
195 163 135
(M-C4H70) + (195-CH3OH) (153-H20) (195-CO + CH3OH) (M-CIoHI60) (M-C I OH 1703) (C 12H 1902) (C13H1903)
C 14H ! 902 C 11HI5 O CIOHI5
i,
i
,,i
i
266 248
114 81 * 71 43
,
Ion Fragment 15 eV
i
70 eV
C6HI002 C6H9 C4H70 C3H 7 i
i
||l
m
Bolded masses represents ions unique to the low energy spectra. An asterix denotes the base peak. Other references: 180, 183, 184.
BEDE and TOBE
404
T a b l e 3.
Proton Nuclear Magnetic Spectra of Juvenile Hormone III i
i
i
ill
i
. . . .L$:~ i s )
.
i
.
.
x.v~
-,_, tul
t
' "
218(d,J-15Hz} "
9
,,
ii
" ..
-
o:;
.
2 . 7 4 (t, J = 6 H z ) 8 1.2 1.63
Assign men t double peak=two methyl groups attached to epoxide s, 3H, C-7
5 1.27 & 1.32 1.65
Assign ment s, 2 x 3H, CH3 at C-I I
8 1.26 1.3 1.62
s, 3H, CH 3 at C-7
1.7
2.1 2.5
epoxide proton
2.18 2.74
3.61
carboxyl methyl vinyl protons C-6 vinyl protons C-2
3.72 5.22 5.75
Assignment s, 3H, CH3 at C-I 1 s, 3H, H-12 s, 3H, CH3 at C-7 m, 4H, H-8, H-9 m, 4H, H-4, H-5
d, sH, J--I.5 Hz, CH 3 at C-3 t, J=6 Hz, IHz, IH, H-10 2.70t, J=6 Hz, IH, H-
10 5.12 5.59
s, 3H, OCH3 m, IH, H-6 m, IH, H-2
CCI 3
Solvent:
[271
Reference: ,
,
_
3.69 5.14 5.67
s, 3H, OCH3 t, J=6 Hz, IH, H-6 br. s, IH, H-2
CDC! 3
CDC! 3
[381
[1831
,,,
_
Chemical shifts are reported at 8 values in ppm. Tetramethylsilane was the internal standard. Other references: 180, 182
T a b l e 4. l,
,,,,,
13C Nuclear Magnetic Spectra of Juvenile Hormone ,,
I I I ( C D C I 3 ) [183]
,,,
8 50.9 167.2 I 15.7 159.9 41.1 26.1 123.8 135.5 26.5 27.7 64.2 58.2 25 25 16.2 18.9
,,
Assignment OCH 3 C-l C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C- l 0 C-I l C-12 CH 3 at C-l l CH 3 at C-7 CH 3 at C-3
i
Chemical shifts are reported as 8 values in ppm. Tetramethylsilane was the internal standard. Numbering of juvenile hormone III as illustrated in Appendix I, Table 3.
i
,t
JUVENILE HORMONES
Table 5.
405
References for Juvenile H o r m o n e III
Extraction and Chromatography: Radioimmunoassay: Radiochemical assay: Chemical synthesis:
12, 15, 37, 185-197. 167, 198-201. 45, 46, 47, 49, 50. 38, 180-184, 202-211.
APPENDIX II. JUVABIONE AND ANALOGS Table 1.
C h e m i c a l Data for Juvabione, (+)-4(R)-[l'(R)-5'-Dimethyl-3'-oxohexyl]-lcyclohexene-l-carboxylic acid methyl ester [87, 90, 92, 101, 102, 212, 213]
Beilstein registry number: 2125665 MW 266.38 C16H260 3 Ultraviolet spectroscopy (ethanol): ~,= 222 nm, e = 13 600 Infrared spectroscopy (carbon disulfide):1722, 1712, 1645 cm "I. Mass spectroscopy (m/z): 266.2 (M+), 234, 206, 167, 166, 139, 135, 134 (base peak), 127, 107, 105, 91, 85, 79, 77, 69, 67, 57, 43, 41. H+-NMR (CDCI 3, I00 Hz): 8 (ppm) 6.96 IH, multiplet,H2 3.72 3H, singlet, methyl ester 0.90 6H, doublet, J = 6 Hz, C-5' methyls 0.86 3H, doublet, J 6 Hz, C-I' methyl 2.8-1.1 13H 13C NMR: 209.8, 167.3, 138.9, 129.8, 52.1, 51.0, 47.4, 32.3, 29.4, 24.9, 24.2, 22.3, 16.2. Chemical synthesis (references): 212, 213, 214. =
Table 2.
Chemical Data of Juvabione and Related Compounds
Compounds (+)-Todomatuic acid (+)-Juvabione (+)-Epijuvabione (+)-Dehydrojuvabione (+)-Dehydroepijuvabione (+)-Dihydrojuvabione Juvabiol Isojuvabiol Epijuvabiol
References 102, 215. 87, 89, 90, 92, I 01, 214. 95,214. 88, 89, 90. 89, 90, 95. 92. 89, 90, 91. 89, 90, 91. 89, 90.
406
BEDE and TOBE
Table 3.
Juvabione and Structurally Related Compounds Isolated from Conifers
(+)-Todomatuic acid
Japanese fir (Abies sachalinensis Schmidt )1 Douglas fir (Pseudotsuga menziesii (Beissn.) Franco) 10
(+)-Juvabione (4R, I'R)
Balsam fir (,4. balsamea (L.) Miller) 2 Douglas fir (P. menziesii) 3,9 Alpine fir (A. lasiocarpa (Hook.) Nutt.) 4 Silver fir (.4. alba Mill.) 5
(+)-Epijuvabione (4R, I'S)
Balsam fir (A. balsamea) (Czechoslovakian) 6
(+)-Dehydrojuvabione (4R,I'R)
d. balsamea 7 A. lasiocarpa 4 A. aiba 5
(+)-Dehydroepijuvabione (4R,I'S)
A. balsamea (Czechoslovakian) 6 A. lasiocarpa 4 ,4. alba 5
(+)-Dihydrojuvabione
P. menziesii 3
Juvabiol (4R, I'R,3'S)
A. balsamea 8 A. lasiocarpa 4 A. alba 5
Isojuvabiol (4R, I'R, 3'R)
A. balsamea 8 A. alba 5
Epijuvabiol (4R, I'S, YS)
A. lasiocarpa 4 A. alba 5
References: I. 215
6.
95
2.
87
7.
88
3.
92
8.
91
4.
90
9.
!01
5.
89
10.
102
JUVENILE HORMONES
407
APPENDIX HI. PLANT JUVENILE HORMONE MIMICS Table 1.
Chemical Data for Juvocimene I, 1-Methoxy-4-[6-methyl-4-(2-methylpropenyl)-octa-l,5,7-trienyl]-benzene [ 105, 106] ,,
MW
282.42
|
C20H260
Ultraviolet spectroscopy (ethanol):
~, = 237 nm, e = 23 000 ~. = 260 nm, e = 22 000
Infrared spectroscopy (cm'l):
3010, 2910, 1640, 1610, 1580, 1513, 1440, 1375, 1295, 1250, 1175, 1105, 1035, 985, 965, 890, 835.
Mass spectroscopy (m/z):
282 (M+), 147 (base peak), 135, 93.
H + N M R (CDCI 3, 100 Hz):
8 (ppm)
p-Methoxycinnamyl:
7.25
2H, doublet, aromatic proton
6.85
2H, doublet, aromatic proton
6.32
I H, doublet, alkenyl proton
6.00
I H, doublet, alkenyl proton
2-Methylbutadienyl:
Isobutenyl:
13C NMR:
3.81
3H, singlet, methoxy
2.42
2H, broad triplet, aikenyl proton, aromatic ring
6.38
I H, broad doublet, vinylic moiety
5.37
I H, broad doublet
5.11
I H, broad doublet, vinylic moiety
4.96
I H, broad doublet, vinylic moiety
1.78
3H, doublet, methyl group
5.04
I H, double septet
3.37
1H, broad quintet, methine proton
!.72
3H, doublet, allylic methyl group
! .66
3H, doublet, allylic methyl group
158.6 (C), 141.7 (CH), 136.1 (CH), 132.6 (C), 131.3 (C), 130.7 (C), 130.3 (CH), 127.0 (CH), 126.4 (CH), 113.9 (CH), 110.8 (C8), 55.3 (CH30), 39.8 (C3), 38.3 (C4), 25.8 (CH3), 18.2 (CH3), 12.1 (CH3)ppm.
408
Table 2.
BEDE and TOBE
Chemical Data for Juvocimene II, 3-[l-[3-(4-Methoxyphenyl)-2-propenyl]-3methyl-2,4-pentadienyl]-2,2-dimethyl oxirane [105] ,,,
MW
,
298
Mass spectroscopy (m/z):
~, = 227 nm, e = 29 000 k = 262 nm, e = 25 000
298 (M+), 207, 147 (base peak), 91.
H+-NMR (CDCI 3, 100 Hz): p-Methoxycinnamyl:
8 (ppm) 7.26 6.85 6.4O 6.08 3.81 6.41 5.39 5.16
2-Methyl-trans- 1,3-bu ta dienyl:
2H, doublet, aromatic proton 2H, doublet, aromatic proton I H, doublet, alkenyl proton 1H, doublet, alkenyl proton 3H, singlet, methoxy I H, broad doublet, vinylic moiety I H, broad doublet I H, broad doublet, vinylic moiety 1H, double septet 3H, doublet, methyl group I H, doublet, epoxy proton 2 x 3H, singlet, methyl groups 3H, singlet
5.03 1.76 2.71 1.33 1.30
1,2-Epoxy-2-methylpropyl:
,l,llll
i
,
,
Chemical Data for (+)-Bakuchiol [107, 216] ,
MW
,
C20H260 2
Ultraviolet spectroscopy (ethanol):
Table 3.
,
256
,,,
,
,,
CI8H240
Ultraviolet spectroscopy (ethanol): ~. = 260 nm, e - 18400 Infrared spectroscopy (cm'l): 3350, 1530, 1245, 980, 822.
Mass spectroscopy (m/z):
H+-NMR:
256 (M+), 213, 174, 173 (base peak), 158, 145, 107, 93, 83, 79, 77. See paper for fragmentation schematic.
5 (ppm) 6.70
4H, AA'BB' quartet, J = 8.5 Hz Aromatic H's
5.90
2H, quartet, J = 16 Hz, A7,8
4.60-5.93
3H, multiplet, AI 6,17
4.9 1.60 1.51 1.13
IH, olefinic, A2,3 3H, singlet, C-2 methyl 3H, singlet, C-2 methyl 3H, singlet, C-6 methyl i
i
ii,
,
JUVENILE HORMONES
Table 4.
MW
409
Chemical Data for Echinolone, ( E ) - 1 0 - H y d r o x y - 4 , 1 0 - d i m e t h y i - 4 , 1 1 dodecadien-2-one [109, 114, 115]
224.2
C14H240 2
Infrared spectroscopy (cm"I):
3440, 3060, 1710, 1632, 1440, 1360, 1230, 1160, 900, 715.
Mass spectroscopy (m/z):
148, 133, 111. 8 (ppm) 5.95 5.32 5.20 5.00 3.05 2.07 1.62 1.22
H+-NMR (CDCI3):
i H, doublet, J = 10, 18 Hz, CH=CH2 I H, triplet, J - 7 Hz, vinylic proton IH, doublet, J = 1, 18 Hz, CH2=CH IH, doublet, J = I, 10 Hz, CH2=CH2 2H, singlet, CH2COCH 3 3H, singlet, CH3CO 3H, singlet, methyl group at double bond 3H, singlet, tertiary CH 3
APPENDIX IV. PLANT JUVENILE HORMONE ANTAGONISTS Table 1.
Chemical Data for Precocene I, 7-Methoxy-2,2-dimethyl-2H- chromene [129, 217-2211
Beilstein Registry Number: 133917 MW
190.24
C12H140 2
Ultraviolet spectroscopy (ethanol):
~. -- 279 nm, e = 5 670 ~. = 322 rim, e = 6 750 3050, 1630, 1600, 1450 cm "I. 1640, 1615, 1570, 1500, 1025 cm "I. 1610, 1470, 1380, 1200, 1050 cm "I.
Infrared spectroscopy
(neat): (neat): (CHCI3):
Mass spectroscopy (m/z):
190 (M+), 175 (base peak).
H+-NMR (CDCI3):
13C NMR (CDCI3):
(ppm) 6.6-6.8 6.19 5.49 3.77 1.40
3H, multiplet, benzene ring H 1H, doublet, J - 10 Hz, 4-H IH, doublet, J - 10 Hz, 3-H 3H, singlet, OCH 3 6H, singlet, 2 x CH 3
76.15 (C-2), 127.67 (C-3), 121.81 (C-4), 126.80 (C-5), 106.47 (C-6), 160.58 (C-7), 101.95 (C-8), 154.08 (C-8a), 114.50 (C-4a), 27.89 (2CH3).
410
BEDE and TOBE
Table 2.
C h e m i c a l Data for Precocene II, 6,7-Methoxy-2,2-dimethyl-2H- c h r o m e n e
[129, 218, 220-224] ,,
, |
,,|
,
i
,,
i
,
Beilstein Registry Number: 160683 MW
220.27
CI3H160 3
Ultraviolet spectroscopy (ethanol):
= 278 nm, e = 3 580 ~. = 322 nm, e = 6 750
Infrared spectroscopy (neat): (CHCi3):
1640, 1613, 1575, 1502, 1350, 1360, 1010, 1610, 1470, 1370, 1260, 1210 cm "1.
Mass spectroscopy (m/z):
220 (M +)
H+-NMR (CDCI3):
8 (ppm) 6.42, 6.54 6.25 5.48 3.83, 3.84 1.42
13C NMR (CDCI3):
750 cm "1.
2H, each singlet, benzene ring H I H, doublet, J = 10 Hz, 4-H I H, doublet, J = 10 Hz, 3-H 6H, singlet, 2 x OCH 3 6H, singlet, 2 x CH 3
75.94 (C-2), 128.19 (C-3), 121.94 (C-4), 110.09 (C-5), 149.84 (C-6), 147.35 (C-7), 101.18 (C-8), 143.20 (C-8a), 113.12 (C-4a), 27.67 (2CH3), 55.9 (OCH3), 56.5 (OCH3).
Synthesis of precocene !, precocene I1 and general chromenes (references): 147, 217, 219-240. Radiolabelling of precocene II (references): 147, 148, 230, 241.
ABBREVIATIONS:
BSA CA DMADP FDP FW GC IDP JH MS MF MVA NAD § NADPH
Bovine serum albumin Corpora allata Dimethyl allyl diphosphate Famesyl diphosphate Fresh weight Gas chromatography Isopentenyl diphosphate Juvenile hormone Mass spectroscopy Methyl famesoate Mevalonate Nicotinamide adenine dinucleotide (oxidized form) Nicotinamide adenine dinucleotide phosphate (reduced form)
JUVENILE HORMONES
411
REFERENCES [1]
Kafka, F. In The Metamorphosis, in the Penal Colony and Other Stories; W. Muir and E. Muir, Tranls.; Schlocken Books: New York, 1975; pp. 67-132. [2] Slfima, K. In Herbivores, Their Interactions with Secondary Plant Metabolites; G.A. Rosenthal and D.H. Janzen, Eds.; Academic Press: New York, 1979; pp. 683-700. Bowers, W.S. In Herbivores, Their Interactions with Secondary Plant [31 Metabolites. Vol I. The Chemical Participants; G.A. Rosenthal and M.R. Berenbaum, Eds.; Academic Press: New York, 1991; pp. 431-456. [4] Harboume, J.B. In Introduction to Ecological Biochemistry; Academic Press: New York, 1993; pp. 117-120. [5] Gilbert, L.I., Rybczynski, R., Tobe S.S. In Metamorphosis; L.I. Gilbert, J.R. Tata and B.G. Atkinson, Eds.; Academic Press: New York, 1996; pp. 59-107. [6] Rosenthal, G.A.; Janzen, D.H. In Herbivores, Their Interactions with Secondary Plant Metabolites, Academic Press: New York, 1979. [7] Rosenthal, G.A.; Berenbaum, M.R. In Herbivores, Their Interactions with Secondary Plant Metabolites. Vol I. The Chemical Participants, Academic Press: New York, 1991. [8] Wigglesworth, V.B. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 1-24. [9] Rhoades, D.F. In Herbivores, Their Interactions with Secondary Plant Metabolites; G.A. Rosenthal and D.H. Janzen, Eds.; Academic Press: New York, 1979; pp. 1-54. [lO] Futuyma D.J.; Keese, M.C. In Herbivores, Their Interactions with Secondary Plant Metabolites. Vol II. Ecological and Evolutionary Processes; G.A. Rosenthal and M.R. Berenbaum, Eds.; Academic Press: New York, 1991; pp. 439-475. [ll] Hay, M.E., Steinberg, P.D. In Herbivores, Their Interactions with Secondary Plant Metabolites. Vol II. Ecological and Evolutionary Processes; G.A. Rosenthal and M.R. Berenbaum, Eds.; Academic Press: New York, 1991; pp. 371-413. [12] Schooley, D.A. In Analytical Biochemistry of Insects; R.B. Turner, Ed.; Elsevier Science: Amsterdam, 1977; pp. 241-287. [13] Tobe S.S.; Feyereisen, R. In Endocrinology of Insects; R.G.H. Downer and H. Laufer, Eds.; A.R. Liss: New York, 1983; pp. 161-178. [14] Meyer, A.S.; Schneiderman, H.A.; Hanzmann, E. Ko, J.H. Proc. Natl. Acad. ScL U.S.A., 1968, 60, 853. [151 Judy, K.J.; Schooley, D.A.; Dunham, L.L. ; Hall, M.S.; Bergot, B.J.; Siddall, J.B. Proc. Natl. Acad. Sci. U.S.A., 1973, 70, 1509. [16] Sparagana, S.P.; Bhaskaran, G.; Dahm, K.H.; Riddle, V..I. Exp. Zool., 1984, 230, 309. [17] Bergot, B.J.; Jamieson, G.C.; Ratcliff, M.A.; Schooley, D.A. Science, 1980, 210, 336. [18] Bergot, B.J., Baker, F.C., Cerf, D.C., Jamieson, G., Schooley, D.A. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds., Elsevier Science: Amsterdam, 1981; pp. 33-45.
412
[19]
BEDE and TOBE
Schooley, D.A., Baker, F.C., Tsai, L.W., Miller, C.A., Jamieson, G. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones; J.A. Hoffmann and M. Porchet, Eds.; Springer-Verlag: Berlin, 1984; pp. 373-383. [20] Lefcvere, K.S.; Lacey, M.J.; Smith, P.H.; Roberts, B. Insect Biochem. Molec. Biol., 1993, 23, 713. [21] Bowers, W.S.; Thompson, M.J.; Uebel, E.C. Life Sciences, 1965, 4, 2323. [22] Trost, B.M. Accounts of Chemistry, 1970, 3, 120. [23] Liedtke, R.J.; Djerassi, C. J. Org. Chem., 1972, 37, 2111. [24l Dunham, L.L.; Henrick, C.A.; Smith, D.H.; Djerassi, C. Organic Mass Spect., 1976, 11, 1120. [25] Silverstein, R.M., Bassler, G.C., Morrill, T.C. In Spectrometric Identification of Organic Compounds 4th Ed., John Wiley and Sons: New York, 1991, pp. 15-16. [26] Thomas, A.F.; Willhalm, B.; Mfiller, R. Organic Mass Spect., 1969, 2, 223. [27] Meyer, A.S.; Hanzmann, E.; Schneiderman, H.A.; Gilbert, L.I.; Boyette, M. Arch. Bcem. Bphys., 1970, 13 7, 190. [28] Kuhnz, W.; Rembold, H. Org. Mag. Res., 1981, 16, 138. [29] Nakanishi, K.; Goto, T.; It6, S.; Natori; S. Nozoc. S. In Natural Products Chemistry, Vol. 1, Academic Press: New York, 1974, pp. 72-78. [30] Meyer, A.S.; Hanzmann, E.; Murphy, R.C. Proc. Natl. Acad. Sci. U.S.A., 1971, 68, 2312. [311 Nakanishi, K.; Schooley, D.A.; Koreeda, M.; Dillon, J. Chem. Comm., 1971, 1235. [32] Giese, Ch.; Spindler, K.D.; Emmerich, H. Z. Naturforsch., 1977, 32c, 158. [33] Kramer, K.J.; Dunn, P.E.; Peterson, R.C.; Law, J.H. In The Juvenile Hormones; L.I. Gilbert, Ed.; Springer-Verlag: Berlin, 1976; pp. 75-95. [34] van Tamelen, E.E. Ace. Chem. Res., 1968, 111. [35] Mumby, S.M.; Hammock, B.D.J. Agric Food Chem., 1979, 27, 1223. [36] Schooley, D.A.; Judy, K.J.; Bergot, B.J.; Hall, M.S.; Jennings, R.C. In The Juvenile Hormones; L.I. Gilbert, Ed.; Springer-Verlag: Berlin, 1976; pp. 101117. [37] Bergot, B.J.; Ra/cliff, M.; Schooley, D.A.J. of Chromatography, 1981, 204, 231. [38] Anderson, R. J.; Henrick, C.A.; Siddall, J.B.; Zurflilh, R. J. Am. Chem. Soc., 1972, 94, 5379. [39] Dunham, L.L.; Schooley, D.A.; Siddall, J.B.J. Chromat. Sci., 1975, 13, 334. [40] Croston, G.; Abdel-Aal, Y.A.I.; Gee, S.J.; Hammock, B.D. Insect Bcem., 1987, 17, 1017. [41] Pfiffner, A. In Aspects of Terpenoid Chemistry and Biochemistry; T.W. Goodwin, Ed.; Academic Press: New York, 1971; pp. 95-135. [42] Wakabayashi, N.; Waters, R.M. In CRC Handbook of Natural Pesticides, Vol III; E.D. Morgan and N.B. Mandava, Eds.; CRC Press: Boca Raton, 1985; pp. 87-151. [43] Szibbo, C.M.; Rotin, D.; Feyereisen, R.; Tobe, S.S. Gen and Comp. Endocrin., 1982, 48, 25. [44] Engelmann, F. Advances in Insect Physio., 1979, 14, 49. [451 Tobe, S.S.; Stay, B. Adv. in Insect Physio., 1985, 18, 305. [46] Tobe, S.S.; Pratt, G.E. Biochem. J., 1974, 144, 107. [47] Pratt, G.E.; Tobe, S.S. Life Sci., 1974, 14, 575. [48] Smith, A.F.; Yagi, K.; Tobe, S.S.; Schal, C. J. Insect Physiol., 1989, 35, 781.
JUVENILE HORMONES
[49]
413
Feyereisen, R.; Tobe, S.S. Anal. Biochem., 1981, 111, 372.
[50] Tobe, S.S.; Clarke, N. Insect Biochem., 1985, 15, 175. [5~1 Roth, L.M. Ann. Rev. Ent., 1970, 15, 75. [52]
[53] [54] [55] [56] [57]
[58] [59] [60] [61] [62] [63]
[64] [65]
[66] [67] [68] [69]
[70] [71] [72] [73] [74] [751 [76] [77] [78] [79]
[80]
Tobe, S.S. In Insect Biology of the Future; M. Locke and D.S. Smith, Eds.; Academic Press: New York, 1980; pp. 345-367. Weaver, R.J.; Pratt, G.E.; Finney, J.R. Experientia, 1975, 31, 597. Weaver, R.J.; Pratt, G.E. Phys. Ent., 1977, 2, 59. Tobe, S.S.; Stay, B. Gen. Comp. Endocrin., 1977, 31, 138. Gadot, M.; Chiang, A.-S.; Schal, C. J. Insect Phys., 1989, 35, 537. Tobe, S.S.; Ruegg, R.P.; Stay, B.A.; Baker, F.C.; Miller, C.A.; Schooley, D.A. Experientia, 1985, 41, 1028. Lanzrein, B.; Gentinetta, V.; Abegglen, H.; Baker, F.C.; Miller, C.A.; Schooley, D.A. Experientia, 1985, 41, 913. Monger, D.J. In Methods in Enzymology, Vol. 110; J.H, Law and H.C. Rilling, Eds.; Academic Press: New York, 1985; pp. 51-58. Sutherland, T.D.; Feyereisen, R. Mol. and Cell. Endocrin., 1996, 120, 115. Svoboda, J.A.; Kaplanis, J.N.; Robbins, W.E.; Thompson, M.J. Ann. Rev. Ent., 1975, 20, 205. Feyereisen, R.; Koener, J.; Tobe, S.S. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds.; Elsevier Science: Amsterdam, 1981; pp. 81-92. Baker, F.C.; Mauchamp, B.; Tsai, L.W.; Schooley, D.A.J. Lipid Res., 1983, 24, 1586. Schooley, D.A.; Baker, F.C. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 363-389. Cusson, M.; Yagi, K.J.; Ding, Q.; Duve, H.; Thorpe, A.; McNeil, J.N.; Tobe, S.S. Insect Bcem., 1991, 21, 1. Feyereisen, R.; Pratt, G.E.; Hamnett, A.F. Eur. J. Bcem., 1981, 118, 231. Wang, Z.; Ding, Q.; Yagi, K.J.; Tobe, S.S.J. Insect Phys., 1994, 40, 217. Reibstein, D.; Law, J.H. Bcem. Bphys. Res. Comm., 1973, 55, 266 Hammock, B.D. Life Sciences, 1975, 17, 323. Hammock, B.D.; Mumby, S.M. Pest. Biochem. Physiol., 1978, 9, 39. Law, J.H. In Biosynthesis of Isoprenoid Compounds, Vol. 2; J.W. Porter and S.L. Spurgeon, Eds.; John Wiley and Sons: New York, 1981; pp. 507-534. Brindle, P.A.; Baker, F.C.; Tsai, L.W.; Reuter, C.C.; Schooley, D.A. Proc. Natl. Acad. Sci. U.S.A., 1987, 84, 7906. Brindle, P.A.; Baker, F.C.; Tsai, L.W.; Schooley, D.A. Arch. Insect Bcem. Physiol., 1992, 19, 1. Peter, M.G.; Shirk, P.D.; Dahm, K.H.; R/Slier, H. Z. Naturforsch., 1981, 36c, 579. Sparagana, S.P.; Bhaskaran, G.; Barrera, P. Arch. Insect Bcem. Phys., 1985, 2, 191. Hammock, B.D. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 431-472. Rotin, D.; Feyereisen, R.; Koener, J.; Tobe, S.S. Insect Bcem., 1982, 12, 263. Mundall, E.C.; Tobe, S.S.; Stay, B. J. Insect Phys., 1981, 27, 821. Riddiford, L.M. In Insect Juvenile Hormone: Chemistry and Action; J.J. Menn and M. Beroza, Eds.; Academic Press: New York, 1972; pp. 95-l 11. Masner, P.; Shima, K.; Landa, V. Nature, 1968, 219, 395.
414
[81]
[82] [83] [84]
[85] [86] [87]
[88] [89]
[90] [911 [92] [93] [94] [95] [96] [97]
[98] [99] [lO0] [lO1] [102] [103] [104]
[1o5] [106] [107] [108] [109]
[llO] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120]
BEDE and TOBE
Saxena, B.P.; Koul, O.; Tikku, K. Indian J. Exp. Biol., 1978, 16, 703. Steele, C.L.; Lewinsohn, E.; Croteau, R. Proc. Natl. Acad. ScL U.S.A., 1995, 92, 4164. Slfima, K.; Williams, C.M. Proc. Natl. Acad. Sci. U.S.A., 1965, 54, 411. Slfima, K.; Williams, C.M. Biol. Bull., 1966, 130, 235. Slfima, K.. Ent. Exp. Appl., 1969, 12, 721. Mansingh, A.; Sahota, T.S.; Shaw, D.A. Can. Ent., 1970, 102, 49. Bowers, W.S.; Fales, H.M.; Thompson, M.J.; Uebel, E.C. Science, 1966, 154, 1020. Manville, J.F. Can. J. Chem., 1975, 53, 1579. Manville, J.F.; Book, K.; von Rudloff, E. Phytochem., 1977, 16, 1967. Manville, J.F.; Kriz, C.D. Can. J. Chem., 1977, 55, 2547. Manville, J.F. Can. J. Chem., 1976, 54, 2365. Rogers, I.H.; Manville, J.F.; Sahota, T. Can. J. Chem., 1974, 52, 1192. Ibaraki, A.; Sahota, T.S. Can. Forestry Bi-Monthly Res. Notes., 1976, 32, 3. Retnakaran, A. Can. Ent., 1970, 102, 1592. v CereS', V.; DoleJ~. L.; Lfibler, L.; Sorm. F.; Slfima, K. Coil Czech. Chem. Comm., 1967, 32, 3926. v Slfima, K.; Such~', M.; $orm, F. Biol. Bull., 1968, 134, 154. $uch~'. M; Slfima, K.; Sorm. F. Science, 1968, 162, 582. Slfima, K.; Williams, C.M. Nature, 1966, 210, 329. Puritch, G.S.; Nijholt, W.W. Can. J. Bot., 1974, 52, 585. Blount, J.F., Pawson, B.A., Saucy, G. Chem. Comm., 1969, 715, 1016. Sakai, T.; Hirose, Y. Chem. Letters, 1973, 491. Sakai, T.; Hirose, Y. Chem. Letters, 1973, 825. McDonough, L.M. In Isopentenoids in Plants: Biochemistry and Function; W.D. Nes, G. Fuller and L.-S. Tsai, Eds.; Marcel Dekker: New York, 1984; pp. 81101. Bowers, W.S.; Nishida, R. Science, 1980, 209, 1030. Nishida, R.; Bowers, W.S.;Evans, P.H.J. Chem. Ecol., 1984, 10, 1435. Mestres, R.; Mufloz, E. Syn. Comm., 1996, 26, 1309. Mehta, G.; Nayak, U.R.; Dev, S. Tetrahedron, 1973, 29, 1119. Bhan, P.; Soman, R.; Dev, S. Agric. Biol. Chem., 1980, 44, 1483. Jacobson, M., Redfem, R.E.; Mills Jr., G.D. Lloydia, 1975, 38, 473. Nishida, R.; Bowers, W.S.; Evans, P.H. Arch. lnsect Bcem. Physiol., 1983, 1, 17. Barton, G.M., MacDonald, B.F., Sahota, T.S. Can. For. Serv. Bi-monthly Res. Notes, 1972, 28, 22. Saxena, B.P.; Saravastava, J.B. Ind. J. Exp. Biol., 1973, 11, 56. Damodaran, N.P.; Dev, S. Tetrahedron, 1973, 29, 1209. Jacobson, M.; Redfem, R.E.; Mills Jr., G.D. Lloydia, 1975, 38, 455. Cooke Jr., M.P. Jr. Org. Chem., 1979, 44, 2461. Orsini, F.; Peizzoni, F. J. Org. Chem., 1980, 45, 4726. Saxena, B.P.; Srivastava, J.B. Experientia, 1972, 28, 112. Prabhu, V.K.K.; John, M. Ent. Exp. Appl., 1975, 18, 87. Prabhu, V.K.K.; John, M. Experientia, 1975, 31, 913. Williams, C.M. Sci. American, 1967, 217, 13.
JUVENILE HORMONES
415
[121] Sparks, T.C.; Hammock, B.D. In Pest Resistance to Pesticides; G.P. Georghiou and T. Saito, Eds.; Plenum Press: New York, 1983; pp. 615-668. [122] Dowd, P.F.; Smith, C.M.; Sparks, T.C. Insect Bcem., 1983, 13, 453. [123] Dyte, C.E. Nature, 1972, 238, 48. [124] Cerf, D.C.; Georghiou, G.P. Nature, 1972, 239, 401. [125] Vinson, S.B.; Plapp Jr., F.W. ,I. Agr. Food Chem., 1974, 22, 356. [126] Bowers, W.S.; Ohta, T.; Cleere, J.S.; Marsella, P.A. Science, 1976, 193, 542. [127] Kaneko, C.; Tsuchiya, T.; Ishikawai, M. Chem. Pharm. Bull., 1963, 11,271. [128] Chang, C.-F.; Isogai, A.; Kamikado, T.; Murakoshi, S.; Sakurai, A.; Tamura, S. Agr. Biol. Chem., 1975, 39, 1167. [129] Suga, T.; Shishibora, T.; Kosela, S.; Sood, V.K. Phytochem., 1975, 14, 308. [130] Proksch, P.; Proksch, M.; Towers, G.H.N.; Rodriguez, E. J. Natl. Products, 1983, 46, 331-334. [131] Tamayo-Castillo, G.; Jakupovic, J.; Bohlmann, F.; Castro, V.; King, R.M. Phytochem., 1989, 28, 139. [132] Ferracini, V.L.; Roewer, I.; Gao, F.; Mabry, T.J. Phytochem., 1989, 28, 1463. [133] Castro, V.; Tamayo-Castillo, G.; Jakupovic, J. Phytochem., 1989, 28, 2415. [134] Siebertz, R.; Proksch, P.; Witte, L. Phytochem., 1990, 29, 2135. [135] Zdero, C.; Bohlmann, F., Niemeyer, H.M. Phytochem., 1991, 30, 693. [1361 Gonz~ilez, A.G.; Aguiar, Z.E.; Grillo, T.A.; Luis, J.G.; Rivera, A.; Calle, J. Phytochem., 1991, 30, 1137. [137] Staal, G.B. Ann. Rev. Ent., 1986, 31,391. [138] Masner, P.; Bowers, W.S.; K~ilin, M.; Miihle, T. Gen. Comp. Endocrin., 1979, 37, 156. [139] Zamorano, G.; Carrau, T.M.; Enguix, A.L.; Martinez-Pardo, R.; Cachaza, A.N. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds.; Elsevier Science: Amsterdam, 1981; pp. 311-314. [140] Unnithan, G.C.; Nair, K.K. Ann. Ent. Soc. Amer., 1979, 72, 38. [141] Bowers, W.S.; Martinez-Pardo, R. Science, 1977, 197, 1369. [142] Milller, P.J.; Masner, P.; K~ilin, M.; Bowers, W.S. Experientia, 1979, 35, 704. [143] Pratt, G.E; Bowers, W.S. Nature, 1977, 265, 548. [144] Unnithan, G.C.; Nair, K.K.; Bowers, W.S. Insect Phys., 1977, 23, 1081. [145] Liechty, L.; Sedlak, B.J. Gen. Comp. Endocrin., 1978, 36, 433. [146] Sedlak, B.J. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol 7; G.A. Kerdut and L.I. Gilbert, Eds.; Pergamon Press: New York, 1985; pp. 25-60. [147] Ohta, T.; Kuhr, R.J.; Bowers, W.S.J. Agric. Food Chem., 1977, 25, 478. [148] Soderlund, D.M.; Messeguer, A.; Bowers, W.S.J. Agric. Food Chem., 1980, 28, 724. [149] Soderlund, D.M.; Feldlaufer, M.F.; Takahashi, S.Y.; Bowers, W.S. In Juvenile Hormone Biochemistry; G.E. Pratt and G.T. Brooks, Eds., Elsevier Science: Amsterdam, 1981; pp. 353-362. [150] Bowers, W.S.; Evans, P.H.; Marsella, P.A.; Soderlund, D.M.; Bettarini, F. Science, 1982, 217, 647 [~51] Feldlaufer, M.F., Bowers, W.S. Gen. Comp. Endocrin., 1982, 46, 377. [152] Srivastava, R.P.; Proksch, P. Naturwissenschaften, 1990, 77, 438. [153] Satoh, A.; Utamura, H.; Ishizuka, M.; Endo, N.; Tsuju, M.; Nishimura, H. Biosci. Biotech. Biochem., 1996, 60, 664. [154] Proksch, P.; Rodriguez, E. Phytochem., 1983, 22, 2335. [155] Toong, Y.C.; Schooley, D.A.; Baker, F.C. Nature, 1988, 333, 170.
416
BEDE and TOBE
[156] Iwamura, J.; Komaki, K.; Komai, K.; Hirao, N. Nippon Nogeikagaku Kaishi, 1978, 52, 379. [157] Iwamura, J.; Kameda, M.; Komai, K.; ttirao, N. Nippon Kagaku Kaishi, 1978, 11, 1552 (Chem. Abstr. 90: 69086h). [158] Iwamura, J.; Komaki, K.; Komai, K., Hirao, N. Nippon Nogei Kagaku Kaishi, 1978, 52, 561 (Chem. Abstr. 90: 135072d). [159] Iwamura, J., Komaki, K., Komai, K.; Hirao, N. Nippon Kagaku Kaishi, 1978, 53, 255 (Chem. Abstr. 90: 164723x). [160] Iwamura, J. Nippon Nogei Kagaku Kaishi, 1979, 53, 343 (Chem. Abstr. 92: 1162321). [161] Komai, K.; Sugiquaka, Y.; Sato, S. Kinki Daigaku Nogakubu Kiyo, 1981, 14, 57 (Chem. Abstr. 95: 162961c). [162] Versini, G., Rapp, A.; Dalla Serra, A.; Pithier, U., Ramponi, M. Vitis, 1994, 33, 139. [163] Kijjoa, A.; Pinto, M.M.M.; Pinho, P.M.M., Tantisewie, B., Herz, W. Phytochem., 1990, 29, 653. [z64] Jaeobs, H.; Ramadayal, F.; McLean, S.; Perpiek-Dumont, M.; Puzzuoli, F.; Reynolds, W.F..I. Natl. Products, 1987, 50, 507. [165] Waterman, P.G.; Muhammad, I. Phytochem., 1985, 24, 523. [166] Tane, P.; Ayafor, J.F.; Sondengam, B.L. Phytochem., 1988, 27, 3986. [167] Goodman, W.G.; Orth, A.P.; Toong, Y.C., Ebersohl, R.; Hiruma, K.; Granger, N.A. Arch. Insect Bcem. Bphys., 1995, 30, 295. [168] Bede, J.C.; Goodman, W.G.; Tobe, S.S. Pure Appl. Chem., 1998, in press. [169] Meneses, C.R.; Garcifi de la Osa, Centro Agrieola, 1988, 15, 90 (CABS 07910772). [170] Dela Cruz, C.G. International Rice Res. Newsletter, 1986, 11, 26 (CABS 07404038). [171] Naresh, J.S.; Smith, C.M. Ent. Exp. Appl., 1984, 35, 89. [172] Wardle, K.; Short, K.C. Bcem. Phys. Pflanzen, 1982, 177, 210. [173] Hasan, A. In Allelopathy: Basic and Applied Aspects; S.J.H. Rizvi and V. Rizvi, Eds.; Chapman and Hall: London, 1992; pp. 413-441. [~74] Davey, K.G. In Endocrinology of Selected Invertebrate Types; H. Laufer and R.G.H. Downer, Eds.; Alan R. Liss" New York, 1988; pp. 63-86. [175] Rogers, W.P. Comp. Bcem. Physiol., 1978, 61A, 187. [176] Glassburg, G.H.; Teffl, P.M.; Bone, L.W. Proc. Helminthol. Soc. Wash., 1983, 50, 62. [177] Meerovitch, E. Can. J. Zool., 1965, 43, 81. [178] Johnson, R.N., Viglierehio, D.R. Experimental Parasitology, 1970, 27, 301. [179] Adler, J.H.; Grebenok, R.J. Lipids, 1995, 30, 257. [18o] McCormick, J.P.; Schafer, T.R.J. Org. Chem., 1977, 42, 387. [181] Ichinose, I.; Hogogai, T., Kato, T. Synthesis, 1978, 605. [182] Kleijn, H., Westmijze, H.; Meijer, J.; Vermeer, P. J. Roy. Neth. Chem. Soc., 1981, 100, 249. [~83] Rodriguez, J.B.; Gros, E.G.Z. Naturforsch., 1990, 45b, 93. [184] Meyer, W.; Spiteller, G. Liebigs Ann. Chem., 1993, 1253. [185] Trautmann, K.H.; Schuler, A.; Such ~, , M.; Wipf, H.-K. Z. Naturforsch, 1974, 29c, 161. [186] van Broekhoven, L.W.; van der Kerk-van Hoof, A.C.; Salemink, C.A.Z. Naturforsch, 1975, 30c, 726.
JUVENILE H O R M O N E S
417
[187] Lanzrein, B.; Hashimoto, M.; Parmakovich, V.; Nakanishi, K. Life Sciences., 1975, 16, 1271. [188] Peter, M.G.; Dahm, K.H.; R611er, H. Z. Naturforsch., 1976, 31c, 129. [1891 Bergot, B.J.; Sehooley, D.A.; Chippendale, G.M.; Yin, C.-M. Life Sciences, 1976, 18, 811. [190] Hamnctt, A.F.; Pratt, G.E.J. Chromatography, 1978, 158, 387. [191] Hagenguth, H.; Reinhold, H. Z. Naturforsch., 1978, 33c, 847. [192] Hagenguth, H.; Rembold, H. J. Chromatography, 1979, 170, 175. [193] Mauchamp, B.; Lafont, R.; Jourdain, D. J. Insect Phys., 1979, 25, 545. [194] Huibregtse-Minderhout, L.; van tier Kerk-van Hoof, A.C.; Wijkens, P.; Biessels, H.W.A.; Salemink, C.A.J. Chromatography, 1980, 196, 425. [195] Rembold, H.; Hagenguth, H.; Rascher, J. Anal. Bcem., 1980, 101, 356. [196] KSrtv~lyessy, G.; Sz6r,'tdi, S.; Sztruhar, I.; Lad,-tnyi, L. J. Chromatography, 1984, 303, 370. [197] Rembold, H.; Lackner, B. J. Chromatography, 1985, 323, 355. [198] Strambi, C.; Strambi, A.; De Reggi, M.L.; Hrin, M.H.; Delaage, M.A. Fur. J. Bcem., 1981, 118, 401. [199] Granger, N.A.; Goodman, W.G. In Immunological Techniques in Insect Biology; L.I. Gilbert and T.A. Miller, Eds.; Springer-Vcrlag: New York, 1988; pp. 215251. [200] Goodman, W.G.; Coy, D.C.; Baker, F.C.; Xu, L.; Toong, Y.C. Insect Bcem., 1990, 20, 357. [2011 Goodman, W.G.; Huang, Z.-H.; Robinson, G.E.; Strambi, C.; Strambi, A. Arch. Insect Bcem. Phys., 1993, 23, 147. [202] Johnson, W.S.; Li, T.-T.; Faulkner, D.J.; Campbell, S.F.J. Am. Chem. Soc., 1968, 90, 6225. [203] Loew, P.; Siddall, J.B.; Spain, V.L.; Werthemann, L. Proc. Natl. Acad. Sci. U.S.A., 1970, 67, 1462. [204] Locw, P.; Siddall, J.B.; Spain, V.L.; Werthemann, L. Proc. Natl. Acad. Sci. U.S.A., 1970, 67, 1824. [205] van Tamelen, E.E.; McCormick, J.P.J. Am. Chem. Soc., 1970, 92, 737. [206] Loew, P.; Johnson, W.S.J. Am. Chem. Soc., 1971, 93, 3765. [207] Hcnrick, C.A.; Schaub, F.; Siddall, J.B.J. Am. Chem. Soc., 1972, 94, 5374 [208] Ohki, M.; Mori, K.; Matsui, M. ,4gric. Biol. Chem., 1974, 38, 175. [209] Sehooley, D.A.; Bergot, B.J.; Goodman, W.G.; Gilbert, L.I. Bcem. Bphys. Res. Comm., 1978, 81, 743. [210] Adams, P.H.J. Labelled Cmpds. Radiopharm., 1988, 25, 395. [211] Messeguer, A.; S,~nehez-Baeza, F.; Casas, J.; Hammock, B.D. Tetrahedron, 1991, 47, 1291. [212] Pawson, B.A.; Cheung, H.-C.; Gurbaxani, S.; Saucy, G. Chem. Comm., 1968, 1057. [2131 Trost, B.M.; Tamaru, Y. Tetrahedron Letters, 1975, 44, 3797. [214] Pawson, B.A.; Cheung, H.-C.; Gurbaxani, S.; Saucy, G. J. Am. Chem. Soc., 1970, 92, 336. [215] Tutihasi, R.; Hanazawa, T. J. Chem. Soc. Japan, 1940, 61, 1041 (CA (1943) 37: 258). [216] Prakasa Rao, A.S.C.; Bhalla, V.K.; Nayak, U.R.; Dev, S. Tetrahedron, 1973, 29, 1127. [2171 Mann, J.; Kane, P.D. Tetrahedron Letters, 1985, 26, 4677.
418
BEDE and TOBE
[2~8] T'm~ir, T.; Seb6k, P.; KSv6r, K.; J~tszber6nyi, J.C. dcta Chim. Hung., 1988, 125, 303. [219] Miranda, M.A.; Primo, J.; Tormos, R. Heterocycles, 1988, 27, 673. [220] Iyer, M.; Trivedi, G.K. Syn. Comm., 1990, 20, 1347. [221] Bissada, S.; Lau, C.K.; Bemstein, M.A.; Dufresne, C. Can. J. Chem., 1994, 72, 1866. [222] Uchiyama, M.; Overeem, J.C.J. Roy. Neth. Chem. Soc., 1981, 100, 408. [223] Miranda, M.A.; Primo, J.; Tormos, R. Heterocycles, 1991, 32, 1159. [224] Solladi6, G.; Boeffel, D.; Maignan, J. Tetrahedron, 1996, 52, 2065. [225] Smith, L.I.; Ruoff, P.M.J. Am. Chem. Soc., 1940, 62, 145. [2261 Livingstone, R.; Watson, R.B. J'. Chem. Soc., 1957,1509. [227] Sehweizer, E.E., Shaffer, E.T.; Hughes, C.T.; Berninger, C.J.J. Org. Chem., 1966, 31, 2907 [228] Mechoulam, R.; Yagnitinsky, B., Gaoni, Y. Jr. Am. Chem. Soc., 1968, 90, 2418. [229] Anderson, W.K.; LaVoie, E.J.; Whitkop, P.G.J. Org. Chem., 1974, 39, 881. [230] Ohta, T.; Bowers, W.S. Chem. Pharm. Bull., 1977, 25, 2788. [23~] Bongini, A.; Cardillo, G.; Orena, M.; Porzi, G.; Sandri, S. Tetrahedron Letters, 1979, 2 7, 2545. [232] Sartori, G.; Casiraghi, G.; Bolzoni, L.; Casnati, G. J. Org. Chem., 1979, 44, 803. [2331 Tsukayama, M.; Sakamoto, T.; Horie, T.; Masumura, M.; Nakayama, M. Heterocycles, 1981, 16, 955. [234] Kawase, Y.; Yamaguchi, S.; Horita, H.; Takeno, J.; Kameyama, H. Bull. Chem. Soc. Jpn., 1982, 55, 1153. [235] Strunz, G.M.; Brillon, D.; Gigu6re. Can. J. Chem., 1983, 61, 1963 [236] Cort6s, M.J.; Haddad, G.R.; Valderrama, J.A. Heterocycles, 1984, 22, 1951. [237] Pandey, G., Krishna, A. 3. Org. Chem., 1988, 53, 2364. [238] Tiabi, M.; Zamarlik, H. Tetrahedron Letters, 1991, 32, 7251. [2391 Kulkami, S.A.; Paradkar, M.V. Syn. Comm., 1992, 22, 1555. [240] Aukrust, I.R.; Noushabadi, M.; Skatteb~l, L. Polish J. Chem., 1994, 68, 2167. [241] Hsai, M.T.S.; Grossman, S.; Schrankel, K.R. Chem.-Biol. Interactions, 1981, 3 7, 265.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, VoL 22
419
9 2000 Elsevier Science B.V. All rights reserved
ANTIULCER AND GASTROPROTECTIVE ACTIVITY OF FLAVONIC COMPOUNDS: MECHANISMS INVOLVED M.J. M A R T I N * ; C. A L A R C O N DE LA LASTRA; V. MOTILVA; C. LA CASA
and
Dept. of Pharmacology, Faculty of Pharmacy. University of Seville, Prf Garcia Gonz6lez s/n, 41012-Seville, Spain A B S T R A C T : The flavonoids comprise a large group of unique compounds that are widely distributed in the plant kingdom. In recent years, they have been reviewed for their wide range of biological activities, focusing in particular on the potential therapeutic use of this class of molecules as antiinflammatory, antiallergic, antiviral, anticancer or immunostimulant drugs. This pharmacological potential is probably due to the capability of flavonoids to interact with important cellular processes in which key enzymes such as cyclooxygenase, lipooxygenase, phospholipase A2, NADH-oxidase or glutathion reductase are involved. Other interesting studies also reported the capacity of some flavonoids to interact with oxygen activated species since they are strong scavengers of lipid radicals. These properties, including the antiinflammatory and antioxidant mechanisms and the capacity to inhibit cellular apoptosis, could also be related with an important antiulcerogenic and protective effect on gastric mucosa. Numerous authors have demonstrated the ulcer-protecting properties of these substances against different experimental models, such as restraint-stress, absolute ethanol, reserpine, acetic acid and pyloric occlusion. This review deals with the gastroprotective effects of flavonic compounds, the mechanisms involved, and the possible structure-activity relationships.
INTRODUCTION O f the substances identified in plants, the flavonoids represent one of the most important and interesting classes of biologically active compounds. They are present in a wide variety of plants, and are especially common in leaves, flowering tissues, pollens and fruits. These compounds are also abundant in w o o d y parts such as stems and bark, and they are an important part of human nourishment. Flavonoids have low molecular weight and occur naturally as aglycons, glycosides and methylated derivatives. The aglycons generally consist of a benzo-(-pyrone which in the position 2 or 3 is substituted by a phenyl ring (Fig. 1).
420
MART|N et aL
1
Flavans
0 X Y D A
0 Dihydroxhalcones
oHZ) Flavan-3-ols (Catechtns)
T I
0 N
0
0 Chalcones
Flavanones
.OAD OH Flavan-3-dlols (Leucocyanidins)
Flavylium salts
~ CH-~ Aurones
0 Flavones
421
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
{Fig. I). contd.....
0 0 X Y D A T I 0 N
%
0
Flavanolols {Dihydroflavonols}
Anthocyanldlns
L E V
0
L
0
Flavonols Fig. (1). Principal structural groups of natural flavonoids (from Bombardelli & Morazzoni) [33].
Hydroxylation occurs naturally in position 3, 5, 7, 3', 4' and 5'. Glycosidation with L-rhamnose, D-glucose, glucorhamnose, galactose or arabinose in position 3 or 7 is frequent in nature. Methyl ethers and acetyl esthers of the alcohol groups are known to occur naturally. The pharmacological profile of these agents presents a wide range of activities affecting different biological systems. The empirical use of flavonoids as drugs has acquired scientific confirmation in the last few years. Numerous studies describe its capability to interact with important cellular processes in which keyenzymes are involved such as cyclooxygenase [ 1-5] lipoxygenase [2, 4-10], xanthine oxidase [ 11-14], phospholipase A2 [15, 16], cyclic nucleotide phosphodiesterases [17], proteinkinase C [18], hyaluronidase [19], reverse transcriptase [20, 21 ], mitochondrial succinoxidase [22], NADH-oxidase [23, 24], glutathione reductase [25], glutathione S-transferases [26]. A considerable body of research work deals with the action of flavonoids upon cell membrane permeability [27], biosynthesis of prostaglandins [28-31], their ability to
422
MARTIN et aL
capture free oxygen radicals [32-34], and to modify the synthesis and liberation of histamine [35], or the neutrophil function [36]. The bibliographic research shows that flavonoids possess widespread biological activities, including antimierobial [37-39], antihelmintie [40], mutagenie [41-42], carcinogenic [40, 43, 44], antieareinogenie [42, 45, 46], antioxidant [34, 38, 47, 48], antiinflammatory [49-53], antiallergie [54], antiviral [55, 56], endocrine [57], antihyperlipidemie [58], and antidiarrheie [59-61 ] properties. In the last few years, many papers have been published on the antiuleer effects of numerous flavonoids. Several of these compounds prevent gastric mueosal lesions produced by different methods of experimental ulcers, and they are able to reduce the number and intensity of the lesions [65-69]. Peptic ulcer disease is one of the most common pathological processes of the gastrointestinal tract. It is associated with chronic inflammation of the gastric mueosa and, in the ease of duodenal ulceration, with the duodenal mueosa. It is characterized by frequent recurrence and high incidence. Approximately 10% of the Western population develops this disease, which is associated with high management costs and a substantial reduction in the quality of life of patients. Numerous pharmacological strategies have been used for the treatment and prevention of this pathological process, including antiaeid, antieholinergie or H2antihistaminergie drugs, and more recently proton pump inhibitors. However the gastroduodenal phytotherapy has not developed in a similar way. In the past century, opium extract was frequently used in the treatment of severe pain associated with peptic ulcer disease. Other sedative remedies prescribed included belladonna, hyoseiamus, eonnium, and cannabis, drugs with analgesic or antieholinergie properties. Over the years, numerous plants have been used in folk medicine against gastroduodenal disorders. The development of phytoehemieal techniques has allowed the identification and isolation of numerous principles from some of them, such as alkaloids [70], sesquiterpen laetones [71, 72], or polyphenolie compounds [66-69]. These antiuleer properties have also been related to flavonie extracts of different species, in which phytoehemieal study has demonstrated that these polyphenols are the prevailing compounds (Fig. 2). FLAVONOIDS WITH ANTIULCER ACTIVITY A traditional remedy frequently used in gastrointestinal phytomedicine has been licorice root, Glizyrrhiza glabra. Its gastroprotective effects are attributable to its flavonoids, the flavone liquiritoside and chalcone isoliquiritoside (1-1.5%), which induce defensive mechanisms of the gastric mucosa such as the stimulation of mucous secretion and also
ANTIULCERAND GASTROPROTECTIVEACTIVITY
Rhamnoglucosyl-
423
0
OH
~OCH
3
O
Neohesperidin
Hesperidin OH
O
.,yOH
,~OH
0 Llqulritoslde
OH
0 Narlngenln
OH Rhanmoglucosyl-
~
OH
H
O Narlngin
OH
O Apigenln
OH
OH
OH
OH H
H
I OH OH
O Luteollne
Quercetin
MARTINet aL
424
(Fig. 2). contd .....
OH OH H
H
I OH
O ~ O H Genlsteln
~=
O-Rhanmoglue~yl
Rutln
/OH
CH-~~~--OH
~
C
HO~ ~ oH Sulphuretln
H__~O -"0
H0 H
l--z
MarltlmeUn (3',4',6,7-Tetrahydroxyaurone) OH
H
H
(2',4'-Dlhydroxychalcone)
lsollqulritoslde
0 Sofalcone
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
425
(Fig. 2). contd.....
Y OH
O !
Sophoradin
Fig. (2). Flavonoids with antiulcer activity.
enhance the healing of the lesions [73-75]. More recently, glycyrrhetic acid has also been implicated and its structure has been used as the starting compound for the synthesis of carbenoxolone, an antiulcer substance with cytoprotective properties (Fig. 3). Precisely the steroid structure of both compounds is also responsible for the side effects, edema, hypertension and electrolytic imbalance. COOH
COOH
)H-CH 2
Glyclrretlc acid
Carbenoxolone
Fig. (3). Structural relation between glycirretic acid and carbenoxolone.
Another isoprenyl chalcone, sophoradin, isolated from the root of
Sophora subprostata [76], exhibits an antiulcer effect in both Shay's
pylorus ligated rats [77], and water-immersed and restraint-stressed rats [78]. In addition to sophoradin, isoliquiretin and isoliquiritigenin have been reported chalcones as possessing antiuleerogenic action, and some reports show that an isoprenyl unit enhances this activity [79]. Kyogoku et al. [80] have synthesized thirty new chalcones which are sophoradin analogues. Several of theses having one or two isoprenyl groups, with or
426
MART|N et al.
without a carboxymethyl group, were found to possess antiulcer activity in both experimental models, and their potencies were equal to that of sophoradin (Table 1). In the course of its screening, they succeeded in finding a new amiulccr compound, 2'-carboxymethoxy-4,4'-bis(3-methyl2-butenyloxy)chalcone (Su-88), sofalcone, which has been used clinically in Japan since 1984 with satisfactory results. The main metabolites of this agent had been detected in human plasma and urine [81 ]. Thus, Hatayama et al. [82] undertook the synthesis and the study of the antiulcer effect of these metabolites. This activity was examined in ulcers provoked by Shay's pyloric ligature, water immersion restraint stress, and histamine induction. Two of the main metabolites in human plasma showed a considerable protective effect, and the compound excreted in human urine showed a somewhat weaker activity. Table 1.
Isoprenyl Chalcones with Antiulcer Activity (Kyogoku et al., 1979) 3
4'
6' 6
3'
0
C-2'
C-Y
C-4'
C-5'
C-2
H
H
i
Pr
OH H
I
OH
Pr
OH
H
OH
C-3 |
C-4
C-5
OPr
H
Inhibitory ratio Shay's rats Stress rats i
/ill
H
ill
+++
+++
,,,
OPr
H
++
+++
OPr
H
+++
++
OPr
H
+++
+++
OPr
H
++
+++
H
H
+++
++
i
OH
Pr
OPr
OH
H
..
H
OPr
H
OPr
H
!
i !
OR H
Soforadin
OH
OPr
i
P
H
OPr
H
H
OPr
H
Pr
OH
H
H
OPt
H
+++
+++
OR
H
H
H
+++
+
H
Pr
OH
Pr
+++
+++
I
!
!
|
,
/ Pr:
/ - ~ ~
R: C H 2 C O H
+: 1 1 - 4 0 %
++: 4 1 - 7 0 %
+++: 7 1 - 1 0 0 %
i
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
427
Another synthetic derivative of sophoradine, solon, was studied by Konturek et al. [83] in order to verify the antisecreting and gastroprotective effects on different experimental models: stress and water immersion, acidified aspirin, and absolute ethanol. They found an important cytoprotective effect which was maximum between 60 and 90 minutes after administration. Other authors have shown the antiulcerigenic effect of 2 ' , 4 ' - d i h y d r o x y e h a l e o n e , 2',4'-dihydroxy-3'methoxyehaleone and neohesperidin dihydroehaleone [84]. Bidens aurea is a herbal plant commonly used for its digestive and sedative properties. The antiulcer efficiency of a flavonic fraction obtained from the flowering tops of this species on gastric damage induced by restraint stress, acetic acid, and absolute ethanol has been demonstrated [85-87]. Oral treatment with the ether fraction of the flavonic extract gave a high level of gastric protection, and its effectiveness was comparable to that of ranitidine and omeprazol. The phytochemical analysis of this fraction showed the presence of polyphenolic compounds, mainly aurones and chalcones, and the genins maritimetin and sulfuretin were identified [88, 89]. Thus, it is possible to attribute the gastroprotective effect to auronic and chalconic compounds. Apart from chalconic derivatives, other flavonic compounds, such as flavones, flavanones, bioflavonoids and anthocyanidins, also exhibit antiulcer activity. C i n n a m o m u m cassia (Chinese cinnamon), a species containing numerous flavonic derivatives (epieateehol, epieateehol-O-glueoside and dieyelie-O-glueosides), has been used in traditional Chinese medicine for its analgesic, antipyretic and tonic properties [90]. The aqueous extract showed an effect comparable to cimetidine, a potent antisecretory agent, preventing the ulceration induced by stress and cold, and contrarily to cimetidine, it inhibited the ulceration induced by serotonin. It also reduced the secretion of acid and pepsin and increased the mucosal blood flow. Similar gastroprotective results were obtained in other experimental models, such as in the lesions induced by phenylbutazone and oral administration of ethanol. The species Chamomilla recutita (chamomile) is rich in phenolic compounds, cumarines, phenolic acids and, especially, flavonoids such as 13-D-glyeosyl-7-apigenine and their acetylated derivatives, luteolin glucosides, quereetin heterosides and their methylated genines [90]. Apart from its known antispasmolytic activity, it also protects against ulceration mediated by indomethacin, stress and absolute alcohol. The leaves of Catha edulis also have a high content of polyphenolic compounds, tannins and flavonoids. The isolated flavonic fraction showed a significant antiulcer activity against the lesions induced by phenylbutazone and pyloric ligature in rats [91]. It is proven that the compounds responsible for this activity are the major constituents of the
428
MARTIN et aL
ether extract: kaempferol, quercetin, myricetin and dihydromyricetinrhamnoside. A study on the antiulcer activity of genistein, a flavonoid appearing in several species of the genus Genista, showed its effectiveness in preventing ulcer caused by reserpine and phenylbutazone [92]. This result is in agreement with those obtained by Rainova et al. [93], who found that the mixture of flavonoids extracted from Genisto rumelico (luteolin, luteolin-7-glucoside and genistein) exhibited dose-dependent protection in rats. In a study of five models of classic ulcer (pyloric ligature, stress, reserpine, phenylbutazone and 5-hydroxytryptamine), genistein was the most active. The studied compounds did not show antisecretory effect nor did they induce changes in pH or in the pepsine concentration of the gastric juice. The floral sumity of Dittrichia viscosa, a species abounding in the Mediterranean region, has been frequently used in folk medicine over the years for the treatment of gastroduodenal symptoms. Some studies have demonstrated the antiulcerogenic effect of this species against different experimental models of acute or chronic gastric lesions [94]. Ether extraction showed that the major constituents were quereetin, 3-0methylquereetin, naringenin, and 7-O-methylaromadendrine [95, 96], and a further study was undertaken to determine its gastroprotective effect. The gastric lesions induced by the oral administration of necrotizing agents (100% ethanol, HC1 0.6 N and 30% NaCI) were reduced sharply following an oral pretreatment with the flavonic extract and this protective effect could be related to an increase in gastric mucus as well as to the extract's glycoprotein contents [97]. Reyes et al. [98] have investigated the antiulcerogenicity of the flavonoid fraction (ethylacetate extract) of Erica andevalensis, a species endemic to southern of Spain. Oral treatment with the purified extract of the major flavonoid, myrieetin-3-O-D-galaetoside, was found to be effective against gastric ulceration induced by cold-restraint stress, pyloric ligature, and absolute ethanol in rats. The complex flavonoid-lignan silymarin, a hepatoprotective agent from Sylibum marianum, was effective in preventing gastric ulcer induced by experimental stress (restraint and cold), pyloric ligature and ischemiareperfusion in rats [99, 100].The animals treated with silymarin, showed a significant reduction in both number and severity of the lesions; however, the volume of the gastric secretion was not altered, although the concentration of histamine decreased remarkably. Nevertheless, in ethanolinduced ulcers, pretreatment with s i l y m a r i n did not prevent the formation of lesions. Quereetin, a flavone which has numerous pharmacological effects, including antioxidant [101 ], antiinflammatory [ 102], antithrombotic [ 103], antitumoural [ 104, 105], and antibacterial [ 106, 107] ones, has also been tried in the prevention of ulcers induced by some experimental models
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
429
[108, 109]. The authors showed the cytoprotective properties of this flavonoid and the participation of its antioxidant properties in the prevention of gastric lesions induced by absolute ethanol [110]. Some studies revealed the protective properties of naringenin against the different types of experimental lesions [108, 111, 112]. A time-effect study showed the maximum effectiveness of this flavone to be after 4 hours of oral administration. Rutin, quercetin-3-rhammnosylglucoside, is known for its antiinflammatory and vasoactive properties, diminishing capillary permeability and exerting a vasoconstrictive effect on the peripheral blood vessels [38]. Seeing that gastroduodenal pathology is accompanied by edema and vascular alterations, a new experiment was designed to verify the possible influence of the carbohydrate side chain on the gastroprotective effect [113]. The authors showed that administering rutin before the necrotizing agent, ulceration was essentially prevented, as with quereetin. By contrast, in the same experimental model, ulceration was also prevented by naringin, the glucoside of naringenin, but the effective doses were significantly higher than those of the genine, showing that the inclusion of the sugar side chain in the molecule decreases the gastroprotective activity [ 114]. Another flavonoid, hypolaetin-8-glueoside, found in many species of the genus Sideritis, is traditionally used in Spain for its antiinflammatory and digestive properties [115, 116]. It was isolated from Sideritis leucantha and its antiulcerogenic properties studied by Alcaraz and Tordera [117]. The authors found that this compound significantly reduces the gastric lesions induced by absolute ethanol, acetyl salicylic acid and, Shay's pyloric ligature. Many other flavonic compounds have been reported to exhibit an interesting antiulcer effect: mecyadanol, (+)-cyanidanol-3, (+)-catechin, 3O-methyl-(+)-catechin, 3-methoxy-5,7,3',4'-tetrahydroxyflavan, apigenin7,4'-diorthomethylether, hesperidin, and amentoflavone [ 118-121 ]. MECHANISMS INVOLVED IN THE ANTIULCEROGENIC EFFECT OF FLAVONOIDS Recently there have been extraordinary advances in the understanding of the pathophysiology and treatment of gastrointestinal disorders. However, many questions regarding acid-related diseases remain to be answered. Traditionally, ulceration of the gastric mucosa was regarded as being caused by excessive gastric acid secretion. The significance of HCI in peptic ulcer disease had already been recognized in 1910, by Schwartz, "without acid gastric juice, no peptic ulcer" [122], and became the basis upon which ulcer therapy was designed: anticholinergic drugs, histamine H2 receptor antagonists, antiacids, and more recently, proton pump inhibitors. But also it was admitted that the enhancement of acid secretion
430
MARTIN et aL
alone is unlikely to produce lesions, because many patients with ulcers secrete acid at normal rates. The discovery, in 1979, by Robert [123] of the remarkable ability of prostaglandins (PG) to increase the resistance of the gastric mucosa to damage started research on the role of defensive factors, such as the mucus-bicarbonate barrier, mucosal blood flow, neutralization of free radicals, stabilization of lisosomal membranes or nitric oxide on the gastric mucosal integrity. Numerous causes can contribute to the genesis of peptic ulcer, so the imbalance between the aggressive and defensive factors plays an important role. Aggressive factors include both endogenous (acid, pepsin, gastrin, leukotrienes, free radicals or Helicobacter pylori) and exogenous (food, NSAID, nicotine or stress) mediators (Table 2). Table 2.
Aggessive and Defensive Factors of Gastric Mucosa i
Aggessive factors i
i
,,
i
,
,
Defensive factors i|
9 Acid secretion
9 Mucus-bicarbonate barrier
9 Pepsine
9 Prostaglandins
9 Helicobacter pylori
9 Endogenous antioxidants
9 Electrolytic balance
9 Mucosai blood flow
9 Leukotrienes
9 Stabilization of the mastocyte membrane
9 Oxygen free radicals
9 Regulation of gastrointestinal motility
9 Neutrophil activation and adherence
9 Nicotine
9NSAID
9 Nitric oxide
9 Stress
9 Epithelial restitution i
A series of papers has been published in the last few years about the mechanisms involved in the antiuleerogenicity of flavonoids. These compounds can modify both aggressive and defensive factors.
Aggressive Factors
Acid Secretion Stimulation of parietal cells for gastric acid secretion is a complex process mediated both centrally by vagal parasympathic fibers and peripherically by release of histamine from the fundic mucosal enterochromaffin-like
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
431
cells. Other mediators include at least acetylcholine from the vagus and gastrin from the G cells of the antrum. H2 receptor antagonists are extremely effective inhibitors of acid secretion during pentagastrin stimulation and are able to inhibit partially the secretion due to vagal stimulation. Since neither gastrin nor acetylcholine binds to H2 receptor, it is apparent that histamine must therefore operate on the pathway of stimulation initiated by these mediators. Thus it seems likely that histamine rather than gastrin is the final mediator of acid secretion. Histamine has been involved in the pathogenesis of gastric ulcers produced by restraint stress, pyloric ligature and other methods because it increases vascular permeability and acidity [124, 125]. Senay and Levine [ 126], and more recently Marazova et al [ 127], related the ulcerogenesis induced by immobilization and cold to an increase of histamine synthesis, resulting from stimulation of histidin-decarboxylase activity. This enzyme was found at high concentrations in the glandular mucosa of the rat. In other experimental models, the mechanisms involved in mucosal lesion production are, among others, an enhancement of vagal activity leading to hyperacidity, and an increase of mast cell degradation with a high liberation of histamine. However, the role of this amine against ulcerogenic agents such as ethanol and other necrotizing agents is not clear. Some authors [128] consider the possibility of a gastroprotective effect of histamine, mediated by stimulation of PG synthesis, because it prevents the appearance of the hemorrhagic bands caused by these agents. Palitzsch et al. [129] suggest that these gastroprotective processes might be induced through H3 receptor. Some flavonoids have been reported to possess antisecretory activity in vivo [ 130]. Yamahara et al. [ 131 ] also found that vexibinol, a flavonol obtained from Sphora significantlyn, inhibits basal and 2-deoxy-D-glucosestimulated acid secretion in rats but possesses no or a much weaker effect on acid secretion stimulated by histamine, carbachol or pentagastrin. Saziki et al. [132] reported that sofaleone significantly reduced the volume and acidity of gastric juice in pylorus-ligated rats. Similar results were obtained by Alcaraz and Tordera [117] with hypolaetin-8-glueoside. In the same experimental model, the flavonic extract of Stachytarpheta cayennensis also inhibited the basal acid secretion as well as that induced by histamine and bethanecol, and the authors suggest that this inhibition could be due to the level of histamine release and H2 receptor interaction [ 133]. Other agents such as quereetin, naringenin, (+)-eyanidanol-3 and m e e y a d a n o l are able to inhibit the enzyme histidin-decarboxylase, decreasing the histamine levels in the gastric juice [134-136]. Konturek et al. [83] and later Martin et al. [108] found that this inhibition was not accompanied by a parallel reduction of acidity. Similar results were obtained by Alarc6n et al. [99] when they assayed the antiulcerogenic
432
MARTI[N et aL
effect of silymarin on pylorus-ligated gastric secretion and lesions. The authors suggest that mechanisms other than antisecretory ones are involved in the preventive effect of these agents. The last mediator of gastric secretion in the parietal cell is an H§ +ATPase (proton or acid pump) which is a member of the phosphorylating class of ion transport ATPases. Hydrolysis of ATP results in ion transport. This chemical reaction induces a conformational change in the protein that allows an electroneutral exchange of cytoplasmic H § for K § The pump is activated when associated with a potassium chloride pathway in the canalicular membrane which allows potassium chloride efflux into the extracytoplasmic space, and thus results in secretion of hydrochloric acid at the expense of ATP breakdown. The activity of the pump is determined by the access of K § on this surface on the pump. In the absence of K § the cycle stops at the level of the phosphoenzyme [137]. The high efficacy of proton pump inhibitors (PPI) in the abrogation of acid secretion is based on the fact that their inhibition is independent of the pathway of stimulation. In fact, these agents block acid production in response to all stimulants. It is this specific blockade of the final stage in the complex chain of acid production that enables PPI to avoid many of the clinical problems noted with other antisecretory drugs. Murakami et al. [138], showed that some flavonie compounds, such as the chalcone derivatives xanthoangelol and 4 - h y d r o x y d e r r i c i n from the root of Angelica keiskei are potent inhibitors of the proton pump in vitro. The same authors [139] studied the antisecretory mechanisms of sophoradin, its synthetic chalconic derivative sofalcone, and chalcone. The order of potency in inhibiting the enzyme was sophoradin>sofalcone>ehaleone. This is compatible with the antisecretory activity shown in pylorusligated rats in vivo, in which sophoradin was more effective than sofalcone in reducing the acid output, while chalcone had little effect on acid output [132]. These compounds were also shown to be effective in the in vitro proton transport mediated by H+,K+-ATPase. The ATP hydrolytic sites for the proton pump are located at eytosolic sites and the high affinity K + sites are on luminal face across the membrane [140]. The enzyme is phosphorylated at cytosolic sites by ATP in the presence of Mg 2+. Then the enzyme-phosphate complex is dephosphorylated by luminal K § The kinetic studies carried out by Murakami et al. [ 139, 141 ] demonstrated that the inhibition of the gastric pump by sofalcone, chalcone and quercetin was competitive with respect to ATP and noncompetitive with K § In this way, Beil et al. [142] showed that quercetin, flavone and flavanone locked acid formation in parietal cells in response to histamine and cAMP stimulation, flavanone being the most potent inhibitor. H § K+-ATPase was inhibited by all of them, and this inhibition increased with lowering ATP concentration. The steady-state phosphorylation level of the enzyme was also dose-
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
433
dependently reduced by quereetin. The authors suggest that these flavonoids may bind to ATP sites competitively with ATP and inhibit the formation of intermediate phosphoenzymes, thereby inhibing H+,K+ATPase activity. Although it is unlikely that flavonoids cause inhibition of acid secretion in vivo by blocking the proton pump activity, these results suggest that this mechanism could be involved at least partly.
Helicobacter pylori Since Warren and Marshall, in 1983, isolated Helicobacterpylori from the human stomach, this microorganism began to be considered an important factor in gastric disorders [143]. Now it is accepted to be the cause of chronic active type B gastritis [ 144], and strong evidence suggests that H. pylori infection is a major causative factor for peptic ulcer disease [146, 147]. In fact, H. pylori is present in over 90% of patients with duodenal ulceration. The prevalence of infection in gastric ulcer patients is somewhat lower and more variable, 70-90% [148, 149]. However, probably the most compelling evidence for a causal relationship between H. pylori and peptic ulcer disease is the prolonged remission from reulceration that follows successful eradication of the organism [150]. The exact mechanism by which H. pylori causes peptic ulceration remains unclear, although it is thought to involve mucosal damage mediated by the virulence factors of the organism, mucosal inflammatory response to tissue damage and H. pylori antigens, disturbances in gastrin, somatostatin and acid secretion, and the development of duodenal gastric metaplasia [ 149, 150]. The acceptance that H. pylori infection has a causal role in peptic ulcer pathogenesis has already had a considerable impact in the management of this disease. Today, a combination of antibacterial (amoxicillin, clarithromycin, tetracyclin, metronidazole, tinidazole or bismuth compounds) and antisecretory agents (H2-receptor antagonists or PPI) constitutes the most important pharmacological strategy in the treatment of this disease. The use of these drugs combined in a regime of triple or quadruple therapy achieves eradication rates of around 90% [151, 152]. A recent study carried out by Beil et al. (1995) [142] shows that flavone, flavanone and quereetin inhibited H. pylori growth in a concentration-dependent manner. The most potent compound was flavone, with an antibacterial activity similar to that reported by colloidal bismuth subcitrate. Therefore, from the known digestive properties of flavonie agents, antisecretory action and inhibition of H.pylori growth, it appears that these agents could have a therapeutic potential, ideal for treatment of gastrointestinal disorders associated with H.pylori infection, e.g. type B gastritis and duodenal ulcer.
434
MARTIN et as
Leukotrienes
Leukotrienes (LT) are a class of eicosanoids which derive from the arachidonic acid cascade, via 5-1ipoxygenase (5-LOX), and they are involved as possible mediators of gastric damage and ulceration [53, 154]. Recent investigations have indicated that these agents play an important role as mucosal aggressive factors. In rats, LT have been shown to be released in response to necrotizing agents such as absolute ethanol [ 155( and hydrochloric acid [156]. The exogenous LT have been reported to exert strong action on mucosal microcirculation, they cause potent vasoconstriction, vascular stasis and an increase in vascular permeability [157], and may contribute to the maintenance of the chronic injury, especially if there is inflammatory infiltration. Furthermore, LTC4 and LTB4, although not ulcerogenic themselves, aggravated mucosal injury induced by various noxious agents or non steroidal anti inflammatory drugs, NSAID [ 154, 158, 159]. A number of reports have suggested that LT synthesis inhibitors, LTB4 receptor antagonists, and LTD4 receptor antagonists might protect against the formation of gastric lesions, and therefore they may be considered as possible clinical agents [158,160, 161 ]. Several flavonoids have been shown to inhibit 15-LOX activity from soybean, 12-LOX from blood platelets [2] or 5-LOX from leukocytes [1, 162]. Some authors have established that the 3',4'-diol substitution in the B ring (catechol) is the most important requirement for the inhibition by flavonoids of arachidonate lypoxygenases [163, 164]. These conclusions were supported by the results obtained by Moroney et al. [1], and they confirm that an additional hydroxyl substituent in the body of the molecule confers the most potent and selective inhibitory effect of 5-LOX. By contrast, the glycosylation considerably reduces potency. In this study, quereetin (3',4',5,7-tetrahydroxyflavonol), rutin (quercetin-3glucoside), n a r i n g e n i n (4',5,7-trihydroxyflavanone), h y p o l a e t i n (5,7,8,3',4'-pentahydroxyflavone), and hypolaetin-8-glueoside were found to be selective 5-LOX inhibitors, and the order of potency was quereetin>hypolaetin>naringenin>rutin>hypolaetin-8-glueoside. All of them exhibit antiulcerogenic effect. Thus, it is possible that this property could be due, at least in part, to inhibition of LT synthesis. However this supposition requires further investigation. Oxygen Free Radicals
Oxygen-generated free radicals have been shown to be implicated in many pathophysiological conditions and in the toxicity of xenobiotics.They provoke severe changes at cellular level leading to cell death, because owing to their extreme reactivity, they attack the essential cell constituents, such as nucleic acids, proteins or lipids. They also induce peroxidation of the
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
435
membrane lipids and this action lead to the formation of a series of toxic compounds, such as epoxides, aldehydes, and new free radicals. An important intermediate in biological actions is the superoxide anion radical, which is formed in vivo during the reduction of molecular oxygen. This species can produce hydrogen peroxide (H202) a highly toxic product, which in turn gives rise to the hydroxyl radical (.OH) by reaction with transition metal ions in the body. The (.OH) radical is very reactive and one of the strongest oxidizing agents [165]. On the other hand, gastric mucosal injury has also been associated with periods of ischemia in several different clinical settings, including trauma [166], ethanol exposure [167], acetic acid [168], NSAID [169], or ischemia-reperfusion [170]. When a tissue is subjected to ischemia, a sequence of chemical reactions is initiated which may ultimately lead to cellular dysfunction and necrosis [ 171]. Although no single process can be identified as the critical event in ischemia-induced damage, most studies indicate that depletion of cellular energy stores and accumulation of toxic metabolites may contribute to cell death. Sources of reactive oxygen metabolites include the xanthine oxidase system, which is modified during ischemia, since it produces the superoxide anion radical and H202 during reperfusion. These oxygen radicals may then be converted to the highly cytotoxic hydroxyl radical by the iron-catalyzed Haber-Weiss reaction. This initiates the process of lipid peroxidation which, in turn, results in the production and release of substances that recruit and activate polymorphonuclear leukocytes [172]. The substances which are able to hinder their formation or with capacity to capture the formed free oxygen radicals are thus potential antiulcerogenic agents. It has been known for a long time that most of the flavonoids and their derivatives are principles capable of capturing free oxygen radicals, peroxides of fatty acids as well as hydroxyl groups. The capture of superoxide free radicals has been evidenced by the formation of trans-trans hydroperoxides of linoleic acid by flavonoids [173, 174]. The capture of hydroxyl radicals responsible of numerous cell degradations is what indicates its relation with factors of cellular protection. Letan [175] proposed a series of structural requirements that flavonoids had to fulfil in order to have antioxidative action: one free OH group in position 3 one double bond between carbons 2 and 3 one keto group in position 4 of the pyrone ring free OH groups in positions 5 and 7 free OH groups in positions 3' and 4' In this context, the sugar moiety masks the antioxidant activity of flavonoids. A possible explanation for this could be that the lower
436
MARTIN et aL
lipophilicity of the glycoside prevents its access to the lipid membranes, the site of lipid peroxidation. This was evident when Rekka and Kourounakis investigated the effect of 7-mono-,7,4'-di-,7,3',4'-tri and 5,7,3',4'-tetra-hydroxiethyl rutosides and 7,3',4'-tri-hydroxiethyl quercetin on non-enzymatic lipid peroxidation in vitro. In this study quereetin was found to be the most antioxidant [ 176]. Interactions between a number of flavonoids, and superoxide anion have been described by many authors [177, 178, 179]. In contrast, only a few data are available relating to the hydroxyl radicals scavenging activity of flavonoids. Cillard and Cillard in 1988 [180] reported the percentage of scavenging of the hydroxyl radical produced by the decomposition of hydrogen peroxide or peroxides of fatty acids such as linoleic acid identified by HPLC. In that study, some antiulcer flavonoids as quereetin and naringenin, exerted a scavenging of about 48% and 36% respectively. More recently, interesting studies also reported the strong hydroxyl radical scavenging capacity of rutin [181 ] and silymarin [48]. There are many studies about the relation between the free oxygen radical scavenging activities of many flavonoids and their effects on the enzymes which take part in the metabolism of arachidonic acid, cyclooxygenase (COX) and lipoxygenase (LOX). Duneic [182] shows that the antiradical action affects the COX activity in several ways. In most of the cases, at high substrate concentrations the enzymatic activity was intensified and at low concentrations it was inhibited. Apparently, the influence of the antiradical properties on the activity of enzymatic metabolism of the arachid0nic acid in vitro might also be due to the effect of these agents on the active center of the enzymes. Robak et al. [ 183, 184] studied a good amount of flavonoids, analyzing their influence on COX and LOX activity, and evaluating their antioxidant properties. They showed that most of them stimulated COX. Rutin and quercetin exerted a strong stimulating action, while naringenin, naringin and hesperidin did not stimulate COX. In addition, quereetin, naringenin and silymarin were found to be relatively selective inhibitors of 5-LOX [ 1, 185-187] inhibiting therefore the biosynthesis of LT. A number of papers show that among flavonoids there are strong scavengers of lipidic radicals [33, 34]. For example, quercetin and Gingko biloba extract show an important antilipoperoxidant action or a weak effect (rutin and kaempferol), moreover hesperidin increases the rate of autoxidation. The discrepancy between the activity of these flavonoids seems to be important in explain the different effects of, for example, quercetin (inhibitor) and rutin (stimulator) on the cell COX activity. It is, in fact, important to remember that COX activity includes lipoperoxidative steps and thus it is inhibited by antilipoperoxidant agents. Chelating activity gives another possibility to flavonoids to interact with the oxidation of membrane lipids, and it is considered important for
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
437
their inhibitory action on microsomal lipid peroxidation induced by an iron dependent system [33]. On the other hand, there is growing evidence for the role of the nitric oxide radical (.NO) in vascular endothelial damage. It is speculated that (.NO) scavenging activity may contribute to the therapeutic effects of the flavonoids. On the basis of this hypothesis,recently the (.NO) scavenging capacity of certain flavonoids, as hydroxyethylrutosides, has been investigated by Van Acker et al. [ 188]. Neutrophil Activation and Adhesion Free oxygen radicals produced in gastric pathology not only come from the affected tissue, but they can also be originated in several biological reactions, pointing up the activation of neutrophils in the extracellular space. Recently, the degree of neutrophil infiltration in the gastric mucosa has been related to the genesis of the lesions [189, 190]. Neutrophils contain an NADPH oxidase which reduces molecular oxygen to the superoxide anion radical and these leukocytes are the primary mediators of reperfusion-induced increases in microvascular permeability. The leukocytes are attracted by the chemotactic factors liberated in the states of ischemia. These chemotactic factors favor the gastrodamaging process and the way they release phagosomes full with oxygen free radicals into the environment. These endogenous agressors stimulate the mucosal parenchyma and many types of cell to produce the inflammatory damage: Mastocytes are stimulated and release: histamine, leukotrienes (LT), interleukin-1 (IL-1), platelet activating factor (PAF), proteases and peroxidases. The endothelial cells of the capillaries liberate LT, nitric oxide (NO) and endothelin- 1. The neutrophils adhere to the endothelium, block the capillaries and damage the endothelial integrity. Activated neutrophils produce reactive oxygen metabolites and release a variety of cytotoxic proteins, e.g. proteases, lactoferrin, and they also secrete the enzyme myeloperoxidase (MPO) which catalyzes the formation of potent cytotoxic oxidants such as hypochlorous acid (HOCI) from H202 and chloride ions and N- chloramines [172]. The inflammatory mediators cause mucosal ischemia and an increase of the capillary permeability affecting the fluid which is extravasated to the interstices, causing mucosal edema. Neutrophils and erythrocytes are moved to the extravascular compartment. The epithelial surface appears hemorrhagic. The mucosal ischemia extends, microcirculation gets
438
MARTIN et aL
congested with stagnant blood, the tissue becomes hypoxic and necrotic cells appear even in the deepest layers of the mucosa. It is evident that agents capable of inhibiting the activation of these granulocytes could be also evaluated as potentially antiulcerogenic. Recent investigations seem to confirm this hypothesis. As shown by Alarc6n de la Lastra et al. [100] using a model of gastric injury induced by ischemia-reperfusion in rats, pretreatment with silymarin, the hepatoprotective principle of Silybum marianum L. prevented post-ischemic injury. These protective effects were specifically related to reduction of MPO activity as index of polymorphonuclear leukocyte infiltration after injury. These findings indicated that inhibitory effects of silymarin on neutrophil function seem to contribute significantly to its gastroprotective actions. By contrast, the inhibition of neutrophil infiltration did not appear to be involved in the antiulcer effect of quercetin in gastric mucosal injury induced by 50% ethanol, an experimental model in which there is a considerable leukocyte influx into the gastric mucosa [ 110]. Treatment of human endothelial cells with cytokines such as interleukin-1, tumoral necrosis factor-alpha (TNF-alpha), or interferongamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation may support a role in maintaining gastric mucosal integrity. A recent experimental study indicated that one of the most potent flavones, apigenin, exhibited a dose and time-dependent, reversible effect on adhesion protein expression, as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited TNF-alpha- induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression [ 191]. By the same token, a flavonoid, 2-(3-amino-phenyl)-8-methoxychromene-4-one, markedly inhibited TNF-alpha induced vascular cell adhesion molecule-1 (VCAM-1) in a concentration-dependent fashion of human aortic endothelial cells, but had no effect on intercellular adhesion molecule- 1 (ICAM- 1) [ 192]. A number of experimental studies have been performed looking at the gastroduodenal damaging effects of non-steroidal antiinflammatory drugs (NSAID) such as indomethacin [193]. In these studies, orally administered 5-methoxyflavone inhibited indomethacin-induced leukocyte adherence to mesenteric venules, suggesting a role of inhibition of leukocyte adherence in gastroprotective activity of this flavonic compound.
Defensive mechanisms In addition to antisecretory mechanisms, the enhancement of the defensive mucosal factors seems to play an important role in the antiulcer effects of
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
439
the flavonoids. The term "mucosal defense" refers to the factors that permit the mucosa to withstand frequent exposure to substances with a wide range of pH, osmolarity and temperature, and to noxious agents and bacterial products capable of causing local and systemic inflammatory reactions. However, the mucosa can repair such injury quickly, thereby limiting it to the most superficial layer of cells and preventing entry into the systemic circulation of substances detrimental to the organism. Their resistance can also be enhanced when irritants are present in the stomach. Thus, the ability of the mucosa to resist significant injury is attributable to a dynamic process rather than to a static barrier. Although numerous flavonic substances present gastroprotective properties, the mechanisms involved in mucosal defense, are not sufficiently explained and, naturally, they are not the same for each flavonoid. Mucus-bicarbonate Barrier
Morris et al. [194] demonstrated that a primary effect of many cytoprotective agents may be stimulation of mucosal secretion, resulting in the formation of a barrier that may attenuate the damaging effects of necrotizing agents. The role of the so-called mucus-bicarbonate barrier in protecting the mucosa from injury induced by acid and pepsin is one of the most controversial aspects of mucosal defense. The mucous gel is proposed to form a continuous "blanket" over the mucosal surface, which traps bicarbonate secreted by the epithelium and therefore acts as a layer in which luminal acid that diffuses toward the epithelium is neutralized. In addition, the mucus plays an important role in restricting the bacterial movement to the surface epithelium; bacteria become trapped in the gel and are eventually excreted in feces. It is also capable of acting as an antioxidant, and so can reduce mucosal damage mediated by these factors produced by bacteria or immunocytes [ 195, 196]. An additional element in mucosal defense is the coupling of defensive and aggressive factors, so that mucosal protective mechanisms are enhanced when acid secretion is occurring. In this way, some authors [ 197, 198] attributed mucus hypersecretion in cases of severe damage to an autodefense mechanism of increased synthesis in the intact glandular zones, to aid restitution of the lesioned areas. The viscous and gel-forming properties of mucus secretions are derived from mucin glycoprotein constituents (5%); they also contains protein, lipid and nucleic acid, much of it derived from dislodged epithelial cells and bacteria [ 199]. These constituents have been shown to enhance the viscous properties of mucins in vitro [200, 201], and such interactions may be significant towards determining mucus gel properties in vivo. Considering the recorded data, we can assert that the role played by gastric mucus in the cytoprotection mediated by flavonic substances is
440
MART|N et at
variable. Quercetin, naringenin, hypolaetin-8-glucoside, solon and sofalcone [202, 203] provoke a clear stimulation of mucus synthesis and secretion, also enhancing the glycoprotein content with an increase of total proteins, hexosamines, neutral glycoproteins and sulfated macromolecules [109, 112, 117]. The same results were obtained from the flavonic fraction of Bidens aurea [86, 89] and Dittrichia viscosa [97]. A rheological study showed that preparations of adherent gastric mucus gel scraped gently from the mucosal surface of rats treated with naringin had significantly increased viscoelastic properties [114]. These findings seem to indicate that the enhancement of mucus characteristics is related to the gastroprotective effects of these compounds. Concerning ulcers induced by absolute ethanol, pretreatment with silymarin stimulated mucus secretion, but did not modify the concentration of total proteins and hexosamines [99]. By contrast, in the same experimental model, Reyes et al. [98] found that mucus amount was not modified by the ethylacetate extract of Erica andevalensis, although there was an increase in the concentration of its components. P6rezGuerrero et al. [113] showed that rutin, the glycoside of quercetin, has a protective effect, although it did not induce any changes in the amount of mucus or in its glycoprotein content. These results confirm that the mucus layer alone is incapable of protecting the underlying mucosa [204]. It is necessary that other defensive mechanisms, such as bicarbonate secretion by the non-parietal cells [205], the restoration of vascular factors [206], or the inhibition of leukocyte adherence [198], come into play. Prostaglandins
In comrast to LT, many natural prostaglandins (PG), particularly of the E series, inhibit gastric acid secretion. However, over and above antisecretory activity, these agents also share the property of "cytoprotection". This term was proposed by Robert in 1979 [123] to account for the ulcer-reducing effect of prostaglandins by a mechanism other than inhibition of gastric secretion. Gastric secretory inhibition is in itself somewhat protective, so separation of these two effects is important. Numerous studies evidence the role and the influence on the gastric defensive mechanisms of these mediators [92, 207-209]. There is evidence that PG may have a physiological role in regulating mucosal mucus; moreover they increase gastric non-parietal cell secretion and duodenal bicarbonate secretion, and improve the lysosomal and vascular integrity [210]. The stabilizing effects of PG on the microvasculature may be particularly important, since it appears that one of the first abnormalities in the pathogenesis of mucosal injury is an increase in microvasculature permeability. Finally, it has been shown that PG limit the depth of mucosal destruction in injury induced by absolute ethanol,
ANTIULCER AND GASTROPROTECTIVE ACTIVITY'
441
permitting more rapid restitution of the damaged superficial epithelium, and they normalize the gastric secretion which was inhibited after administering the necrotizing agents. These findings, described by Robert et al. [211 ], show that prostaglandins maintain the morphologic integrity of parietal cells even in the presence of this class of agents. The antiulcer effect of some flavonoids might be related to their ability to increase the mucosal levels of these prostanoids. This is the case of solon, which inhibits the metabolizing enzyme 15-OH-PG-DH 15hydroxy-prostaglandindehydrogenase [83]. The cytoprotective mechanism would be similar to the one of carbenoxolone, a drug frequently used in experimental models because of its ability to stimulate the mucosal defenses. Some flavonoids have been shown both to inhibit and to stimulate production of prostaglandins in vitro [28, 212]. Flavone and flavanone induced PGE2 production in isolated gastric mucosal cells, but since these compounds did not stimulate the prostanoid production in gastric cells exposed to arachidonic acid, it is likely that both flavonoids enhance prostaglandin formation by acting as cofactors of cyclooxygenase [ 142]. Other flavonoids that are able to enhance the luminal release of PG in vivo are the glucoside of hypolaetin [ 117], the anthocyanidin pigment IdB 1027 [213], and the flavonic extract of Bidens aurea [214]; therefore their antiulcer effect could be related to an enhancement of PGE2 levels in gastric mucosa and also to a generalized stimulation of the defensive mucosal mechanisms. Alarc6n et al. [ 109] designed an assay to verify the possible role of the mucosal prostaglandins in the antiulcerogenic effect of quereetin on ethanol-induced gastric lesions in rat. The animals were pretreated orally with this flavonoid before administering the ulcerogenic agent, and the degree of alcohol-ulceration was notably reduced. The total amount of mucus as well as its content in glycoproteins increased.When indomethacin was administered subcutaneously to the animals pretreated with quereetin, the protective effect reverted partially, the intensity of the lesions increasing and the synthesis of mucus and concentration of hexosamines decreasing. This may point to a possible participation of prostaglandins in this effect. However, Beil et al. [142] found that quereetin was not effective in stimulating PGE2 production in vitro. These data are in agreement with those of Alcaraz and Hoult [6], who showed that this flavonoid has no effect on prostaglandin formation in fragments of rat caecum. Similar results were obtained with naringenin on mucosal lesions induced by necrotizing agents [112]. Therefore, the antiulcerogenic effect does not seem to be mediated through the cyclooxygenase pathway, because in this experimental model, this flavonoid does not increase the synthesis of mucosal PGE2. Rather, the gastroprotection induced by quereetin and narigenin might be related to
442
MARTIN et aL
an enhancement of mucus secretion and enrichment of its glycoprotein content. Endogenous Sulfh ydryls
Sulfhydryl compounds (SH), like PG, are endogenous cytoprotective agents. SH-containing compounds, and also agents that modify SH groups, oxide SH groups or bind SH groups, prevent the acute hemorrhagic erosions caused by ethanol, nonsteroidal antiinflammatory drugs or stress in animal models [215, 216]. Depletion of GSH results in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased GSH consumption. In contrast, a rise in gastric SH levels limits the production of oxygen-derived free radicals, and could be related with cellular protection [217]. In addition to free-radical scavenging activity, SH compounds, as well as PG, may maintain a good blood flow that allows the energydepen-dent rapid restitution to cover initial epithelial surface damage [218, 219]. Some experimental studies have demonstrated that flavonoids, such as quereetin [ 110] and the flavonic extract from Bidens aurea (Aiton) Sherff [89] prevented the gastric necrosis induced by 50% v/v ethanol, and produced a significant enhancement of gastric mucosal SH content. Therefore, it was assumed that endogenous SH contributed to the functional mechanisms of protection by flavonoids in this experimental model. Blood Flow and Nitric Oxide
Gastric mucosal flow plays a central role in preventing damage to the gastric lining.The remarkable effectiveness of this vascular defensive system occurs at all levels of gastric tissue organization, from the gross arterial anastomotic network to the ultrastructure of mucosal capillaries and collecting venules. This is because it supports the defensive mechanisms by supplying oxygen and fuel to the mucosal cells involved in maintaining the transepithelial barrier against HCI. Blood flow also removes metabolic waste and CO2 resulting from mucosal metabolism and from neutralization of influxing acid. In addition, mucosal blood flow is critically important in helping control intratissue pH, because it is the vehicle by which bicarbonate is transported into the superficial mucosa and by which excess protons are removed from the tissue [220]. Besides protecting mucosal tissue from infiltrating gastric acid, mucosal blood flow constitutes an important aspect of tissue resistance to nonspecific chemical assaults [221]. Toxic agents directly injure the mucosal cells and also stimulate the contraction of smooth vascular muscle, slowing the mucosal blood flow and congesting the
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
443
microcirculation at this level. The available data show that stasis of blood flow and increased vascular permeability reflect early pathogenic factors in the development of toxic agents such as ethanol on gastric hemorrhagic erosions [167, 222]. In less than 3 minutes after a necrotizing insult, two circulatory responses become evident. These responses are (a) vasospasm of venules and dilation of arterioles reflecting vascular congestion and slowing or stasis of blood flow, and (b) increased capillary permeability to the macromolecules of, plasma, which causes a net flux of fluid out of the plasma into the interstitium [223]. It is obvious that agents capable of preventing an increased microvascular permeability and of restoring an adequate blood flow in the affected areas, might contribute to regeneration of the lesions. This is the case of the flavonoids quereetin and naringenin which, in a model of chronic ulcer induced by 5% acetic acid in rats [ 111 ], induced an important proliferation of blood vessels in the internal area of the ulcerative formation, revealing that the enhancement of angiogenesis could restore the damaged microcirculation. Recently, Blank et al. [193], using laser doppler flowmetry have demonstrated the vascular mechanisms of 5-methoxyflavone-induced protection against acute gastric damage induced by a nonsteroidal antiinflammatory drug (indomethacin) in rats. The finding that this flavonoid significantly increased gastric vascular perfusion suggested that these effects could contribute to the flavonoid's gastroprotective activity. In this regard, several investigators have proven that some flavonoids show interesting effects upon the wall of the smallest blood vessels and particularly on the perivascular tissue. This is the case of Erica andevalensis, whose ethyl acetate extract showed a marked dosedependent effect on the mucosal vascular permeability against the proinflammatory mediator histamine [98]. Furthermore, pretreatment with the flavonic extract of Oxyris quadripartita significantly diminished the increase in the mucosal permeability induced by bradykinin in rats, evidencing a marked antiedema and vasoprotective action of this compound. These results confirmed that Oxyris quadripartita exerted protective mucosal activity through vascular mechanisms [224]. In addition, recent experimental studies indicated that the flavonic extract obtained from the flowers of Bidens aurea exerted antiulcer activity on ethanol-induced mucosal damage through a complex mechanism involving a significant inhibition of the enhanced vascular permeability in the gastric mucosa [89]. Thus, data are available which explain the critically important role played by gastric blood flow in the mechanisms of flavonoid-induced gastroprotection in experimental gastric ulcer. Furthermore, it is abundantly clear that significant advances have been made in the knowledge of the physiology of nitric oxide over the last few years. There
444
MARTIN et aL
are numerous experimental studies revealing the important role of this endogenous vascular-active substance in the mechanisms of gastroprotection [225, 226]. Nitric oxide itself, as well as known releasers of nitric oxide, protects significantly against ethanol- and HCl-induced gastric damage, and this effect is not altered by indomethacin pretreatment, suggesting mechanisms independent of those involving prostaglandins which are also known vasodilators [223]. Nevertheless the role of nitric oxide in flavonoids antiulcer activity has not yet been investigated, and further studies are required in this line. FLAVONOIDS AND APOPTOSlS Apoptosis is a controlled form of cell death that serves as a molecular point of regulation for biological processes. Cell selection by apoptosis occurs during normal physiological functions as well as in toxicities and diseases. Apoptosis is the counterpart and counterbalance to mitosis in cell population determination. Complex patterns of cell signaling and specific gene expression are clearly involved in the control of cell fate. Exposure to an apogen, a trigger of apoptosis, can significantly increase apoptotic cell loss during homeostatic processes as well as in acute or chronic toxicities. Examples of apogens are numerous, and include endogenous regulatory proteins and hormones as well as xenobiotic chemicals, oxidative stress, anoxia, and radiation. This can lead to inappropriate survival and pathological accumulation of aberrant cells [227]. Alternately, suppression of apoptosis through, for example, interference in cell signaling, can result in pathological accumulation of aberrant cells and diseases such as tumors [228]. The process of controlled cell death during development has been recognised for over a century [229], and is often described as "gene-directed cell death" because it is an integral component of development involving gene-directed steps [230]. Researchers originally distinguished apoptosis as a mode of cell death distinct from necrosis based on morphological criteria [231 ]. Apoptosis was recognised to occur under certain pathological conditions e.g., viral hepatitis as well as under physiological ones e.g., atrophy of the postlactational breast. In contrast, necrosis never occurs under physiological conditions and is a common consequence of the severe insults frequently studied by toxicologists. Although apoptosis was defined two decades ago, necrosis is the mode of cell death most familiar to biological scientists, except perhaps embryologist. Diseases characterized by the accumulation of cells include cancer, autoimmune diseases, and certain viral illnesses. Cell accumulation can result from either increased proliferation or the failure of cells to undergo apoptosis in response to appropriate stimuli. Although much attention has been focused on the potential role of cell proliferation in these so-
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
445
called proliferative disorders, increasing evidence suggests that alterations in the control of cell survival are important in their pathogenesis [232]. It is well known that many diet products, including flavonoids, reduce the risk of cancer, and recently some authors have tested the effect of flavonoids on various cell lines. Quereetin is one of the most abundant natural flavonoids, being present in various common vegetables and fruits [232]. As a semi-essential metabolite in plant life, quercetin was considered to be an inert, nonessential component of the human diet. It was assumed that quercetin had no specific effect on human health. However, following the discovery in 1977 that quercetin was mutagenic in the Ames and other short-term in vitro tests, interest in this agent increased. Growing concern for human safety regarding dietary quercetin was prompted a few years ago by reports that the flavonoid caused an increase in the incidence of different tumors. A number of long-term, well controlled feeding experiments with rats, mice and hamsters followed. These studies did not confirm the reported carcinogenity of quercetin. Instead, they indicated that quercetin might exert a protective effect in reducing the number of spontaneous tumors or the incidence of chemically induced ones [234]. Meanwhile, quercetin, together with other flavonoids, became the subject of intensive investigations not only by toxicologists and nutritionists, but also by immunologists, enzymologists, pharmacologists, and other medical scientists. In several in vitro experiments various flavonoids showed growth-inhibitory effects on cells from various human cancers: colon, breast, ovarian, gastrointestinal, and leukemic cells [104, 235-239]. It appears that a number of the biological effects of quercetin and other flavonoids may be explained by their antioxidative activity and ability to scavenge free radicals. Some flavonoids may act extracellularly, others intracellularly, and still others by both mechanisms and simultaneously at various sites. More than a dozen different mechanisms for the protective effect of flavonoids as anticarcinogens have been suggested. Because they are antioxidants, it is likely that their antioxidative property is, at least in part, responsible for their reported anticarcinogenic potential. Other mechanisms include their capacity to scavenge free radicals, to chelate, to block or trap ultimate carcinogen electrophiles by forming innocuous products in a nucleophilic chemical reaction, to inhibit the promotion phase of carcinogenesis, and to modulate the balance between activation and inactivation processes of specific enzymes in the liver [42]. Other authors have demonstrated that flavonoids inhibit the growth of malignant cells through mechanisms including inhibition ofglycolysis, macromolecule synthesis and enzymes, freezing the cell cycle, and interaction with estrogen type II binding sites synthesis [240, 241 ]. Wei et al. [242] have recently indicated that quercetin displays antitumor activity by triggering apoptosis and the agent was found to
446
MARTIN et aL
increase the amount of cells at G~ and S without affecting total cell quantity. Other studies have shown that flavonoids specifically inhibit the synthesis of heat shock proteins at the level of mRNA accumulation and transcription without affecting the other synthetic processes [241 ]. Heat shock proteins are known to play an important role in the protein metabolism and survival of cells [243]. The data indicated that quercetin induces apoptosis in tumor cells through inhibition of heat shock proteins and expression. Several reports have indicated that topoisomerase-II inhibitors induce apoptosis in thymocytes and other cell types [244]. Many agents have been reported to trigger the apoptotic program in thymocytes. Schneider et al. [234] found that the isoflavonoid compound genistein induces apoptosis in a distinctive human subpopulation through the inhibition of topoisomerase-II. At the gastrointestinal level, detailed studies by Potten and colleagues of epithelial cell kinetics in the mouse gastrointestinal tract have shown that apoptosis is rare in normal epithelium one apoptotic body being seen every 5 th crypt [245]. The paucity of apoptotic cells on histological section reflects the rapid kinetic of apoptosis and the removal of apoptotic cells by phagocytosis. This, and an absence of inflammatory response, probably explain why apoptosis went unnoticed for so long. Apoptosis has been shown in a number of diseases of the gastrointestinal tract 9 Excessive apoptosis 9Melanosis coli, Shigella flexneri dysentery, graft versus host disease, AIDS, inflammatory bowel disease. Defective apoptosis 9carcinogenesis. Neither is it known whether apoptosis plays an important part in disease pathogenesis nor whether apoptosis is merely part of the normal clearance of damaged cells. However, the main importance of apoptosis in intestinal disease concerns carcinogenesis, and hence the potential of treatment for cancer. It is now realised that, in general, anticancer agents do not kill by necrosis but rather by causing sensitive cancer cells to commit suicide by the induction of apoptosis [246]. Recently the effect of different flavonoid compounds (bioehaninA,
daidzein, genistein, genistin, pruneetin, puerarin and pseudobaptigenin) on cell proliferation of various cancer cell lines derived from the gastrointestinal tract has been demonstrated [247]" Stomach cancer: HSC-39K6, HSC-40A, HSC-41E6, HSC-42H, HSC-43C1, HSC-45M2, SH101-P4. Esophageal cancer : HEC-46R1. Colon cancer: HCC-48B2, Hcc-50D3. Fibroblast 9ST-Fib, ST-Fib2.
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
447
The results suggest that two of them, bioehanin A and genistein, inhibit the growth of stomach cancer cell lines in vitro through activation of a signal transduction pathway for apoptosis. The authors hypothesized about the mechanisms involved. It was shown that tyrosine kinase inhibitors, such as erbstatin and tyrophostins, had an antineoplasic effect against human tumors of mammary, oesophageal, and maxillary origins in xenograph systems [248, 249]. In the study, they examined the antineoplasic effect of biochanin A or genistein upon athymic nude mice bearing human stomach tumors. Biochanin A significantly inhibited HSC45M2 and SH10 l-P4 tumor growth. Data suggested that biochanin A may serve as useful anticancer drug. However, growth of HSC-41E6 tumor was not suppressed by the flavonoid, suggesting that action of this compound is cell-type specific. Much more needs to be learnt about cell death in the gastrointestinal tract. A major challenge is to understand fully the factors that regulate apoptosis and to develop therapies that can manipulate apoptosis for the treatment of cancer. CONCLUSIONS The flavonoids comprise a large group of naturally occurring low molecular weight substances found in essentially all plant parts. They have been extensivel studied and reported to possess widespread biological activities. They possess a high capability to interact with important cellular processes mediated by different classes of enzymes. Other interesting studies also reported the capacity of some flavonoids to interact with active-oxygen species since they are strong scavengers of free radicals. These properties, together with the anti-inflammatory activity, could be involved in the antiulcerogenic effect of flavonoids. Some of them are inhibitors of aggressive mechanisms of the gastric mucosa, such as acid secretion, H. pylori, leukotrienes or neutrophil infiltration, and a parallel enhancement of defensive factors, such as enrichment of mucus gel, release of prostaglandins and endogenous sulfhydryls, restoring the mucosal blood flow or preventing the increase of microvascular permeability. The data reported in this review show that the inclusion of the sugar side chain in the molecule reduces both antioxidant and gastroprotective activities. Therefore, from the known digestive properties of flavonic agents, it appears that they could have a therapeutic potential for treatment of gastrointestinal disorders. At the same time, it has been reported recently that some flavonoids inhibit the synthesis of heat shock protein, which plays an important role in the protein metabolism and survival of cells. Quercetin, genistein and other flavonic agents induce apoptosis in different tumor cells through inhibition of heat shock protein and expression. Although these findings could suggest a possible anticancer therapy, it is necessary to learn much
448
MARTIN et aL
more about the effect of flavonoids on cell death in the gastrointestinal tract. ACKNOWLEDGEMENTS The autors are grateful to B.Berenguer for helpful assistance in the elaboration of the manuscript. RE~REN~ [1]
Moroney, M.A.; Alcaraz, M.J.; Forder, R.A.; Carey, F.; Hoult. R.S.d. Pharm. Pharmacol., 1988, 40, 787. [2] Robak, J.; Shridi, F.; Wolbis, M.; Kr61ikowska, M. Pol. J. Pharmacol. Pharm., 1988, 40, 451. [3] Laughton, M.J.; Evans P.J.; Moroney, M.A.; Hoult, J.R., Halliwell, B. Biochem. Pharmacol., 1991, 42, 1673. [41 Grupe, R. Agents Act., 1991, 32, 79. [5] Hoult, J.R., Moroney, M.A.; Pay~t, M. Methods Enzymol. 1994, 234, 443. [61 Alcaraz, M.J.; Hoult, J.R.S. Arch. Int. Pharmacodyn., 1985, 278, 4. [71 Ban, M.; Tonal, T.; Kohno, T.; Matsumoto, K.; Horie, T.; Yamamoto, S.; Moskowitz, M.A.; Levine, L. Stroke, 1989, 20, 248. [8] Ferrandiz, M.L.; Ramachandran Nair, A.G.; Alcaraz, M.J. Pharmazie, 1990, 45, 206. [91 Kalkbrenner, F.; Wurm, G.; Bruchhausen, F. Pharmacology, 1992, 44, 1. [lo] Abad, M.J.; Bermejo, P.; Villar, A. Gen. Pharmacol., 1995, 26, 815. [11] Ito, M.; Moriyama, A.; Matsumoto, Y.; Takaki, N.; Fukumoto, M. Agric. Biol. Chem., 1985, 49, 2173. [12] Ito, M.; Ono, Y.; Kai, S.; Fukumoto, M. J. Nutr. Sci. Vitaminol., 1986, 32, 635. [13] Constantini, L.; Albasini, A.; Rastelli, G.; Benvenuti, S. Planta Med., 1991, 58, 342. [141 Chang, W.S.; Lee, Y.J.; Lu, F.J.; Chiang, H.C. Anticancer Res., 1993, 13, 2165. [151 Gil, B.; Sanz, M.J.; Tereneio, M.C.; Ferrandiz, M.L.; Bustos, G.; Payfi, M.; Gunasegaran, R.; Aicaraz, M.J. Life Sci., 1994, 50, 333. [16] Gil, B.; Sanz, M.J.; Terencio, M.C.; Gunasegaran, R.; Paya, M.; Alcaraz, M.J. Biochem. Pharmacol., 1997, 53, 733. [17] Cazenave, J.P.; Beretz, A.; Anton, R. In Flavonoids and Bioflavonoids; L. Farkas; M. Gabor; F. Kallay, Ed.; Elsevier : Amsterdam, 1986; pp. 373 -380. [18] Ferriola, P.C.; Cody, V.; Middleton, E. Jr. Biochem. Pharmacol., 1989, 38, 1617. [19] Kuppusamy, V.R.; Khoo, H.E.; Das, N.P. Biochem. Pharmacol., 1990, 40, 397. [20] Vlietink, A.J.; Vanden Berghe, D.A.; Haemers, A. In Plant Flavonoids in Biology and Medicine II. Biochemical, Cellular and Medicinal Properties; V. Cody; E. Midldleton Jr.; J.B. Harbome; A. Beretz, Ed.; Alan R.Liss: Now York, 1988; pp.283 -299.
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
[21] [221 [23] [24]
[25] [26] [27]
[28] [29] [30] [311 [32] [33]
[34] [351 [36] [37]
[38] [39]
[40] [41] [42] [43] [44]
[45] [46] [47]
[48] [49]
[50] [52]
[53] [541
449
Ono, K.; Nakane, I.; Fukushima, M.; Chermann, J.C.; Barr(~-Sinoussi, F. Eur. J. Biochem., 1990, 190, 469. Hodnik, W.F.; Kung, F.S.; Roettger, W.J.; Bohmont, C.W.; Pardini, R.S. Biochem. Pharmacol., 1986, 35, 2345. Tauber, A.L.; Fay, J.R.; Marietta, M.A. Biochem. Pharmacol. 1984, 33, 1367. Hodnik, W.F.; Bohmont, W.J.; Capps, R.S.; Pardini, R.S. Biochem. Pharmacol., 1987, 36, 2873. Elliot, A.J.; Scheiber, S.A.; Thomas, C.: Pardini, R.S. Biochem. Pharmacol., 1992, 44, 1603. Zhang, K.; Das, N.P. Biochem. Pharmacol., 1994, 47, 2063. Gabor, M. Flavonoids and Bioflavonoids, Farkas, I.; Gabor, M.; Kallay, F. Ed. Szeged, Budapest, 1985. Bauman, J.; Bruchhausen, F.; Wurm,G.; Nauyn -Schmiedeberg, S. Arch. Pharmacol., 1979, 307, 73. Slater, T.F.; Eakins, M.N. In New trends in the therapie of liver disease. Bertr A. (Ed.), Karger: Basel, 1984, pp. 84 -88. Elia, G.; Santoro, M.G. Ann. N.Y. ,4cad. Sci., 1994, 744, 323. Hodnick, F.W.; Kung, F.S.; Roettger, W.J.; Bohmont, C.W.; Pardini, R.S. Biochem. Pharmacol., 1986, 14, 2345. AIcaraz, M.J.; Mas, J.A.; Ubeda, A.; M,~fiez. Planta Med. 1991, 57 (Suppl. 2), All0. Bombardr E.; Morazonni, P. Chimicaoggi, 1993, 11, 25. Wolf, B.; Michel, C.; Saran, M. Meth. Enzymol., 9194, 234, 420. Hayes, N.A.; Foreman, J.C.J. Pharm. Pharmacol., 1987, 39, 466. Midleton, E.; Drzewiecki, G.; Tatum, J. Planta Med., 1987, 325. Havsteen, B. Biochem. Pharmac., 1983, 32, 1141. Robak, J.; Gryglewski, R.J. Pol. J. Pharmacol., 1996, 48, 555. Middleton Jr., E. Trends Pharmacol. Sci., 1984, 5, 335. McClurr J.W. In The Flavonoids; J.B. Harbome; T.J. Mabry; H. Mabry, Ed.; Academic Press: New York, 1975; Vol. 2, pp.1011 -32. Czeczot, H. ,4cta Biochim. Pol., 1994, 41, 144. Stavric, B. Clin. Biochem., 1994, 27, 245. Brown, J.P. Mutation Res., 1980, 75, 243. Gaspar, J.; Rodrigues, A.; Laires, A.; Silva, F.; Costa, S.; Monteiro, M.J.; Monteiro, C.; Rueff, J. Mutagenesis, 1994, 9, 445. De Crosse, J.J. Cancer N.Y., 1982, 50, 1982. Yamamoto, S.; Aizu, E.; Jiang, H.; Nakadate, T.; Kiyoto, Y., Wang, J.C.; Kato, R. Carcinogenesis, 1991, 12, 317. Stadler, R.H.; Markovic, J.; Turesky, R.J. Biol. Trace Elem. Res., 1995, 47, 299. De Groot, H.; Dehmlow, C.; Raven, U. Meth. Find. Exp. Clin. Pharmacol. 1996, 18 (Suppl. B), 25. Alcaraz, M.J.; Hoult, J.R.S. Biochem. Pharmacol., 1985, 34, 2477. Gabor, M.; Razga, Z. Acta Physiol. Hung., 1991, 77, 197. Borissova, P.; Valcheva, S.; Belcheva, A. ,4cta Physiol. Pharmacol. Bulg., 1994, 20, 25. Tordera, M.; Ferrandiz, M.L.; Alcaraz, M.J.Z. Naturforsch., 1994, 49, 235. Da Silva, J.A.; Oliveira, A.B.; Lapa, A.J.J. Pharm. PharmacoL, 1994, 46, 118. Nassis, C.Z.; Haebisch, E.M.; Giesbrecht, A.M. Braz. J. Med. Biol. Res., 1992, 25, 929.
450
MARTIN et aL
[55] Brinkwoth, R.I.; Stoermer, M.J.; Fairlie, D.P. Biochem. Biophys. Res. Comm., [56] [57]
[581 [59]
[601 [611 [62] [63]
[641 [65] [66]
[67] [681 [691
[701 [71] [72] [73] [74] [75] [76] [77] [781 [79]
[80] [81]
[821 [83] [84]
1992, 188, 631. Ohnishi, E.; Bannai, H. Antiviral Res., 1993, 22, 327. Baker, M.E. Proc. Soc. Exp. Biol. Med., 1995, 208, 131. Farboodniay, M.A.; Ray, A.B.J. Nat. Prod., 1993, 56, 989. G~ilvez, J.; Crespo, M.E.; Jim6nez, J.; Su,Srez, A.; Zarzuelo, A. J. Pharm. Pharmacol., 1993, 45, 157. G61vez, J.; S,-tnchez de Medina, F.; Jim6nez, J.; Torres, M.I.; Fern6ndez, M.I.; Nuflez, M.C.; Rlos, A.; Gil, A.; Zarzuelo, A. Planta Meal, 1995, 61, 302. Sanchez de Medina, F., Gfilvez, J.; Romero, J.A.; Zarzuelo, A. J. Pharmacol. Exp. Ther., 1996, 278, 771. Morazonni, P.; Bombardelli, E. Fitoterapia, 1995, 66, 3. Ferrandiz, M.L.; Bustos, G.; Pay6, M.; Gunasegaran, R.; Alcaraz, M.J. Life Sci., 1994, 55, 145. Closa, D.; Torres,M.; Hotter, G.; Bioque, G.; Leon, O.S.; Gelpi, E.; Rosell6, C.; Catafau, J. Prostaglandins Leukot. Essent. Fatty Acids, 1997, 56, 331. Maschenko, N.; Lipkan, G.N.; Voitenko, G.N. Pad. Fiziol. Eksp., 1981, 6, 47. Barnaulov, O.D.; Manicheva, O.A.; Kamissarenko, N.F. Khim.Farm. Zh., 1983, 17, 945. Manicheva, O.A.; Barnaulov, O.D. Rastit. Resur., 1984, 20, 256. Barnaulov, O.D.; Manicheva, O.A.; Yasinov, R.K.; Yakoulev, G.P. Rastit. Resur., 1985, 21, 85. Khrenova, D.H.; Dargaeva, T.D.; Nikolaev, S.M.; Fedotovskika, N.N.; Brutko, L.I.; Farmatsiya, 1986, 35, 46. Martin, M.J.; Alarc6n, C.; Motilva, V.; La Casa, C.; Villegas, I.; Keijjoa, A. Meth. Find. Exp. Clin. Pharmacol., 1997, 19 (suppl. A), 164. Guardia, T.; Guzm~n, J.A.; Pestchanker, M.J.; Guerreiro, E.; Giordano, O.S.J. Nat. Prod., 1994, 57, 507. Maria, A.O.; Wendel, G.H.; Guardia, T.; Guzmfin, J.A.; Pestchanker, M.J.; Guerreiro, E.; Giordano, O.S. Biol. Pharm. Bull., 1995, 18, 1784. Nomura, H. Fukuoka IshL 1959, 50, 354. Takagi, K.; Ishii, Y. Arzneim. Forsch., 1967, 17, 1544. Bruneton, J. Elementos de Fitoqulmica y Farmacognosia, Acribia: Zaragoza, 1991. Komatsu, M.; Tomomori, K.; Hatayama, K.; Mikuriya, N. Chem. Phar. BulL, 1970, 18, 602. Sasajima, M.; Nakane, S.; Saziki, R.; Saotome, H.; Hatayama, K.; Kyogoku, K.; Tanaka, I. Folia Pharmacol. Jpn., 1978, 74, 897. Takagi, K.; Okabe, S. dpn. s PharmacoL, 1968, 18, 9. Adami, E.; Marrazzi-Ubert, E.; Turba, C. Arch. Int. Pharmacodyn., 1964, 147, 113. Kyogoku, K.; Hatayama, K.; Yokomori, S.; Saziki, R.; Nakane, S.; Sasajima, M.; Sawada, J.; Ohzeki, M.; Tanaka, I. Chem. Pharm. Bull., ! 979, 27, 2943. Hayashi, Y.; Umehara, T. Yakuri to Chiryo, 1982, 10, 2697. Hatayama, K.; Yokomori, S.; Kawashima, Y.; Saziki, R.; Kyogoku, K. Chem. Pharm. Bull., 1985, 33, 1327. Konturek, S.J.; Radecki, T.; Brozowski, T.; Drodowiitz, D. Eur. J. PharmacoL, 1986, 125, 185. Su~.rez, J.; Herrera, M.D.; Marhuenda, E. Phytoter. Res., 1996, 10, 616.
ANTIULCERANDGASTROPROTECTIVEACTIVITY
[85] [86] [87]
[88] [89]
[901 [91] [921 [93]
[94] [95] [96] [971 [98] [99]
[~00] [101] [102] [103] [104]
[io5] [106] [107]
[~o8] [109] ill0] [lll] [112] [113] ill4]
451
Ayuso, M.J.; Marhuenda, E.; Martin, M.J. Plant. Mdd. Phytothdr., 1986, 20, 255. Alarc6n de la Lastra, C.; Martin, M.J.; La Casa, C.; Motilva, V. J Ethnopharmacol., 1994, 42, 16 I . Martin Calero, M.J.; La Casa, C.; Motilva, V.; L6pez, A.; Alarc6n de la Lastra, C. Z. Naturforsch., 1996, 51c, 570. Pardo, M.P.; Fernandez, C.I. Pharm. Mediterranea, 1978, 12, 259. La Casa, C.; Martin, M.J.; Alarc6n de la Lastra, C.; Motilva, V.; Ayuso, M.J.; Martin, C.; L6pez, A. Z. Naturforsch., 1996, 50c, 85. Rombi, M. 100 Plantes Medicinales. Composition, mode d'action et interet therapeutique, Romart: Nice, 199 I. Ageel, A.M.; Parmar, N.S.; AI -Meshal, I.A., Tariq, M. FIP Abstract, 1984, 43. llarionov, I.; Rainova, L.; Nakov, N. Farmatsiya, 1979, 26, 39. Rainova, L.; Nakov, S. Phytother. Res., 1988, 2, 137. Martin, M.J.; Alarc6n de la Lastra, C.; Jim6nez, B.; Marhuenda, E.; Moreno, J. Pharm. Mediterranea, 1986, 16, 573. Grande, M.; Piera, F.; Cuenca, A.; Tones, P.; Bellido, S.I. Planta Med., 1985, 35, 414. Martin, M.J.; Alarc6n de |a Lastra, C.; Marhuenda, E.; Delgado, F.; Torreblanca, J. Phytother. Res., 1988, 2, 183. Alarc6n de la Lastra, C.; L6pez, A.; Motilva, V. Planta Med., 1993, 59, 497. Reyes Rutz, M.; Martin-Cordero; Ayuso, M.J.; Toro, M.V.; Alarc6n de la Lastra, C. Phytother. Res., 1996, I 0, 303. Alarc6n de la Lastra, C.; Martin, M.J.; Marhuenda, E. J. Pharm. Pharmacol., 1992, 44, 929. Alarc6n de la Lastra, C.; Martin, M.J.; Motilva, V.; Jimenez, M.; La Casa, C.; L6pez, A. Planta Med., 1995, 61, I 16. Bors, W.; Michel, C.; Saran, M. Meth. Enzymol., 1994, 234, 420. Mascolo, N.; Pinto, A.; Capasso, F. J. Pharm. Pharmacol., 1987, 40, 293. Landolfi, R.; Mower, R.L., Steiner, M. Biochem. Pharmacol., 1984, 33, 1525. Scambia, G.; Ranevett, F.; Panici, B.; Piantelli, M.; Bonniammo, G.; De Vizenzo, R.; Ferrandina, G. Br. J. Cancer, 1991, 62, 942. Argullo, G.; Garnet, L.; Besson, C.; Demige, C.; Remesy, C. Cancer Lett., 1994, 87, 55. Waage, S.; Hedin, P. Phytochemistry, 1985, 24, 243. Panasiak, W.; Wleklik, M.; Oraczewska, A.; Luozak, M. Acta Microbiol. PoL, 1989, 38, 185. Martin, M.J.; Motilva, V.; Alarc6n de la Lastra, C. Phytother. Res., 1993, 7, 147. Alarc6n de la Lastra, C.; Martin, M.J.; Motilva, V. Pharmacology, 1994, 48, 56. Martin, M.J.; La Casa, C.; Alarc6n de la Lastra, C.; Cabeza, J.; Villegas, I.; Motilva, V. Z. Naturforsch., 1997, 53C, 82. Motilva, V.; Alarc6n de la Lastra, C.; Martin, M.J. Phytother. Res., 1992, 6, 168. Motilva, V.; Alarc6n de la Lastra, C.; Martin, M.J.J. Pharm. Pharmacol., 1993, 45, 89. P6rez-Guerrero, C.; Martin, M.J.; Marhuenda, E. Gen. Pharmacol., 1994, 25, 575. Martin, M.J.; Marhuenda, E.; P6rez-Guerrero, C.; Franco, J.M. Pharmacology, 1994, 49, 144.
452
MARTIN et aL
[1~5] [ll6] [117] [118]
Villar, A.; Gasc6, M.A.; Alcaraz, M.J.J. Pharm. Pharmacol., 1984, 36, 820. Villar, A.; Gasc6, M.A.; Alcaraz, M.J.J. Pharm. Pharmacol., 1987, 39, 502. Alcaraz, M.J.; Tordera, M. Phytother. Res., 1988, 2, 85. Wendi,P.; Reimann, H.J.; Swobwda, K.; Hennings, G.; Blumet, G. Ach. PharmacoL, 1980, 313 (SuppL), 238. [1191 Albertani -Borowski, N. Drug of the Future, 1983, 8, 209. [120] Parmar, N.S. Int. J. Tiss. Res., 1983, 4, 415. [121] Gambhir, S.S.; Goel, R.K.; Das Gupta, G. Indian. J. Med. Res., 1987, 85, 689. [122] Schwartz, K. Beitr. Klin. Chir., 1910, 67, 96. [123] Robert, A. Gastroenterology, 1979, 77, 761. [124] Takeuchi, K.; Furukawa, O.; Nishiwaqui, H.; Okave, S. Gastroenterology, 1987, 93, 1276. [125] Takeuchi, K.; Matsumoto, J.; Ueshima, K.; Ohuchi, T.; Okabe, S. Digestion, 1992, 51, 203. [126] Senay, E.C.; Levine, R.J. Proc. Soc. Exper. Biol. Med., 1967, 125, 122 I. [127] Marazova, K.; Lozeva, V.; Belcheva, A. Agents Actions, 1993, 38, C301. [128] Arakawa, T.; Satoh, H.; Fukuda, T. Hepato -Gastroenterol., 1990, 13, 166. [129] Palitzsch, K.D.; Horales, R.E.; Kronauge, J.F.; Szabo, S. Gastroenterology, 1989, 26, 381. [130] Albinus, N.; Friseh, G.; Henningns, G. Agents and Actions, 1983, 13, 249. [131] Yamahara, J.; Moehizuki, M.; Chisaka, T.; Fujimura, H.; Tamai, Y. Chem. Pharm. Bull., 1990, 38, 1039. [132] Saziki, R.; Arai, I.; Kobayashi, N.; Mutoh, K.; Sasajima, M.; Ohzeki, M. Pharmacometrics, 1976, 18, 7690. [1331 Vela, S.M.; Souccar, C.; Lima-Landman, M.T.; Lapa. A.J. Planta Med., 1997, 63, 36. [134] Parmar, N.S.; Ghosh, M.N. Eur. J. Pharmacol., 1981, 68, 1981. [135] Parmar, N.S. Int.J.Reac., 1983, 5, 415. [136] Hayes, N.A.; Foreman, 1L Jr. Pharm. Pharmacol., 1987, 39, 466. [137] Modlin,I.M. From Prout to the Proton Pump, Schnetztor -Verlag GmbH" Konstanz, 1995. [138] Murakami, S.; Isobe, Y.; Kijima, H.; Muramatsu, M.; Aihara, H.; Otomo, S.; Baba, K.; Kozawa, M. J. Pharm. Pharmacol., 1990, 42, 723. [139] Murakami, S.; Muramatsu, M.; Aihara, H.; Otomo, S. Biochem. PharmacoL, 1991, 42, 1447.
[1401 [141] [142] [143] [1441 [145] [146] [147] [148]
[149] [150]
Ray, T.K.; Nandi, J. Biochem. J., 1986, 233, 231. Murakami, S.; Muramatsu, M.; Otomo, S. J. Enzyme Inhib., 1992, 5, 293. Beii, W.; Birkholz, C.; Sewing, K.-Fr. Arzneim. Forsch., 1995, 45, 697. Warren, J.R.; Marshall, B.M. Lancet, 1983, i, 1273. Sipponen, P.; Sepp/tl/l, K.; Aarynen, M.; Helaske, T.; Kettunen, P. Gut, 1989, 30, 922. Figura, N.; Guglielmetti, A.; Rossolini, A.; Barberi, A.; Cusi, G.; Musnanno, R.A.J. Clin. Microbiol., 1989, 27, 225. Sipponen, P.; Hyv/trinen, H. Scand. J. Gastroenterol., 1993, 28 (suppl 196), 3. M~graud, F. Eur. J. Gastroenterol. Hepatol., 1993, 5 (suppl 1), S17. Hunt, R.H.; Malfertheiner, P.; Yeomans, N.D.; Hawkey, C.J.; Howden, C.W. Eur. J. Gastroenterol. Hepatol., 1995, 7, 658. Tytgat, G.N.J. Aliment. Pharmacol. Ther., 1994, 8, 359. Malfertheiner, P.; Haiter~ F. In The year in Helicobacter pylori, Current Science: London, 1994.
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
453
[151] Lamou|iatte, H.; Cayla, R.; Zerbib, F.; Talbi, P.; M6graud, F. Gut, 1995, 37 (Suppl 2), A 151.
[1521 Goddar, A.F.; Logan, R. Eur. J. Gastroenterol., 1995, 7, 1. [153] Wallace, J.L.; McKnijht, G.W.; Keenan, C.M.; Byles, N.I.A.; McNaughton, W.K. Gastroenterology, 1990, 98, 1178. [1541 Asako, H.; Kubes, P.; Wallace, J.L.; Gaginella, T.; Wolf, R.E.; Granger, D.N. Am. J. Physiol., 1992, 262, G903. [155] Boughton -Smith, N.K. Meth. Find. Exp. Clin. Pharmacol., 1989, 11, 53. [156] Osada, T.; Goto, H.; Tsukamoto, Y.; Nakazawa, S.; Sugiyama, S.; Ozawa, T. Dig. Dis. Sci., 1990, 35, 186. [157] Wittle, B.; Oren -Wolman, N.; Guth, P.H. Am. J. Physiol., 1985, 248, G580. [158] Konturek, S.J.; Brzozowski, T.; Drozdowicz, D.; Beck, G. Dig. Dis. Sci., 1988, 33, 806. [159] Pihan, G.; Rogers, C.; Szabo, S. Dig. Dis. Sci., 1988, 33, 625. [160] Vaananen, P.M.; Keenan, C.M.; Grisham, M.B.; Wallace, J.L. Inflammation, 1992, 16, 227. [161] Miyata, K.; Kamato, T.; Nishida, A.; Takizawa, K.; Takeda, M. Eur. J. Pharmacol., 1995, 276, 165. [162] Vob, C.; Sept~lveda-Boza, S.; Zilliken, F.W. Biochem. Pharmacol., 1992, 44, 157. [163] Yamamoto, S.; Yoshimoto, T.; Furukawa, M.; Horie, T.; Watanabe-Kohno, S. J. Allergy Clin. Immunol., 1984, 74, 349. [~64] Welton, A.F.; Tobias, L.D., Fiedler-Nagy, C.; Anderson, W., Hope, W., Meyers, K.; Coffey, J.W. Prog. Clin. Biol. Res., 1986, 7, 33. [165] Byung, P.Y. Physiol. Rev., 1994, 74, 139. [166] Lucas, C.E.; Sugawa, C.; Riddle, J.; Rector, F.; Rosenberg, B.; Walt, A.J. ,4cta Surg., 1971, 102, 266. [167] Whittle, J.R. Br. J. Pharmacol., 1993, 110, 3. [168] Motilva, V.; Martin, Ma J.; Luque, I.; Alarc6n de la Lastra, C. G en. PharmacoL,1995, 27, 545. [169] Avila, J.R.; Alarc6n de la Lastra,C.; Martin, M.J.; Motilva, V.; Luque, I.; Delgado, D.; Esteban, J.; Herrerias, J.M. Inflamm. Res., 1996, 45, 83. [170] Alarc6n De La Lastra,C.; Motilva, V.; Cabeza, J.; Martin, M.J.J. Pineal Res., 1997, 23, 47. [171] Granger, D. N.; J. Kourthuis Ann. Rev. Physiol., 1995, 57, 31 I. [172] Zimmerman, B.J.; Granger, D.N. Hepato-Gastroenterology, 1994, 41, 337. [173] Torel, J.; Cillard, J.; Cillard, P. Phytochemistry, 1986, 25, 383 [174] Husain, R.S.; Cillar J.; Cillard, P. Phytochemistry, 1987, 26, 2489. [1751 Letan, A. J. Food Sci, 1986, 31, 518. [176] Rekka, E.; Kourounakis, P.N.J. Pharm. Pharmacol., 1991, 43,486. [1771 Robak, J.; Gryglewski, R.J. Biochem. Pharmacol. 1988, 37, 837. [178] Sichel, G.; Corsaro, G., Scalia, M, Di Bilio, A.J.; Bonomo, R.P. Free Radical Biol. Med. 1991, 11, 1. [179] Capatano,A.L. Angiology, 1997, 48, 39. [180] Cillard, J.; Cillard, P. S.T.P. Pharma, 1988,4, 594. [181] Negre -Saivayrr A.; Affany, A.; Hariton, C.R. Pharmacology, 1991,42, 262. [182] Duneic, Z. Folia Med. Cracov, 1987, 2813, 283. [183] Robak, J.; Duneic, Z.; Rzadkowska -Bodalska, H. Pol. J. Pharm. Pharmacol., 1986, 38, 483. [184] Robak, J.; Ryszard, J.; Gryglewski, R.J. Biochem. Pharmacol., 1988, 37, 837.
454
MARTIN et al.
[185] Yoshimoto, T.; Furukawa, M.; Yamamoto, S.; Horie, T.; Watanabe-Kohno, S. Biochem. Biophys. Res. Comm., 1983, 116, 612.
[1861 Koch, H.P., LOftier, E. Meth. Find Exp. Clin. Pharmacol. 1985, 7, 13. [187] Cholbi, M.R., Experientia, 1991, 47, 195. [~88] Van -Ackcr, S.A.; Tromp, M.N.; Haenen, G.R.; Van der Vijgh, W.J.; Bast, A. Biochem. Biophys. Res. Commun,1995, 25, 755. [189] Andrews, F.J., Malcontcnti,C.; O'Brien, P.E. Dig. Dis. Sci., 1994, 37, 1356. [1901 Granger, D. N.;. Kourthuis, J. Ann. Rev. Physiol., 1995, 57, 31. [1911 Gerritsen, M.E.; Carley, W.W.; Ranges, G.E.; Shcn, C.P.; Phan, S.A.; Ligon, G.F.; Perry, C.A. Am. J. Pathol. 1995, 147, 278. [192] Wolle, J.; Hill, R.R.; Ferguson, E.; Devall, L.J.; Trivedi, B.D.;Newton, R.S.; Saxena, U. Arterioscler. Thromb. Vasc. Biol. 1996, 16, 1501. [193] Blank,M.A.; Ems, B.L.; O'Brien, L.M.; Weisshaar, P.S.; Ares, J.J.; Abel, P.W., McCafferty, D.M.; Wallace, J.L. Digestion, 1997, 58, 147. [194] Morris, G.P.; Basu, B.K.; Holitzner, C.A.; Williamson, T.E.J. Clin. Gastroenterol., 1993, 17 (Suppl. 1), $5. [1951 Grisham, M.B.; Von Ritter, C.; Smith, B.F.; La Mont, J.T.; Granger, D.N. Am. J. Physiol., 1987, 253, G93. [196] Allen, A.; Cunliff% W.J.; Pearson, J.P.; Venablcs, G.W.J. Int. Med., 1990, 228 (S upp l 732), 83. [197] Allen, A.; FlemstrSm, G.; Garner, A.; Kivilaakso, A.E. Physiol. Rev., 1993, 73, 823. [1981 Wallace, J.L.; Granger, D.N. FASEB J., 1996, I0, 731. [1991 Bell, A.E.; Sellers, L.A.; Allen, A.; Cunliffe, W.J.; Morris, E.R.; Ross -Murphy, S.B. Gastroenterology, 1985, 88, 269. [200] Murty, V.L.; Sarosiek, J.; Slomiany, A.; Slomiany, B.L. Biochem. Biophys.Res. Commun., 1984, 1221,521. [2011 Mall, A.S.; Hutton, D.A.; Coan, R.M.; Sellers, L.A.; Allen, A. Biochem. Soc. Trans., 1988, 16, 585. [202] Suwa, T.; Fukushima, K.; Kyogoku, K.; Edo, H.; Mori, Y. Japan J. Pharmacol., 1984, 34, 89. [203] Slomiany, B.L.; Liau, Y.H.; Mizuta, K.; Slomiany, A. Biochem. Pharmacol., 1987, 36, 3273. [204] Brooks, F.P.; Cohen, M.D.; Soloway, L.D. Peptic Ulcer, Churchill -Livingstone: New York; 1985. [205] Bahari, H.M.N.; Ross, I.N.; Tumberg, L.A. Gut, 1982, 23, 513. [2o61 Whittle, B. Meth. Find. Exp. Clin. Pharmacol., 1989, I 1, 35. [2071 Walt, R.P. Scand. J. Gastroenterol., 1990, 25 (Suppl 174), 29. [2081 Holtz, J. Z. Gastroenterol. Verh., 1991, 26, 162. [2091 Robert, A.; Leung, F.W.; Guht, P.H. Gut, 1992, 33, 444. [210] Wilson, D.E. In Gastric Cytoprotection a Clinician's Guide; Hollander, D.; Tamawski, A.S., Ed.; Plenum Publishing Co.: New York, 1989; pp 169-183. [211] Robert, A.; Nezamis, J.E.; Lancaster, C. Am. J. Physiol., 1983, 245, G I 13. [212] Baumann, J.; Wurm, G.; Bruchhausen, F. Arch. Pharm. Weinhein., 1980, 20, 627. [2~3] Mertz-Nielsen, A.; Munck, L.K.;Bukhave, K.; Rask -Madsen, J. Ital. d. Gastroenterol., 1990, 22, 288. [214] Alarc6n de la Lastra, C.; La Casa, C.; Motilva, V.; L6pez, A.; Martin, M.J. Phytomedicine, 1996/97, 3, 327. [215] Itoh, M.; Guth, P.H. Gastroenterology, 1985, 88, 1162.
ANTIULCER AND GASTROPROTECTIVE ACTIVITY
455
[216] Takeuchi, K.; Megumu, O.; Hiromichi, N,: Okabem S. ,1. Pharmacol. Exp. Ther., 1988, 248, 836. [217] Konturek, P.K.; Brzozwski, T.; Konturek, S.; Dembinski, A. Gastroenterology, 1990, 98, 909. [218] Szabo, S.; Pihan, G.; Dupuy, D. In New Pharmacology of Ulcer Disease. Szabo, S.; Moszik, G. Eds. Elsevier: New York, 1987, pp. 424 -446. [219] Szabo, S.; Vattay, P.1990 Gastroenterol. Clin. North Am. 19, 67. [220] Oates, P.J. In Gastric Cytoprotection, a Clinician's Guide. Hollander, D. ; Tarnawski, A.S. Eds., Plenum Publishing Corporation: New York, 1989, pp. 125 -164. [221] Szabo, S.; Goldberg, I. Scand d. GastroenteroL,1990, 25 (suppl 174), 1. [222] Giavin, G.; Szabo, S. F,4SEB 3., 1992, 6, 825. [223] Jacobson, E.D. Gastroenterology, 1992, 102, 1788. [224] Gomez, M.E.; Ayuso, M.J.; Toro, M~ V.; Alarc6n de la Lastra, C. Meth. Find. Exp. Clin. Pharmacol., 1994, 16 (suppl 1), 131. [225] MacNaugton, W.; Cirino, G.; Wallace, J.L. Life Sci. 1989, 54, 1869. [226] Konturek, S.J.; Brzozowski, T.; Majka, H.; Pykto, P.J. Eur. 3.Pharmacol., 1993, 239, 215. [227] Williams, G.T. Biochem. Cell. Biol., 1994, 72, 450. [228] Zakeri, Z.; Lockshin, R.A. Ann. N. Y. Acad. Sci., 1994, 71, 9229. [229] Lockshin, R.A.; Zakeri, Z.F. Ann. N. Y. Acad. Sci., 1992, 663, 249. [230] Zakeri, Z.F.; Quaglino, D.; Latham, T.; Lockshin, R.A. FASEB d., 1993, 74, 78. [231] Bellamy, C.O.; Malcomson, R.D.; Harrison, D.J.; Wyllie, A.H. Semin. Cancer. Biol., 1995, 616, 285. [232] Guillouf, C.; Grana, X.; Selvakumaran, M.; De Luca, A.; Giordano, A.; Hoffma N,; B.Liebermann,; D.A. Blood, 1995, 85, 2698. [233] Larocca, L.M.; Piantelli, M.; Teofili, L.; Leone, G.; Ranelletti, F.O. Exp. Hematol., 1996, 24, 496. [234] McCabe, M.J.; Orenius, S.; Biochem. Biophys. Res. Com., 1993, 194, 945. [235] De Vincenzo, R.; Scambia, G.; Benedetti Panici, P.; Ranelletti, F.O.; Bonanno, G.; Ercoli, A.; Delle Monache, F.; Ferrari, F.; Piantelli, M.; Mancuso, S.; Anticancer Drug Res., 1995, 10, 490. [236] Scambia, G.; Ranelletti, F.O.; Panici, P.B.; De Vincenzo, R.; Bonanno, G.; Fer randina, G.; Piantelli, M.; Bussa, S.; Rumi, C.; Cianfriglia, M. Cancer Chemother. Pharmacol., 1994, 344, 64. [237] Larocca, L.M.; Giustacchini, M.; Maggiano, N.; Ranelletti, F.O.; Piantelli, M.; Alcini, E.; Capelli, A.J; Urology, 1994, 152, 1033. [238] Scambia, G.; Ranelletti, F.O.; Panici, P.B.; Piantelli, M.; De Vincenzo, R.; F errandina, G.; Bonanno, G.; Capelli, A.; Mancuso, S. Int. J. Cancer, 1993, 54, 466. [239] Yoshida, M.; Yamamoto, M.; Nikaido, T. Cancer Res., 1992, 52, 6681. [240] Hosokawa, N.; Hirayoshi, K.; Kudo, H.; Takechi, H.; Aoike, A.; Kawai, K.; Nagata, K. Mol. Cell Biol., 1992, 123, 498. [241] Koishi, M.; Hosokawa, N.; Sato, M.; Nakai, A.; Hirayoshi, K.; Hiraoka, M.; Abe, M.; Nagata, K.; Jpnan J. Cancer Res., 1992, 831,222. [2421 Wei, Y.Q.; Zhao, X.; Kariya, Y.; Fukata, H.; Teshigawara, K.; Uchida, A. Cancer Res., 1994, 54, 4957. [2431 Welch, W.J. Physiol. Rev., 1992, 722, 36. [2441 Onishi, Y.; Azuma, Y.; Kizaki, H. Biochem. Mol. Biol. Int., 1994, 32, 112.
456
[245] [246] [247] [248]
MARTf[Net as
Potten, C.S. Cancer Metastasis Rev., 1992, 11, 179. Dive, C.; Evans, C.A.; Whetton, A.D. Semin. Cancer Biol., 1992, 34, 27. Yanagihara, K.; Ito, A.; Toge, T.; Numoto, M. Cancer Res., 1993, 535, 821. Toi, M.; Mukaida, H.; Wada, T.; Hirabayashi, N.; Toge, T.; Hori, T.; Umezawa, K. Eur. J. Cancer, 1990, 267, 24. [249] Yoneda, T.; Lyall, R.M.; Alsina, M.M.; Persons, P.E.; Spada, A.P.; Levitzki, A.; Ziberstein, A.; Mundy, G.R. Cancer Res., 1991, 51, 4435.
Atta-ur-Rahman(Ed.)Studies in Natural Products Chemistly, Vol. 22 9 2000 ElsevierScienceB.V. All rightsreserved
457
SIMPLE FLAVONES POSSESSING COMPLEX BIOLOGICAL ACTIVITY S. TAHARA* and J. L. INGHAMw
Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589, Japan and w of Food Science and Technology, University of Reading, Whiteknights, P.O. Box 226, Reading RG6 2AP, England, U. K. ABSTRACT: Two simple flavones, each of which exhibits distinct biological activity despite their closely related structures, have been recognized by detailed bioassays, and bioassay-orientated isolation procedures. The identity of both flavones has been confirmed by synthesis. One of these compounds, 5-methoxy-6,7-methylenedioxyflavone has been found in an extract of Polygonum lapathifolium L. subsp, n o d o s u m (Polygonaceae) using a screening test devised to detect antidotes against the benzimidazole fungicide, benomyl (or its active principle MBC, l H-benzimidazol-2ylcarbamic acid methyl ester). The other compound, 5-hydroxy-6,7methylenedioxyflavone, is a host-specific signalling substance that exudes from spinach roots and attracts zoospores of the phytopathogenic fungus ,4phanomyces cochlioides the cause of spinach root rot. This review describes the bioassay, isolation and identification of these active compounds, and compares their activity with that of various other related, and unrelated, chemicals of either plant or synthetic origin. The possible ecochemical role and mode of action of flavone and non-flavone antidotes and attractants is briefly discussed.
GENERAL INTRODUCTION The flavonoids, and their close chemical relatives, the isoflavonoids, easily comprise the largest and most widespread group of naturally-occurring secondary compounds. Since the isolation, in the mid-1840's, of pure apiin (now known to be the flavone apigenin-7-O-apiosylglucoside) from seeds of parsley (Petroselinum sativum, syn..4pium petroselinum; Umbelliferae), many thousands of flavonoids and isoflavonoids have been chemically identified. Most of the flavonoids found in green plants are oxygen-ring heterocyclics, with the vast majority being chalcones/dihydrochalcones (non-heterocyclic), flavones (including the 3hydroxyflavones, or flavonols), flavanones (dihydroflavones), aurones and anthocyanidins/anthocyanins. Of these, the flavones, which are based on 2-phenylchromone (1), are the most commonly encountered, being essentially ubiquitous within the families and genera of the higher plants. Most flavones occur in the Plant Kingdom as simple aglycones, with hydroxyl, methoxyl or methylenedioxy substituents, or as O- or C-linked
458
TAHARA and INGHAM
glycosides, but more complex molecules are also regularly, and increasingly, described in the phytoehemical literature.
8
"~
HO.
5'
6' i
7
o.
6
114 O
5
2
.o
o.
luteone
1 2-phenylchromone
~f' ) OH
H
I
OH
<~~
OCH3
O
OCH30
4 betagarin
3 phlorettn
TI~ OCH3
HO
O
OR 5 betavulgarin
6 R=H cochllophllln A 10 R=CH3
R
I
O OH 7 daidzein
8 R-H
genistein
OH
9 R=CHa prunet/n
Although the role of certain flavonoids (e.g. the red and blue anthocyanins, and the yellow or orange chaleones and aurones) as pigments in flowers and fruits has long been recognized, the vast majority of flavonoids were, until comparatively recent times, merely regarded as biosynthetic end-products lacking any specific function in those plant parts in which they occurred. This situation has now changed
SIMPLE FLAVONES
459
dramatically, with flavonoid (and isoflavonoid) compounds almost daily being assigned functions and properties that were unimagined only 20 - 30 years ago. Whilst an in-depth discussion of the biological properties of flavonoids and isoflavonoids is beyond the scope of this review, it is worth mentioning that whereas these compounds are absent from, or of negligible importance in, the fungi, some flavonoids (and a very large number of isoflavonoids) have been reported to protect plants from fungal attack. Thus, in the Leguminosae, certain antifungal isoflavones (e.g. luteone, 2) may occur on leaf or other plant surfaces, where they appear to provide an initial barrier to the growth of potentially pathogenic (disease-causing) fungi [1]. More commonly, however, isoflavonoids (and especially those belonging to the isoflavone, isoflavanone, isoflavan and pterocarpan groups) are produced as phytoalexins, undergoing a rapid de novo accumulation around the point of fungal invasion where they provide a localized and generally effective barrier to further growth and development of the fungus [2]. In contrast to isoflavonoids, relatively few flavonoids have yet been assigned a prominent role in plant defense. However, there is some evidence to suggest that the dihydrochalcone phloretin (3), and its oxidation products, may be involved in the resistance of apple (Malus sp.; Rosaceae) varieties to the scab fungus (Venturia inaequalis) [3]. Additionally, three simple flavan phytoalexins have been obtained from daffodil bulbs (Narcissus pseudonarcissus; Amaryllidaceae) inoculated with Botrytis cinerea [4], whilst in response to the fungus Helminthosporium carbonum, the leaflets of Shuteria vestita (Leguminosae) accumulate two relatively complex flavanone phytoalexins [5]. In other plant-fungus interactions (e.g. Medicago sativa - Ascochyta imperfecta), the levels of flavones have been shown to increase significantly, although the exact role of the compounds involved has not been firmly established [6]. In sugar beet leaves (Beta vulgaris; Chenopodiaceae) infected with the fungus Cercospora beticola (leaf spot), two compounds accumulate rapidly. One of these, 2',5-dimethoxy-6,7-methylenedioxyflavanone (betagarin, 4) has relatively weak antifungal activity, whereas the second, 2'-hydroxy-5-methoxy-6,7-methylenedioxyisoflavone (betavulgarin, 5) is significantly more antifungal, and may by itself be largely responsible for limiting the spread of C. beticola [7, 8]. Although the flavanone betagarin (4) has little antifungal activity, both it and the isoflavone betavulgarin (5) are notable because they possess a 6,7-methylenedioxy substituent, a feature that is relatively uncommon amongst the flavonoids and isoflavonoids. Interestingly, both 4 and 5 also occur in sugar beet roots infected with Rhizoctonia solani, where they are accompanied by three other methylenedioxy-substituted compounds" 5hydroxy-6,7-methylenedioxyflavone (cochliophilin A, 6), 3,5-dihydroxy-
460
TAHARA and INGHAM
6,7-methylenedioxyflavanone and 2',5-dihydroxy-6,7-methylenedioxyisoflavone (desmethylbetavulgarin) [9]. Whilst the function of these various compounds in sugar beet roots has still to be determined, in the root exudates of another member of the Chenopodiaceae (spinach, Spinacia oleracea), it has been found that cochliophilin A (6) acts as a potent chemo-attractant for zoospores of the root-rotting fungus, Aphanomyces cochlioides [10]. Although the isoflavones daidzein (4',7-dihydroxyisoflavone, 7) and genistein (4',5,7trihydroxyisoflavone, 8), and genistein-7-O-methyl ether (prunetin, 9), have been reported as fungal chemo-attractants in the roots of soybean (Glycine max; Leguminosae) and garden pea (Pisum sativum; Leguminosae) respectively [11, 12], such a role for flavones has not previously been recognized. The possibility exists, however, that cochliophilin A (6) may also act as a fungal chemo-attractant in sugar beet roots, providing that it can be detected in non-infected tissues, or in exudates from healthy roots. Apart from chemo-attractant properties, which are discussed in detail in the second part of this review, we have discovered that another flavone, the methyl ether of cochliophilin A (5-methoxy-6,7methylenedioxyflavone, 10), found in Polygonum (Polygonaceae) species, can act as an antidote to several of the benzimidazole fungicides now used to control plant diseases [13]. Although the occurrence of an antidotal flavone in species of Polygonum is unlikely to be of any practical significance, it is conceivable that the presence of similarly active flavones in cultivated plants may reduce the efficiency of certain types of fungicide. As a final point, it is interesting to note that in contrast to cochliophilin A (6), the 5-O-methyl ether has a very low chemo-attractant effect on zoospores ofA. cochlioides, a clear indication that even amongst relatively simple molecules such as the flavones, a small molecular change can drastically alter biological activity. Studies on the antidote effects of flavones and other naturally-occurring compounds [ 14], are dealt with in the first part of this review. ANTIDOTES AGAINST BENZIMIDAZOLE FUNGICIDES Introduction
Because of our general interest in plant secondary metabolites which show some interaction with xenobiotics [15], we have developed a simple bioassay system to find compounds that act as antidotes against the benzimidazole fungicide benomyl. Benomyl [methyl 1(butylcarbamoyl)benzimidazol-2-ylcarbamate, 11], one of the best known systemic fungicides for agricultural use, is believed to suppress the development of numerous species of fungi, except for certain Ascomycetes, Oomycetes and other Phycomycetes [16], by disturbing
S I M P L E FLAVONES
461
cell division when it binds to the protein 13-tubulin, thus interfering with the assembly of microtubular subunits to yield microtubules. It is well known that various chemical agents can interfere with tubulin (which consists of ct-and 13-tubulins), and prevent it from combining effectively with microtubular-associated proteins to afford microtubules. The microtubules themselves are important sub-cellular structures involved in construction of the cytoskeleton, and in cell devision and cell movement. Representative chemicals which bind to tubulin are shown in Table 1. Most of these compounds are potent inhibitors of mitosis, and for this reason some of them appear to be promising chemotherapeutic agents in the treatment of certain cancers (e.g. breast and ovarian carcinomas). Table 1.
Mitosis Inhibitors which Bind to Tubulin 118] i
i
,.
i
A. Tubulin assembly inhibitors* A-I Colchicine group colchicine (Colchicum autumnale) combretastatin (Combretum caffrum) curacin A (from a marine cyanobacterium [19]) podophyllotoxin (Podophyllumpeltatum) steganacin (Steganotaenia araliacea) benzimidazole fungicides (synthetic) A-2 Vinblastine-Maytansine group vinblastine (Catharanthusroseus) maytansine (Maytenus ovatus) dolastatin 10 (from a marine mollusc) phomopsin (from a fungus) rhizoxin (from a fungus) B.
Tubulin disassembly inhibitors** taxol (Taxus brevifolia [20]) epothilones A and B (from a myxobacterium [21]) discode..rmolide (fr0m a Caribbean sponge [22])
. . . . . . . . . . . . . .
* Compounds in groups A-I and A-2 bind to I]-tubulin, but the binding sites are different from group to group.
** The epothilones and diseodermolide competitively inhibit the binding of taxol to tubulin polymers, presumably indicating an overlap between their respective binding sites.
Although research is still at a very early stage, it is possible that studies involving plant-derived antidotes, or synthetic analogues, may lead to the development of new remedies which may be helpful in cases of accidental poisoning by agricultural or medicinal chemicals. The identification of antidotes against benomyl (11) and related compounds may, as described later in this review, also provide a novel chemical approach to understanding the mode of action of benzimidazole fungicides.
462
TAtlARA and INGHAM
Bioassays for Detection of Antidotes Against Benzimidazole Fungicides
Materials The commercially available chemicals, benomyl (11), carbendazim (MBC, 12), thiabendazole (13), thiophanate-methyl (14) and diethofencarb (15), and a sixth compound, nocodazole (16), prepared by a recognized method [17], were used in the bioassay procedures. Benomyl (MW 292) is known to spontaneously decompose in water to give carbendazim (MBC, MW 192) as an antifungal principle, and it is possible, therefore, to use MBC (12) as a direct replacement for benomyl ( l l ) in the bioassays. Cladosporium herbarum AHU 9262, which is highly susceptible to benomyl (11) and MBC (12), was used as the test fungus for detecting antidotes, and was cultured on PYG agar medium (3% glucose, 1% peptone, 0.1% yeast extract and 1.5 % agar) for 5-7 days at 25oC unless otherwise mentioned. Under these conditions, spores were produced in abundance. Details of the medium used for TLC bioassays are given in ref. [23]. Plant materials were collected in and around Sapporo during the summer months. For the screening test, 10 g (fresh weight) of the aerial parts (mainly leaves/stems) were immersed in 150 ml of MeOH for 3-4 days at room temperature. The resulting extract was then reduced to dryness and the residue was partitioned between EtOAc and water. In order to isolate and characterize the antidotes, Polygonum lapathifolium L. subsp, nodosum, P. sachalinense Fr. Schm. and P. thunbergii were collected in large quantities and extracted with MeOH. TL C Bioassays
A small portion of the plant extract (25 lxl out of 2 ml of concentrated EtOAc extract from 10 g of fresh plant material) was chromatographed on silica gel 60 F254 thin-layer plates (20 x 20 cm, thickness 0.25 mm). Two plates, each loaded with the same plant extract were developed (15-16 cm) in hexane-EtOAc (3:2) and CHC13 -MeOH (25:1), respectively, and airdried to remove all traces of the solvents. The TLC plates were then dipped in a 0.3 ppm benomyl (or 0.2 ppm MBC) solution in hexane for 3 sec, dried and sprayed with a suspension of C. herbarum spores according to the procedure of Homans and Fuchs [23]. The amount of benomyl or MBC in the dipping solution was slightly in excess of the minimum inhibitory dose for C. herbarum on TLC plates. When the plant extract contained an antidote to benomyl or MBC, C. herbarum grew on and around that part of the chromatogram corresponding to the location of the active material. The size of the resulting dark (black) zone of fungal growth
SIMPLE FLAVONES
463
was directly related to the activity of the antidote (Fig.l, left). In contrast, around inactive constituents in the extract, and on control ehromatograms (spotted only with EtOAe), the silica gel remained free of fungal growth and appeared white. Although not of direct relevance to the present review, it is worth mentioning that the TLC bioassay also proved to be an easy and rapid method for detecting fungitoxie compounds in plant extracts. Thus, on developed TLC plates, untreated with fungicide, C. herbarum grew extensively except where compounds with antifungal activity were located. These areas appeared as white zones against the black fungal background (Fig.l, right). By slightly modifying the bioassay method it is possible, therefore, to screen the same plant extract for both antidotes to fungicides and for natural antifungal compounds.
Fig. (1). Results of TLC bioassays to detect antidotes and antifungal compounds. Left: a thin-layer chromatogram showing the presence of antidotes (dark zones) Right: a chromatogram showing the presence of antifungai material (white zone) P. lap.: Polygonum lapathifolium L. subsp, nodosum, P. sac.: P. sachalinense, P. thu.: P. thunbergii, *: due to pigments. Note: In Fig. 1 (left), crude plant extracts were used to detect antidote activity. In Table 2 B, the extracts were purer, and this presumably accounts for some observed differences in the position (Rj') and number of compounds detected.
464
TAilARA and INGHAM
Paper Disk Bioassay on Agar Plates
To 9 ml of PYG agar medium (0.9 % agar) at 50~ was added 1 ml of C. herbarum spore suspension and 100 ~tl of a 20 ppm solution of benomyl (or another benzimidazole fungicide) in acetone, and the preparation was allowed to solidify in a 9 cm i.d. Petri dish. A plant extract (25 ~tl), or a test solution (pure compound in acetone), was applied to a paper disk (thickness, 1.3 mm; diam. 8 mm) and dried in vacuo. Control disks were treated with solvent only (acetone or EtOAc). The treated and control disks were placed on the agar and conditioned for 1 hr at room temperature before being incubated at 25~ for 24-48 hr. When the paper disk contained an antidote to the fungicide, the test fungus grew around the relevant disk, the amount of growth being related to the activity of material on the disk. This activity was quantified by measuring the distance (in mm) from the edge of the paper disk to the outer edge of the zone of fungal growth. The procedure and results are illustrated in Fig. 2.
Fig. (2). Paper disk method for detecting antidotes against MBC.
As discussed later in this review (PP. 476 and 477), we have also found that certain compounds (e.g. 5-hydroxy-7-methoxychromone) are antagonistic to the benzimidazole antidotes, and can nullify their effects. This antagonism can easily be detected using a modification of the paper disk bioassay on agar containing C. herbarum spores and a benzimidazole fungicide. In the modified procedure, a paper disk impregnated with the
SIMPLE FLAVONES
465
compound to be tested for antagonistic activity is placed on the agar close to, but not touching, a disk impregnated with a benzimidazole antidote. After an appropriate incubation period, the C. herbarum is seen to have grown normally around the antidote-containing disk except where this is in close proximity to the disk impregnated with the antagonist to the antidote. Here, fungal growth is reduced, or halted, so that the normally circular zone of fungal growth has a concave, or crescent, appearance. When testing for possible antagonistic activity, it is important that the compound under study should first have been shown to lack significant antifungal activity (e.g. by TLC bioassay against C. herbarum), and not act synergistically with the benzimidazole fungicide that is incorporated into the agar. Table 2. ..,,
,,.
Results of Screening Extracts of Higher Plants for Benzimidazole Antidotes
.,
,.
.,
,..
,,
...,
,
.
,.
,,,,,
,
,.
,
,,,
A. First screening test (TLC plate assay; the test sample was equivalent to 250 mg of flesh plant material) Negative (except for weak antidote activity near the solvent front, presumably attributable to cztocopherol): Boraginaceae: Myosotis sylvatica, Caryophyllaeeae: Stellaria media, Chenopodiaceae: Spinacia oleracea, Compositae: Chrysanthemum leucanthemum and Erigeron annuus, Cruciferae: Capsella bursa-pastoris, Equisetaceae: Equisetum arvense, Geraniaceae: Geranium sp., Hippocastanaceae: Aesculus turbinata, Labiatae: Ajuga yezoensis and Lamium album, Liliaceae: Trillium kamtschaticum, Magnoliaceae: Liriodendron tulipifera, Osmundaceae: Osmundastrum cinnamomeum, Papaveraceae: Corydalis ambigua, Polypodiaceae: Onoclea sensibilis, Scrophulariaceae: Veronica arvensis and V. persica, Violaceae: Viola grypoceras, Umbelliferae: Heracleum lanatum subsp.lanatum and Torilisjaponica Positive: Polygonaceae: Polygonum thunbergii jl
.
ii
B. Second screening test using Polygonaceae plants (TLC plate assay: each extract consisted of neutral and phenolic constituents soluble in ethyl acetate; the test sample was equivalent to 125 mg of fresh plant m aterial ) . . . . . . . . . . . . . . . .
9
[ Antidote ' [
,
Locstion'of antidote on
......
activity
TLC plates (RJ)
,] TLC solvent* , ,
Fagopyrum esculentum Moench.
..
:
,
A,B-I , u
F. tataricum (L.) Gaertn.
~
Polygonum arenastrum Boreau
4-
A, B-I i
P. lapathifolium L. subsp, nodosum
+ +
P. Iongisetum De Bruyn
+
P. sachalmense Ft'. Schm
+ +
ill,
,,
,
i
,
,,,,,
,iml
i
.,i
.
0.96
B-2
0.27, 0.32, 0.41
B-2
0.22
C-i
0.82, 0.96
C-2
i
i
,
P. thunbergii Sieb. et Zucc.
+ +
0.26
Rumex acetosella L
+
0.37
B-2 i
R. obtusifolius L.
4-4-
0.31, 0.74, 0.81, 0.90
C-2
*solvents: A, hexane-EtOAc (! :1); B-land B-2, CHCI3-MeOH (25: I) and (70: I); C-I and C-2, hexane-EtOAc-HCOOH (600:600:1) and (400:800:1).
466
TAHARA and INGHAM
Antidotes Against Benzimidazole Fungicides in Species of Polygonaceae A preliminary screening test (TLC bioassay) for benzimidazole antidotes was carried out using 22 plant species, representing 18 plant families. As shown in Table 2 (A), out of the 22 plants tested, benzimidazole antidote activity was initially found only in Polygonum thunbergii (Polygonaceae). An aliquot, equivalent to 125 mg of fresh plant material, of the EtOAc soluble constituents from methanol extracts of P. thunbergiiwas sufficient to clearly show the antidote activity on a thin-layer plate [Table 2 (B)]. Polygonum lapathifolium L. subsp, nodosum aerial parts [12.8 kg) MeOH extracts (40 liters x 2), concd, and partitioned between EtOAc and water EtOAc solubles
....
~-- washed with 5 % sodium bicarbonate and saline Concentrate of EtOAc layer (82 g) SlO 2 Column (500 g)
Eluate with CHCI3-MeOH= I 0: I [from 2nd to 6th fractions, 5 x 1,000 ml; 42 g) SiO 2
Column {400 g)
Eluate with CHCI3-MeOH= 100:1 (from 3rd to 7th fractions, 5 x 400 ml. 8 g) SiO 2 Column (80 g) |
ii,
I
I
Eluate with hexane-EtOAc=5: I (from 5th to 10th fractions, 6 x 100 ml; 710 rag} SIO 2
Eluate with hexane-EtOAc= 1:1 (4th fractilon. 1 x 100 ml) + s u b s e q u e n t elution with EtOAc alone (first 200 ml); combined solutes; 2.5 g
Column ( I 0 g) SIO 2
Eluate with CHCIa-MeOH- 100: I [from 12th to 15th fractions, 4 x 6 . 8 ml)
Column ( I 0 g)
Eluate with CHCI3-MeOH= 100:1 (from 5th to 18th fractions, 14 x 50 rnl; 210 mg)
Preparative TLC In 11 CHCI3-MeOH=40: I {Rf0.50) 2) hexane-EtOAc=5:3 (Rf0.57)
Preparative TLC In
{Rf 0.34) 2) CHCI3-MeOH=40: I {Rf0.42) I) hexane-EtOAc= I: I ii
PN-1 {19) 32 mg S h c e m e 1. Isolation procedures for
PN-2 (I0) 3 mg
Polygonum lapathifolium antidotes.
SIMPLE FLAVONES
467
Since we had found a benomyl antidote in the Polygonaceae, further surveys for active compounds were focused on species belonging to this family. The Polygonaceae (Order:Polygonales) consists of about 30 genera with a total of approximately 750 species. As show in Table 2, extracts of five Polygonum species, and two Rumex species, were shown to contain varying amounts of benzimidazole antidotes. Nothing was found in two cultivated species of Polygonaceae, Fagopyrum esculentum and F. tataricum. Two antidotes from P. sachalinense Fr. Schm. have now been isolated and identified as tx-tocopherol (vitamin E, 17) and the anthraquinone emodin (18), using a combination of chromatography and bioassaydirected monitoring [14]. Samples of authentic a-tocopherol (Eastman Kodak Co.) and emodin (Wako Pure Chemical Ind. Ltd.) were indistinguishable from our isolates by spectroscopy and bioassay. Although, ct-tocopherol (17) is a ubiquitous constituent in green plants, and emodin (18) is found in the Polygonaceae [24, 25], Leguminosae [26], and Rhamnaceae [27], and is known to be a fungal metabolite [28], their unusual activity as benzimidazole antidotes is reported here for the first time. Three further benzimidazole antidotes have also been identified, two in P. lapathifolium subsp, nodosum, and one in P. thunbergii. Isolation and purification procedures for the two active compounds in P. lapathifolium are shown in Scheme 1 [ 13]. In TLC bioassays, the upper running compound (PN-1) from P. lapathifolium was found to be both fungitoxic and a benzimidazole antidote. From high resolution mass spectroscopy, PN-1 had a molecular formula of C~5H~402, whilst its ~H NMR spectrum contained signals attributed to two olefinic protons (2H, J= 16.3 Hz, trans), a phenyl ring (8 7.53: 2H, hr. d-like, J-ca 7.7 Hz; 7.34: 2H, hr. t-like, ,/=ca 7.7 Hz; and 7.24: 1H, br. t, ,/=ca 7.7 Hz), a 1,3,4,5-tetrasubstituted benzene ring (8 6.64: 2H, s), a pair of phenolic OH groups (8 8.17: 2H, hr. s), and an aromatic methyl ( 8 2.10: 3H, s). The symmetric nature of the tetrasubstituted and monosubstituted benzene rings was evident from ~H and 13C NMR data [13]. From this evidence, two structures for PN-1 (19) H| 9~
H3Cw
~
H 7.5:,
!
H
"H H3 ~CH
/g~' ~o b s e r v e d NOE
Fig. (3). NOE experiment on PN-1-di-O-methyl ether (20) in acetone-d 6 at 500 MHz.
468
TAHARAand INGHAM
were possible, one possessing a 2,6-dihydroxy-4-methylated A-ring, and the other an A-ring with 3,5-dihydroxy-4-methyl substitution. The second arrangement was preferred on the basis of an NOE experiment using the di-O-methyl derivative 20 (Fig. 3). Irradiation of the signal at 8 6.87 (2H, singlet, aromatic), attributed to the A-ring, enhanced the signals for both the O-methyl and olefinic protons, a result which clearly ruled out the former structure. All the available data suggested that PN-1 was 3,5-dihydroxy-4-methylstilbene, and this was confirmed by chemical synthesis (Scheme 2) [13], the product being chromatographically and spectroscopically identical with PN-1. Thus the third benzimidazole antidote is 3,5-dihydroxy-4methylstilbene (19), a compound not previously recognized as a natural product.
Ho cooc, _ H.co coo H3 H.CO.fycoa IOH 2) NaOMe'CuCI2 HaC" T OCH3
rtae"
T OCH3
I DMSO (COCI) 2
CH2CI2 H 19~ PyrldlneHCI Ig0~
a C O l ~ ~J
~ _.NaOMe/DMF
HaC~'- T
-OCHo
~
HaC o ~ ~
.... "-~ Hag" PO(OCH2CHa)2
CHO
T OCHa
20 Scheme 2. Syntheticroute to 3,5-dihydroxy-4-methylstiibene(19). The molecular formula C17H1205for PN-2 was also obtained by HR-EI mass spectrometry. UV maxima in methanol at 215 (rel. int., 1.00), 272 (0.69) and 308 nm (0.56) suggested a flavone-type structure for PN-2, as did the IH NMR spectrum run in acetone-d6 which revealed a 1H signal at 8 6.67 (flavone H-3) [29]. As a 2H signal around 8 7.84-7.86, and a 3H signal around 8 c a 7.5 were attributed to a monosubstituted phenyl group (B-ring) [ 10], the remaining protons were reasonably assigned to H-3 (8 6.67), an A-ring proton (8 6.74), a methoxyl group (8 4.13, 3H) and a methylenedioxy group (8 6.07, 2H). Because the flavone H-5 resonates around 8 7.9-8.2 [29], and as almost all known flavones are oxygenated at C-7, the A-ring proton must be located at either H-6 or H-8.
SIMPLE FLAVONES
469
CONH (CH2)aCH 3 NHCOOMe
12 MBC [carbendazim)
I I benomyl
NHCSNHCOOMe
NHCSNHCOOMe
14 thlophanate-methyl
IS TBZ (thlabendazole) H
NHCOOMe
18 nocodazole H3CH2C
NHCOOCH(CH3} 2
OCH2CH 3 15 dlethofencarb
Fig. (4). Synthetic antimitotic fungicides: benzimidazoles derivatives (11-14, 16) and an isopropyl carbamate (15).
The preferred structure for PN-2 was 5-methoxy-6,7-methylenedioxyflavone (10), rather than 5-methoxy-7,8-methylenedioxyflavone, because of known ~H NMR solvent shift effects on a methoxy signal when a methoxyl group is located ortho to an unsubstituted carbon. In these cases the chemical shift value in CDCI3 is lower than that in C6D6 by 0.50.8 ppm [30]. For PN-2, however, the methoxy signal appeared at ~54.13 in CDC13, being only slightly shifted to ~i 4.09 (A~i 0.04) by changing the solvent to C 6 D 6 . This indicates that the carbon ortho to the methoxyl group is substituted. This result is compatible only with the substitution pattern of 10. Compound 10 was chemically prepared, and found to be identical with PN-2 in chromatographic and spectroscopic properties [ 13]. Thus, the second antidote from P. lapathifolium is 5-methoxy-6,7methylenedioxyflavone (10), which has previously been isolated from
TAtlARA and INGHAM
470
Physalis minima (Solanaceae) [31]. Interestingly, this compound is the 5O-methyl ether of cochliophilin A (6) which has been isolated from spinach (Spinacia oleracea) roots, where it is a potent attractant for Me Me
Me
Me
Me Me
Me
17 a-tocopherol
Me
HO
O
MeO
O
10 5-methoxy-6,7-methylenedloxyflavone
18 emodin
Me ~~
M
OMe
e
\oil 19
3, 5-dihydroxy-4-methylstilbene
O 9-1 2.6-dlmethoxy-pbenzoqulnone
Fig. (5). Antidotes against benzimidazole fungicides found in the Polygonaceae.
zoospores of the phytopathogenic fungus Aphanomyces cochlioides [10] (see, PP. 482-500). Cochliophilin A (6) has been synthesized by known methods [32, 33]. Methylation of cochliophilin A (6) in 80 % yield was achieved by the addition of 0.5 ml of CH3I to a mixture of 1 ml of 1,3dimethyl-2-imidazolidione, 43 mg of cochliophilin A (6) and 40 mg of Nail (60 %), followed by stirring for 24 hr at room temperature. [5-OMe14C]-5-methoxy-6,7-methylenedioxyflavone ([5-OCH3-14C]-10) used in the binding test discussed later in this review was prepared with t4CH3I. Finally, a fifth antidote to benzimidazole fungicides was isolated from 13 kg of the aerial parts of P. thunbergii in very poor yield (ca 1 mg). However, comparison of its physicochemical and chromatographic properties with those of authentic 2,6-dimethoxy-p-benzoquinone (21) [ 13] confirmed that the compounds were identical. This benzoquinone has
471
SIMPLE FLAVONES
been isolated from Caesalpiniapulcherrima (Leguminosae) [34], Peddiea fischeri (Thymelaeaceae) [35], and Phyllostachys heterocycla var. pubescens (Gramineae) [36] and is already known to be an antimicrobial agent [36, 37]. The structures of compounds found in the Polygonaceae with antidote effects against benzimidazole fungicides, are shown in Fig. 5. Although,
/
OH MeO
....
Me
~
:
0
Me
OH
OMe
OMe
22 colchlclne
2 3 combretastatln OH
H
OMe
m i
~0"
~'CH a
OMe OMe
25 streptopyrone
2 4 podophyllotoxtn Fig. (6). Natural compounds which inhibit tubulin assembly.
the identified antidotes, tx-tocopherol (17), emodin (18), 3,5-dihydroxy-4methylstilbene (19), 5-methoxy-6,7-methylenedioxyflavone (10), and 2,6dimethoxy-p-benzoquinone (21), are all phenolic compounds, it is not easy to find any part structure which is both common to them and to the benzimidazole fungicides (Fig. 4). However, it is quite probable that the mode of action of the benzimidazole antidotes is closely related to that of benomyl itself, which is believed to exert its antifungal activity through its interaction with 13-tubulin. In fact, as shown in Fig. 6, there are interesting part, and gross, structural similarities between the benzimidazole antidotes and several naturally occurring tubulin-binding agents such as colchicine (22) [38], combretastatin (23) [39, 40], podophyllotoxin (24) [41], and streptopyrone (25) [42].
472
TAHARA and INGtlAM
Structure-activity Relationships of Antidotes to Benzimidazole Fungicides The relative antidote activity of various naturally occurring phenolics (1721) and related compounds was examined using the TLC plate and paper .
,..
,
.,
,,..
,
,_42
,
.
,
.
o
R=H* 19
CHz
,,,
,
lapachol* 27
R=COCHz 26
NOR More active than emodln (181 , ,,,,, ,=,..,
,,...
,
0
,
O
Me(~
.
,
~
H
H3CI
,
,.,
H
~
~," v -OH 0 18 emodln 32 a mixture of 3- and 1or 8- O-dimethylemodin
v
10
OH ~
O
g
o
[ OH
E
v O
33
28
Similar in activity to emodln CH 3
.OMe
,__CH3 17 a-tocopherol
CH 3
20 0
0
RI
R2 2 9 R=H* 30 R=OH*
34 R l =OH, R2=R3=R4--Rs=R6=H
35 R,=R2=R3=~=R6=H.R3=OH Se R,=R2-~=~=R6=H.R4=OH 37 Rl =R2=R3=R4=Rs=H, I~=OH 38 R I =Rz=R4=R5, R2=CH 3, R6=OH ,
,
,
OH 0
OH
Me
CH 3 42
39 R I =R3=R4 =H, I~=CH2OH, RsfR6=OH ..,
OH
Positive but less active than emodin
L
Fig. (7). Relative antidote activity of some classes of phenols on impregnated (20 nmol) paper disks against MBC contained in agar at 0.2 ppm. The data were obtained by the paper disk method using Cladosporium herbarum as the test fungus. .
,.
,.,
,
,
*Asterisked compounds exhibited both antidote and antifungal activity.
SIMPLE FLAVONES
n
473
H R o ~ O
H3C
OMe
O
0
40
43 R=H
41
o
44 R=CH3
Inactive compounds at the dose amount of 100 nmol/dlsk Active compounds
.o
.o
H "T "oH 45 R=OMe. ayameninA 48 R=H, ayamenln B
47 tectorlgenin
48 plscerythrol
Me
Inactive compounds
50 R ~=R2=H, lrllln C 51 R I=H. R2=OMe. lrllln B 52 R~=CH3. R2=OMe. lrilm A
49 alpinone ,,
,
ii
|
i
i
|
,,,
|
--
||
i
i
i . . . . . . . . .
Flavonold compounds qualitaUvely tested for their antidote activity against MBC by TI/2 bloassay Fig. (8). Supplemental data to Fig. 7. C o m p o u n d s 4 5 - 4 7 and 4 9 - 5 2 , see Hanawa et al., Phytochemistry, 1991, 30, 157 & 2197; compound 48, see Tahara et al., Z. Naturforsch., 1991, 46c, 331.
disk bioassay methods. The first experiment, designed to reveal the gross relationship between structure and antidote activity, involved a comparative paper disk study of stilbenoids (19, 20, 26), anthraquinones (18, 32-41), naphthoquinones (27-31), benzoquinones (42-44) and flavonoids (6, 10). The results are summarized in Fig. 7, and suggest that the antidote activity depends on the substituents affecting the polarity of each molecule, rather than on the nature of the molecular skeleton, since appropriately substituted stilbenoids (19, 26), naphthoquinones (27, 28), a flavone (10), and anthraquinones (18, 32, 33) were all highly active. In addition to the semi-quantitative data shown in Fig. 7, a qualitative TLC assay on some other compounds revealed that the coumaronochromones, ayamenins A (45) and B (46), the isoflavone tectorigenin (47), and the
474
TAHARA and INGHAM
coumaranochromanone piscerythrol (48) were all active, whilst a dihydroflavonol, alpinone (49), and the isoflavones, irilins C (50), B (51) and A (52), were inactive (Fig. 8) [43]. '
Test I. Threshold dose {mg/dlsk} for antidotes against 0.2 ppm MBC in agar 200:i00 ' "pg . . . .I00- I0 . Pg . . . I0-5 . Pg ,,
i
i
i
H
~ I Me~)
~)
63
H~)
~
6
i
5-I llg
I-0'2 lag
l
012-0.05 ~g
H H OH
54
Me
,
0
~~,sOMe
MeO
1 10 60 61 62
~[ ~
56
59
57
Test 2. Threshold dose inmol/dlsk)for'antidotes'against 01'4ppm MBC Lnagar . . . . . . . 100-20 nmol . . . . . . . . . . . . . . . . 20-4nmol '~, nmol
O
1
<~ [ ~
M e O ~ , ~~ MeO
O
10
..[
8
6o
O
[ff~~
..L l~eu
Fig. (9). Threshold amounts of some flavones and a chromone exhibiting antidote activity (paper disk method). ,
,.
,,
,,.
,
,.
,,
* Based on the extent of fungal growth, flavone 62 was slightly more active than its regio-isomer61. See also the footnote to Fig. 7.
,
SIMPLE FLAVONES
475
The relationships between structure and antidote activity among 13 synthetic and naturally occurring flavones(l, 6, 10, 53-62), and a chromone (63), were investigated by the paper disk method (see also the results and notes to Fig. 7) [44]. The results are summarized in Fig. 9. Test 1, carried out on agar impregnated with MBC at 0.2 ppm made it possible to select the five most active flavones, which were then further compared with each other, on a molar basis, on agar containing double the previousamount of MBC (0.4 ppm, Test 2). Quite simple flavones with mono-methoxy substitution at position C-5 or C-7 were found to exhibit the highest antidote activity. The results suggest that less polar substituents, especially those located at C-5, increase the activity significantly. This semi-quantitative assay revealed that those compounds with high antidote activity had some, or all, of the following structural features: 1) A complete flavone skeleton 2) Absence of substitution on the B-ring 3) Presence of hydrophobic (lipophilic) substitution on the A-ring Regional isomerism in ring A appeared to have only a slight effect on activity (compare 61 and 62).
Some Characteristic Aspects of Polygonaceae Antidotes Against Benzimidazole Fungicides To further understand the properties of benzimidazole antidotes, the following experiments were carried out using the anthraquinone emodin (18) as a representative antidote found in the Polygonaceae.
Relationships Between Dosal Amount of MBC (12) and the Antidote Activity of Emodin (18) When Cladosporium herbarum was grown on PYG agar medium for 48 hr at 25 ~ a thick mycelium with dark greenish spores was produced. In contrast, mycelial growth was completely inhibited on agar containing 0.25 ppm MBC (MW 191). When the agar medium contained both 0.25 ppm MBC (12) and 0.5 ppm emodin (18, 1.4 mol equivalent to 12), the mycelium of C. herbarum appeared to grow normally, although fewer spores were produced when compared with growth on agar only. Although the thickness of the mycelium was far less than that on MBC free medium, mycelial growth still recovered after the addition of 0.25 or 0.13 ppm of emodin (ca 0.71 or 0.35 mol equivalent to MBC) to the agar. However, as shown in Table 3, at highMBC concentrations (ca. 4-5 times
TAIIARA and INGltAM
476
T a b l e 3.
R e l a t i o n s h i p B e t w e e n M B C (12) C o n c e n t r a t i o n and the A n t i d o t e Activity o f E m o d i n (18) |
MBC (ppm)**
Growth of Cladosporium herbarum (mm)* Emodin nmol/disk 20
0 i
100
ii
"
'
l',
0.2
0
8
0.4
0
3
0.8
0
*Extent of mycelial growth as measured from the edge of each emodin-impregnated paper disk to the outer edge of the growing mycelium. Values are the average of duplicate data. **Concentration in PYG medium.
the minimum inhibitory concentration), not even 1O0 nmol of emodin per disk was sufficient to cause a recovery in the growth of the test fungus, even though MBC was only fungistatie and not fungicidal at 0.8 pprn.
Antidote Activity of Emodin Against Other Benzimidazole Fungicides In the initial screening test, we used benomyl (11), or its active principle MBC (12), as a representative of the benzimidazole fungicides, but subsequently we tested emodin (18), on treated paper disks, for antidote activity against a number of other widely used benzimidazole fungicides. These tests were also carried out on agar containing fungal spores or mycelium, and incorporating a fungicide in amounts in excess of its minimum inhibitory concentration. In addition to C. herbarum, these bioassays also used Botrytis cinerea and Aspergillus nidulans as test fungi mainly because of their differences in susceptibility to each fungicide. As shown in Table 4, emodin exhibited antidote activity against all five benzimidazole fungicides [benomyl (11), carbendazim (MBC, 12), thiabendazole (TBZ, 13), thiophanate-methyl (14), and nocodazole (16)] tested in this experiment. Emodin (18) is thus fully confirmed as a benzimidazole antidote. The other antidotes isolated from the Polygonaceae all had activity closely comparable with that of emodin.
Chromone Antagonism to the Antidote Activity of 5-Methoxy-6,7methylene-dioxyflavone (10) and Emodin (18)Against MBC (12) Although 5,7-dimethoxychromone (63) showed weak antidote activity against MBC (12) as described in PP. 472 and 473 (Fig. 7), another chrornone derivative, 5-hydroxy-7-methoxychromone (64) synthesized in
SIMPLE FLAVONES
T a b l e 4. i
477
A n t i d o t e Activity of E m o d i n (18) A g a i n s t Some B e n z i m i d a z o l e F u n g i c i d e s i
,,
Fungus tested , ,,,,,
=
Fungicide (do~': ppm)
Antidote activity of emodin* (dose: pg/disk)
be"omyl, 11' i0.2) ' ' carbendazim, 12 (0.2) thiabendazole, 13 (0.8)
....
,
Cladosporium herbarum
.....
(5.4)
++
++ (5.4)
++ (2.0) .
Botrytis cinerea
.
.
+
(5.4)
nocodazole, 16 (0.5)
++
(5.4)
Aspergillus nidulans ........... |
.
lhiophanate-methyi', 14 (2.0) ,i
.
.
* Paper disk method; +, growth zone, 2-3 mm from the edge of the paper disks; ++, growth zone, more than 5 mm from the edge of the paper disks.
Antagonistic Effect of 5-Hydroxy-7-methoxychromone (64) on the A n t i d o t e Activity of 5 - M e t h o x y - 6 , 7 - m e t h y l e n e d i o x y f l a v o n e (10) a n d E m o d i n (18) Against M B C (12)
T a b l e 5.
i,
Growth of C herbarum ( m m , 24 hr)*
Antidote (20 nmol/disk)
Concentration of 5-hydroxy-7-methoxy-chromone (64, ppm)
0
0.1
5-methoxy-6,7-methylenedioxy-flavone (10)
8**
7
emodin (18)
9**
8
i
,o1!ooi,ooo 5
0
0
6
0
0
,,
* Paper disk method; growth zone in mm from the edge of paper disks impregnated with antidotes to the outer edge of
the growth zone. MBC (0.2 ppm) was also incorporated in the PYG medium. The growth of C. herbarum was slightly suppressed in PYG medium containing 100 ppm of compound (64) but no MBC (12). **Antidote activity of 18 and 10 in the absence of the antagonist 64.
our laboratory, was found to be antagonistic to the antidote activity of 5methoxy-6,7-methylenedioxyflavone (10) against MBC (12). As shown in Table 5, the antidote activity of the flavone (10) was partly or completely suppressed when the chromone (64) was added to PYG medium containing 0.2 ppm of MBC (12)and spores of C. herbarum. This interesting result was observed in antidote experiments involving both a flavone (10) and an anthraquinone (18), and is strongly indicative of a common mechanism of antidote activity, although the mode of the antidote action has not yet been determined. It was also confirmed that 5-hydroxy7-methoxychromone (64) had no synergistic effect on the fungitoxic activity of MBC. Further experiments using Neurospora crassa revealed that the antidote activity of the naphthoquinone lapachol (27) against MBC was also antagonized by 5-hydroxy-7-methoxychromone (64).
TAHARA and INGHAM
478
Antidote ActivRy o f Emodin (18) Against Diethofencarb (15) in an M B C Resistant Strain ofNeurospora crassa
Recently, the carbamate fungicide diethofencarb (15) which takes advantage of a negatively-correlated cross-resistance phenomenon, has been developed as a potent fungicide against benzimidazole-resistant fungi [45, 46]. Studies on the mode of action of 15 have revealed that ]3-tubulin in the benzimidazole-resistant strain F914 of N. crassa has an affinity for diethofencarb (15) rather than for benzimidazole derivatives, whilst in a wild-type strain of iV. crassa there was an inverse affinity [46, 47]. T a b l e 6.
A n t i d o t e Effect o f E m o d i n (18) on the G r o w t h o f Neurospora crassa in the P r e s e n c e o f M B C (12) or D i e t h o f e n c a r b (15)
Fungicide in agar (ppm)
Emodin
Growth of Neurospora crassa*
(dose:p g/disk) Wild-type
Strain F914
0
0
G
5.4
9.7
G
0
G
0
54
G
10.0
,,,
MBC (0.2)
....
Diethofencarb (0. l)
,
* Paper disk method; G, normal growth; 0, completely inhibited. Other figures indicate the width of the growth zone in mm from the edge of a paper disk with and without emodin (18). Results are the average of duplicated data.
According to the results of Fujimura et al. [45], the minimum inhibitory concentrations (MICs) of MBC (12) and diethofencarb (15) for wild-type N. crassa were 0.2 ppm and > 100 ppm, whereas for N. crassa F914 they were >100 ppm and 0.1 ppm, respectively. Therefore, an experiment using two combination systems, (1) benzimidazole-resistant strain + diethofencarb + emodin, and (2) benzimidazole-susceptible strain + MBC + emodin was carried out in order to obtain data on whether emodin could exhibit antidote activity in both systems. In this experiment, only emodin was used as an antidote because of the relatively weak activity of other antidotes in the interaction of MBC with wild-type N. crassa [44]. The results are summarized in Table 6, and indicate that emodin (18) can also act as an antidote against diethofencarb (15) in a benzimidazole-resistant strain of At. crassa. These and other results (Tables 4 and 6) are indicative of the versatility of emodin (18) as an antidote to the action of certain types of fungicide which possess an affinity for fungal ~-tubulin, and may suggest that the antidote effect of emodin is closely linked to the function of ]3-tubulin.
SIMPLE FLAVONES
479
Interaction Between the Antidote Emodin (18) and MBC-bound Fungal Tubulin in a Cell-free System
Preparation of Tubulin Fraction from Neurospora crassa N. crassa was grown for 14-15 hr at 28 ~
in a shaking liquid PYG medium (peptone-yeast extract-glucose-water=1%, 0 . 1 % and 5 % respectively, w/w). The mycelium at the late logarithmic phase was harvested and washed successively, five times with deionized water and twice with NC buffer I solution [48]. The vacuum-filtered mycelium was frozen in liquid nitrogen, and homogenized with a pestle in an ice-chilled mortar. To the dampish homogenate was added 0.5-1 ml of NC buffer I, and the resulting material was further macerated for 10 min. The resulting homogenate was centrifuged at 4 ~ for 45 min at 48,000 x g to yield a crude extract of N. crassa, which was used for the [2-14C]-MBC binding test. When the extract (protein 28.8 mg) was incubated with 2.8 nmol of [214C]-MBC (sp. act. 1.93 mCi/mmol) and applied to a Sephadex G-25 column (15.4 ml), radio-activity was detected in the eluate (NC buffer II [48]) as two peaks, the first from 5 to 9 ml and the second from 22 to 40 ml. The first peak seemed to be due to bound MBC and the latter to free MBC as described by Davidse [49], the molecular size of the bound MBC complex being estimated at ca 100,000 by gel filtration using Sephadex G150.
Effect of Emodin on theBinding of Tubulin with 12-14C]-MBC The charcoal method [50] was applied to determine free [2-14C]-MBC in the binding test solution which consisted of crude fungal tubulin and [214C]-MBC. This method was successfully used to examine the ability of emodin to compete with [2-14C]-MBC bound to the colchicine site of [3tubulin. As shown in Fig. 10A, the benzimidazole fungicide, nocodazole (16) can release [2-14C]-MBC bound to ~-tubulin because of the property of 16 to compete with MBC. Under the same conditions, however, the antidote emodin (18)could not release the I]-tubulin bound [2-14C]-MBC (Fig. 10B). These results clearly suggest that the benzimidazole antidote emodin does not compete directly with the binding site for MBC in 13tubulin [44].
Concluding Notes It is now well known that the antimitotic benzimidazole fungicides do not prevent spore germination, but act by inhibiting the mycelial growth of many fungi. We have found that antidotes against benzimidazole
480
TAHARA and INGHAM
fungicides, which occur in some species of the Polygonaceae, can overcome the growth suppression of C. herbarum caused by MBC (12). In cultures containing both fungicide and antidote, the growing mycelium is indistinguishable in extent and appearance from that produced in medium without any benzimidazole fungicide. Although we initially considered that antidotes against benzimidazole fungicides may have a direct interaction with ~-tubulin, the antidote emodin (18) showed no competitive effect on the binding of MBC to [3-tubulin in a cell-free system. Further biological and biochemical experiments are therefore required in order to fully elucidate the mode of action of the Polygonaceae antidotes revealed by our studies. [A] H O[~i
,~-NI-IC OOMe
I
O
|
CONTROL
I
2
3
I/[Free MBC] (dpm/500 ~I)l x 104 [B] OH 0
OH ............
..
o
i
I0 gM
! CONTROL
m O
t:h
0
"
0
I
I
"
I
2
"
' ....
I
"
3
4
I/[Free MBC] (dpm/500 ~l) "I x 104 Fig. (10) Effect of nocodazole (16) [A] and emodin (18) [B] on [2-14C]-MBC (12) bound to 13tubulin.
SIMPLEFLAVONES
481
Although the effect of the antidotal flavone (10) on the binding of tubulin with [2-14C]-MBC has still to be investigated, it was recently reported that the flavone centaureidin (5,7,3'-trihydroxy-3,6,4'trimethoxyflavone, 65), obtained from extracts of Polymnia fruticosa (Compositae), could inhibit by 55 % the binding of [3H]-colchicine to purified tubulin [see, ref. 86] . As centaureidin (Fig. 11) has structural features in common with the natural benzimidazole antidote (10), and several other antidotal flavones (Fig. 9), it seems probable that flavone (10) may also interact with tubulin at the colchicine-binding site. As 10 prevents benzimidazole derivatives such as MBC (12) from expressing their normally powerful fungitoxic effects, it can be argued that the mode of action of MBC and related compounds involves in some way the colchicine-binding site(s) on fungal tubulin. H3 C H 2 C O ~
NHCOOCH{CH3)2
Me~
OH
O
HaCH2CO 64
15 dlethofencarb
H-Z
HaCO
~
5-hydroxy-7-methoxychromone
,fOCHa
fN Cl
"
"~
OH
0
/
~OCH2CHa
"OCHa
65 centauretdtn
65a zarllamlde
Fig. (11). Structures of the non-benzimidazole fungicides, diethofenearb (15) and zarilamide (65a) which inhibit tubulin assembly, 5-hydroxy-7-methoxyehromone (64), an antagonist of benzimidazole antidotes, and centaureidin (65), a tubulin-interactive flavone.
Various anthraquinones, including emodin (18), and some flavones closely related to 5-methoxy-6,7-methylenedioxyflavone (10), have also been found to inhibit the enzyme protein-tyrosine phosphokinase which is involved in the regulation of cell division and tumorigenesis [51, 52].
482
TAHARA and INGHAM
Another survey involving antidotes against the fungicide zarilamide (65a, Fig.ll), which is also known to be an antimitotic agent, inhibiting [3tubulin assembly in Oomycetes (e.g. Pythium and Phytophthora spp.), yielded the phytosterols, campesterol, stigmasterol and 13-sitosterol, and their acylated or non-acylated glycosides, as the active antidotes [53]. Interestingly, these sterol antidotes were also found to be antagonistic against the fungitoxicity of taxol to Pythium ultimum [53]. However, we have not been able to detect any common basis for activity between the two classes of antidotes found using the screening systems (1) benzimidazole fungicide-Polygonaceae antidotes-MBC susceptible fungi and (2) zarilamide-sterol antidotes-Pythium ultimum. Finally, some herbicide antidotes are now commercially available [54], and the naturally-occurring isoflavone genistein (8) has been found to act as an antidote against the herbicides, haloxyflop and alloxydim [55]. However, as far as we are aware, no antidotes to benzimidazole-related compounds, or to other types of fungicides, have previously been reported. HOST-SPECIFIC ZOOSPORE ATTRACTANTS OF THE FUNGUS A P H A N O M Y C E S COCHLIOIDES, THE CAUSE OF SPINACH ROOT ROT Introduction
Aphanomyces cochlioides, a soil-borne plant pathogenic fungus, is responsible for a serious root rot disease of spinach (Spinacia oleracea L.), and a damping-off disease of sugar-beet (Beta vulgaris L.). The fungus also affects some other species of Chenopodiaceae, e.g. Chenopodium album L. The flagellate zoospores of Aphanomyces spp. originate from resting oospores in the soil (or in decaying plant material), and from zoosporangia formed asexually in diseased tissue. The zoospores swim in the soil water. It is now believed that the zoospores are initially attracted to the host plant by chemicals exuding from healthy roots [12, 56, 57]. Previous studies have shown that zoospores ofA. raphani Kend.[56], which infects Cruciferae, are attracted to the hypocotyls of cabbage seedlings, and that zoospores of A. euteiches Drechs.[12], which infects Leguminosae, are attracted to the root caps of pea. The attractants of A. raphani and A. euteiches zoospores have recently been identified as indole-3-aldehyde [58] (66) in cabbage seedlings, and prunetin (an isoflavone, 9) [12] in pea seedlings. The compatibility of Aphanomyces cochlioides with several higher plants has been investigated by Ui and Nakamura [59]. According to their results, the pathogen was highly compatible with Chenopodiaceae
SIMPLE FLAVONES
483
Results of a Preliminary Screening Test for Zoospore Attractants in Roots of Species* Compatible and Incompatible with A p h a n o m y c e s cochlioides
T a b l e 7.
,
Plant
,,,,
,
,
Attractable concentration (ppm)** ,,
, m
i
J
,
,,
i
i
Chenopodiaceae Chenopodium album L.
30
Beta vulgaris L. var. saccharifera
100
B. vulgaris L. vat. cicla
30
Kochia scoparia Schrad.
30
Spinacia oleracea L.
30
Amaranthaceae Celosia cristata L.
30
Amaranthus tricolor L.
30
A. inamoenus Willd.
100
Corydalis ambigua Chain. et Schlecht.
NA***
Papaver orientale L.
100
P. rhoeas L,
1000
Portulaca oleracea L.
NA
Polygonum sachalmensis Fr. Schm.
NA
Rumex obtusifolius L..
NA
Trifolium repens L.
NA
Medicago sativa L.
NA
Erigeron annuus L.
100
Taraxacum officinale L.
NA
Lycopersicon esculentum L.
NA
Papaveraceae
Portulacaceae
Polygonaceae
Leguminosae
Compositae
Solanaceae
Solanum tuberosum L. ,m
NA ,
,
,
,
,
,
,
,
*Roots from plants growing outside were extracted with MeOH for 7 days, and the concentrated extract was then shaken with diethyl ether. The organic layer was used as the test solution. Water soluble fractions were also tested and confirmed to be inactive at 1000 ppm. ** Activity was checked by the particle method. The lowest concentration which led to a clear increase of zoospore density around the treated Chromosorb W AW particles was determined. *** NA: inactive at a concentration of 3000 ppm.
484
TAHARA and INGtlAM
(Chenopodium sp., Beta spp., and Spinacia sp.) and Papaveraceae (Papaver sp.), and slightly less compatible with Amaranthaceae (Amaranthus spp. and Celosia sp.). It had low compatibility with the Portulacaceae (Portulaca sp.) and Caryophyllaceae (Dianthus sp. and Stellaria sp.), and was incompatible with members of the Cruciferae (Brassica spp., Raphanus sp., and Rorippa sp.), Commelinaceae (Commelina sp.), Polygonaceae (Polygonum sp.), Compositae (Lactuca sp.), Cucurbitaceae (Citrullus sp. and Cucumis sp.), Solanaceae (Solanum sp. and Lycopersicon sp.), Leguminosae (Pisum sp. and Phaseolus sp.), Linaceae (Linum sp.), and Gramineae (Avena sp.). In our laboratory, the zoospore attractant properties of crude extracts from the roots of 20 species belonging to 8 plant families were checked by the particle bioassay method (see PP. 485 and 486). The results, shown in Table 7, confirm that there is a strong positive relationship between plantpathogen compatibility, and the zoospore attractant activity of root extracts. The crude root extracts from six out of eight species belonging to the Chenopodiaceae and Amaranthaceae were highly attractive to zoospores suspended in water in a Petri dish, when Chromosorb W AW particles coated with a 30 ppm solution of the ether-soluble constituents from methanolic root extracts were added to the suspension. In earlier studies, Rai and Strobel [57] found that zoospores of A. cochlioides were attracted to an organic acid fraction and a neutral fraction (i.e., gluconic acid, fructose and glucose), which exuded from the roots of sugar beet seedlings. Yokosawa et al. showed that NaCI, KNO3 and NaNO3 from sugar beet seedlings also attracted zoospores of A. cochlioides at concentrations between 10-2 and 10.3 M [60], whilst N-[2(4-hydroxy-3methoxyphenyl)-ethyl]ferulamide (69) was isolated as an attractant from young spinach (whole plant, Yokosawa et al., unpublished). More recently, we have found that the roots of adult spinach also contain a substance which shows potent attracting activity for the zoospores of A. cochlioides. In this section, we will first describe the bioassay, isolation and identification of attractants from spinach and pigweed (Chenopodium album) roots, and then discuss the experiments that were undertaken to evaluate the activity. Table 8 lists various compounds which are already known to specifically attract Zoospores of phytopathogenic fungi.
Bioassay Two strains of A. cochlioides (AK-1 and AC-5) isolated from the soil of spinach and beet fields, respectively, were used for the studies described in this review. Both strains of A. cochlioides were grown for 3-4 days on corn meal agar (Difco) at 20 ~ Large pieces of mycelium-covered agar were then placed in Petri dishes containing 40 ml of distilled water [10]. To remove nutrients from the agar, the water (40 ml) in each Petri dish
SIMPLE FLAVONES
T a b l e 8.
485
Chemical Attractants of Phytopathogenic Zoospores Isolated from Plant Species Attractant ,,
,
' ,
|
Host plant '
,,
,,
l~hytopathogen'' and Reference ,,
Brasslca oleracea L. vat. capitata 08 3-indolecarbaldeh~de
i
,
Aphanomyces raphanl Yokosawa & Kunlnaga 1581
....
Aphanomyces euteiches
OH
9 prt~.,eUn
M~
OH
Ptsum satlvurn
O
~ o--R
~ 0
NaNO3
H
KN03
07 R=H 68 R=Me ~ MeO
Yokosawa eta/. | 12l
. . . . . . . . spinach
SpOtacm oleracea
Aphanomgces coc~loides Yokosawa et cd. 1601
OMe O
H
H
Yokosawa et oL unpublished {1989}
69 >
spinach
Aphanomyces cochlloates
Horio et al [ 10]
8 cochliophl~ H
M ~ HO
~
OMe
~ 0
H~ ~ #
amino acids, saccharides unidentified attractants .
.
.
Chenopodflun album
Horio et at [61 |
70
R 0 t~~O 7 R=H: daidzein 8 R-OH: genistein
.
OH
soybean Glycine max
PhytophthorasoJae Morris & Ward [ 11]
H
Py~nspp. Donaldson & Deacon 1621
.
was changed three times at intervals of 30 rain. The Petri dishes, containing a final 25 ml of distilled water, were then allowed to stand for 15-24 hr at 20 ~ to promote the release of zoospores. Immediately before
486
TAtlARA and INGHAM
use, a portion of the liquid was collected and the zoospore concentration adjusted to about 104 spores/ml with distilled water. The spore suspension was then transferred to a small Petri dish for bioassay. Two bioassay techniques, the capillary method [58], and the drop method [63], have already been developed to study the chemotaxis of phytopathogenic zoospores and gametes of marine brown algae. We have devised a new procedure (the "particle method") which is a qualitative, rather than a quantitative, method of detecting an attractant after it has been absorbed on to an inorganic particle. This method has been used to study attractants of A. cochlioides zoospores in the roots of both spinach and pigweed (Chenopodium album). The particle method is carried out as follows. Several particles of Chromosorb W AW (acid washed; a commercially available support for the liquid phase in gas chromatography) were placed on a watch glass, and 5 lal of a diethyl ether or ethyl acetate solution of the test compound, adjusted to an appropriate concentration, was dripped carefully on to these particles. Any excess solution on the watch glass was immediately absorbed with a piece of filter paper, and the particles were then air-dried at room temperature. A few of the treated particles were then dropped into an aqueous suspension of zoospores in a small Petri dish (see above). The behaviour of zoospores around the particles was observed microscopically after a period of 1 min. Control particles were treated with ether or ethyl acetate only. Microscopic observation revealed that around particles treated with a non-active compound, or with an insufficient amount of an active one, the zoospores moved in a monotonous and straightforward fashion, and at a constant speed. In contrast, near particles treated with active substances, the zoospores moved faster, in a complex zigzag or circular manner. When the amount of active material was increased, greater numbers of zoospores collected around the particles. The zoospore density shown in Fig. 12[A] was regarded as the standard against which the activity of different compounds was defined. The minimum concentration required to give this density was determined by consecutive dilutions of a sample solution, with the relative activity of samples being deduced from the results that were obtained (see Tables 9, 12 and 13).
Chenopodiaceae Attractants of A. cochlioides in spinach The fractionation stages, which were monitored by bioassays using zoospores of A. cochlioides AK-1, are illustrated in Scheme 3. The relative activity of each fraction, as determined by the particle method, is shown in Isolation and Identification of Zoospore Attractants from the Table 9.
SIMPLE FLAVONES
487
F i g , (12). A t t r a c t a n t effect o f treated and u n t r e a t e d particles on z o o s p o r e s o f
Aphanomyces
cochlioides The figure shows dark field microscopic photographs of a zoospore suspension containing Chromosorb W AW particles (ca 150 x 200 ~tm) with and without the attractant, cochliophilin A (6). [A]: a particle soaked in !.0 x 10-8 M cochliophilin A, [B]: a control particle treated with solvent alone. The zoospore density around the particle [A] containing the attractant is clearly higher than around the control particle [B]. T a b l e 9.
Procedures for the Zoospore Attractant in Roots of Spinacia
Purification
oleracea |
i
Attractancy (ppm)
from 23 kg of S. oleracea roots (mg)
Fraction
i
,
i
Ether solubles in MeOH ext. ,,,
,
,i
.......
79,200 i|l
30
,
Neutral & phenolic fraction
49,390
CHCi3-MeOH/SiO 2 column frac.
28,810
10
90 % MeOH phase/.-hexane
4,754
1
Sephadex LH-20 column frac.
496
0.1
Toluene-acetone/SiO 2 column frac.
55.3
0.01
Preparative-HPLC
14.7
0.001
10
i
i
i
ill
|l,
,,
,
,
Fresh roots of spinach (Spinacia oleracea L. cv. Solomon) were collected in Sapporo at harvest-time in November. The concentrated methanol extracts were partitioned between water and diethyl ether after being
488
TAHARA and INGtlAM
Fresh roots of Spinacia oleracea (23 kg) I extraction with MeOH MeOH extraqcts solvent partition i
,i
i
I
l
Aqueous layer
Ether layer (60 gl
washing with 5 % NaHCO a i i
i,
ii
l
Neutral and phenolic fractions 49 g
Washings (acidic fraction}
SIO2 column chromatography {CHCIa-MeOH} Eluate (0-1% MeOH in CHCI3}. 29 g I solvent partition
I
'
1
nvHexane layer
90 %-MeOH layer 4.8 g
Sephadex LH-20 column chromatography (Hexane-EtOAc) Eluate 125-100 ~ EtOAc in hexane). 500 mg SIOz column chromatography {toluene-acetone)
Eluate (1-2 % acetone in toluene}, 55 mg reparative HPLC on Inertsil PREP ODS 120X250} mm) H20-CHaCN=3:7, flow rate 5 ml/mln UV at 254 nm. tR 27.0-28.4 mln Cochliophllin A {8}. 15 mg
S c h e m e 3. Isolation procedures for cochliophilin A (6) from the roots o f spinach.
acidified to c a pH 3 with HC1/H20, and the organic layer was washed with 5 % NaHCO3. The concentrated ether layer yielded 49 g of neutralphenolic constituents with a relative activity of 10 mg/l. This fraction was chromatographed over silica gel and eluted using a chloroform-methanol gradient. The bioactive fractions, eluting with 0 - 1 % methanol in chloroform, were combined, concentrated, and partitioned between 90 % aqueous methanol and n-hexane. The active, aqueous methanol layer was concentrated, and the residue was chromatographed on a Sephadex LH-20 column using a hexane-ethyl acetate gradient as the eluent. The active fraction, which eluted with 25-100 % ethyl acetate in hexane, was
SIMPLE FLAVONES
489
rechromatographed on a silica gel column eluted with a toluene-acetone gradient. The active principle was present in the fraction that eluted with 1-2 % acetone in toluene. Final purification was achieved by HPLC on a Cls column (Inertsil PREP ODS, 20 mm i.d. x 250 mm) using a mixture of acetonitrile-water (7:3) as the mobile phase to yield ca 15 mg of pure, active compound 6 (6.5 x 10.5 % overall yield). Because of its ability to attract zoospores of A. cochlioides, the compound was named cochliophilin A. The UV spectrum in methanol (~max, 276 and 317 nm) suggested that cochliophilin A (6) had a flavone structure. In the IH NMR spectrum, the presence of a singlet at ~5 6.68, attributable to the flavone H-3, also supported this conclusion. Signals for aromatic protons at 87.87 (2H, m) and ~5 7.54 (3H, m) indicated that the B-ring was unsubstituted. One proton signal at ~5 12.70 was easily assigned to a chelated hydroxyl group at C-5. The singlet at ~56.10 (2H) was characteristic of a methylenedioxy group, which is a relatively uncommon feature of flavonoid compounds. As cochliophilin A (6) gave a positive response in the Gibbs test, it was concluded that C-8 was unsubstituted [64]. The methylenedioxy group was therefore allocated to C-6/C-7, allowing cochliophilin A to be identified as 5-hydroxy-6,7-methylenedioxyflavone (6) [10]. Cochliophilin A has previously been isolated from sugar beet roots (Beta vulgaris L. var. saccharifera Alefeld) infected with the fungus Rhizoctonia solani [9], where it is accompanied by several other methylenedioxy-substituted flavonoids. A second flavone attractant for zoospores of A. cochlioides has also now been isolated from the leaves of spinach [65] and identified as 5,4'dihydroxy-3,3'-dimethoxy-6,7-methylenedioxyflavone (71). Although this compound has already been found as a glucuronide derivative in spinach [66], the role of the aglycone 71 in the interaction between the host plant and its fungal pathogens has still to be determined.
OH
OH
0
,OH
-~ O
"OCH 3
71 5,4'-dlhydroxy-3,3'-dlmethoxy6.7-methylenedioxyflavone
76 2,3- methylenedloxybenzolc acid
490
TAHARA and INGHAM
Attractants of A. cochlioides in Chenopodium During a survey of zoospore attractants in the Chenopodiaceae, we found that root extracts of pigweed (Chenopodium album) contained both cochliophilin A (6) and a new phenolic amide chemo-attractant, N-transferuloyl-4-O-methyldopamine (70). Fresh roots of C. album (1.2 kg), collected on the Hokkaido University campus, were extracted with methanol. The methanol extracts were then partitioned between diethyl ether and water. The ether layer was washed with 2N HC1, and then with 5 % aq. NaHCO3, and the resulting neutral-phenolic fraction (4.0 g) was chromatographed on a silica gel column using a CHCI3-MeOH gradient. Each column fraction was monitored for zoospore attracting activity. The active fractions (0.2 g), which eluted with 2-5 % MeOH in CHCI3, were applied to a Sephadex LH-20 column (75 ml). Elution with MeOH yielded a highly active fraction (retention vol., VR 0-85 ml, about 0.14 g) which was rechromatographed on a reversed phase column (7 g) eluting with THF-H20 (2:3) to yield 8.9 mg of material (VR 0-23 ml). Final purification was achieved by HPLC [Inertsil ODS 5 ~m (250 x 4.6 mm), MeOH-H20 (3:2), flow rate: 5 ml min -~ and UV monitor set at 320 nm] to give the amide 70 (0.1 mg) as an oil. The molecular formula of 70 was determined to be CI9H21NO5 by HRmass spectrometry. In the ~H NMR spectrum of 70, signals for two methylene groups, two methoxy groups, six aromatic protons, and one disubstituted trans-olefine group were observed. Most of these signals were very similar to those of N-trans-feruloyl-3-O-methyldopamine (69) previously isolated from spinach [67], although chemical shift values for H-2' (5 6.74, ~i, J=2.1 Hz) and H-5' (~i 6.85, ~i, J=8.1 Hz) indicated that the phenethylamine (dopamine) part structure had slightly different aromatic substitution. As an NOE interaction was observed between the methoxy protons (15 3.81, s) and H-5' (~i 6.85, ~, J=8.1 Hz) in the phenethylamine unit, the structure of 70 was considered to be N-trans-feruloyl-4-Omethyldopamine [61 ]. The structures of the main attractants found in spinach and Chenopodium roots were unambiguously confirmed by chemical synthesis. Cochliophilin A (6) was synthesized as shown in Scheme 4. 5,6,7-Trihydroxyflavone (baicalein, 54), prepared by the method of Agasimundin et al. [32], was converted into the corresponding 6,7methylenedioxy derivative using the procedure reported by Iinuma et al. [33]. The physicochemical properties of synthetic cochliophilin A were in good agreement with those of the natural compound (6). The ferulamide derivative 70 was prepared according to the route shown in Scheme 5.3O-Benzylisovanillin (72) was first transformed into 3-benzyloxy-4methoxy-13-nitrostylene (73) by condensation with nitromethane. Reduction of 73 with lithium aluminum hydride yielded 3-O-benzyl-4-Omethyldopamine (74). Aeylation of 74 with ferulic acid in the presence of
SIMPLE FLAVONES
491
N,N'dicyclohexylcarbodiimide afforded N-trans-feruloyl-3-O-benzyl-4O-methyldopamine (75), which was finally deprotected to yield 70. The regio isomer N-trans-feruloyl-3-O-methyldopamine (69) was also synthesized by an analogous route using 4-O-benzylvanillin instead of 3O-benzylisovanillin. The physicochemical properties of synthetic 7t} were indistinguishable from those of the amide isolated from Chenopodium. Compound 70 is the first naturally occurring amide known to possess a 4O-methyldopamine moiety, although 4-O-methyldopamine itself has been isolated from the Cactaceae [68]. In this respect, it is interesting, from a chemotaxonomic point of view, to note that both the Chenopodiaceae and Cactaceae are characterized by the production of nitrogen-containing betacyanin pigments rather than anthocyanins. The isomer N-transferuloyl-3-O-methyldopamine (69), of compound 70, has previously been isolated from both the Chenopodiaceae [67, 69] and Lauraceae [70]. OH ~ C O O E t
O
OH
In Ph20 reflux for 3 hr [32]
(•"•
O OH / 30 %-H202/4 % NaOH l O~ for 6 hr ~ 1321
HO0~,~ [
reflux for 3 hr OH
O
O
CH2Br2, KF/DMF
O
[331
H OH
O
54
Scheme 4. Synthetic route for cochliophilin A (6).
Activity of Chenopodiaceae Attractants and Detection of Cochliophilin A in the Rhizosphere of Spinach Activity of Chenopodiaceae Attractants The attractant activity of cochliophilin A towards zoospores of A. cochlioides (strains AC-5 and AK-1) was tested over the range 10-7-10-ll M by the particle method (Table 10). Zoospores of AC-5 were more sensitive to the attractant at all concentrations, when compared with the closely related strain AK-1. Zoospores of both AK-1 and AC-5 began to
TAHARA and INGHAM
492
OH
02
CHaNO2. Na2OD3
LiAIH4 in THF v
H reflux for I0 hr
-
OBn
OCH3 In MeOH, r.t. for 10 days
OC 3
[72]
OBn
[711 72
73
Bn=benzyl
H2
DCC in THF
OCH 3 m-
,
, _
, ,,
|,,, r
OBn
1701 ~
~
OCH3
a. BF3-Et20. Me2S ,,,,
,|,,,
,
|
, r
in CH2C12, r.t. for 48 hr
H OO_la
75
[73] 6
It
5 A
40CHa
2
OCH a
7O
Scheme 5. Synthetic route for N-trans-feruloyl-4-O-methyldopamine (70).
aggregate a few seconds after dropping Chromosorb W AW particles treated with cochliophilin A (6) into the suspension. Activity was observed at cochliophilin A concentrations of 10 .7-10 -g M (for AK-1) and 10-7-10 .9 (for AC-5). At a concentration of 10-~ M, zoospore aggregation lasted for over 60 min. Even at concentrations as low as 1.0xl0 -9 and 1.0xl0 -I~ M respectively, zoospores of isolates AK-1 and AC-5 showed typical aggregation, although zoospore density around the treated particles was less than that shown in Fig. 12[A]. Interestingly, zoospores of .4. raphani and A. euteiches, which are respectively known to be pathogens of cabbage and pea, showed no positive response to cochliophilin A even at the highest concentration of l xl 0 .7 M. These results strongly suggest that amongst Aphanomyces species, host-pathogen specificity may be closely
493
S I M P L E FLAVONES
linked with the ability of zoospores to successfully recognize, and respond to, chemical stimuli in rhizosphere exudates. Table
10.
Attractant
Effect of Cochliophilin
of Aphanomyces cochlloldes
A (6)on
Strains ofA.
Concentration (M)
cochlioldes
AK-I ,,
,
,
, =
,
1.0 x 10-7
_
AC-5 |
,
,
,
,
++
++
++
++
l.Ox 10 -8
++
4-+
3.0 X 10 -9
++
4-+
3 . 0 x 10-8
1.0X l0 "9
++
3.0 X 10"10
++
1.0 X 10 "10 3.0 x 10"11 i
Attractant activity was determined I min after dropping Chromosorb W AW particles treated with cochliophilin A (6), into the zoospore suspension. ++" indicates activity which is approximately equal to, or stronger than, that shown in Fig. 12 [A]. +: indicates activity which is weaker than that shown in Fig. 12 [A]. -: no observed activity.
The attractant activity of the ferulamide derivatives (70 and 69) towards zoospores ofA. cochlioides (AK-1 and AC-4) was also tested. As shown in Table 1 l, 70 exhibited clear chemo-attraction at l xl 0 .8 M, whilst its regio isomer 69 was only active around 10.5 M. MOiler developed a quantitative bioassay (the "drop method") to evaluate the attractant effect of test compounds in an aqueous medium, by using a water-insoluble fluorinated hydrocarbon as a solvent for attraetants of the gametes of marine brown algae [63]. The drop method, as modified slightly by Takayama [74], was also used to determine the attractant activity of cochliophilin A (6). A droplet of fluorinated hydrocarbon containing 3 x 1 0 "9 M of cochliophilin A, placed in a suspension of Aphanomyces (AC-5) zoospores, showed clear attractant activity. When 1x 10 .6 M of cochliophilin A was used, the zoospores rapidly aggregated on the surface of the droplet, and lost their flagella within 5-30 mins. This effect appeared very similar to that observed for ,4. cochlioides zoospores on the surface of the roots of spinach seedlings [74].
TAHARA and INGHAM
494
A t t r a c t a n t Effect o f the F e r u l a m i d e Derivatives (69 and 70) on Z o o s p o r e s o f
T a b l e 11.
Aphanomyces cochlioides Concentration (M)
69
70
AC-5
AK-I
i
3.0
x
10.5
AC-5 i
I i
II
++
nt J
1.0x 10.5 3 . 0 x 10 -6 l.Ox 10.6
+
nt
.+.
3 . 0 x 10-7 !.0 x 10"7
++
4-+
++
++
3 . 0 x 10-8
4-+
1.0x 10 -8
4-+
3 . 0 x 10-9
nt
1.0x 10.9
nt
I
+.
nt---not tested, see footnotes to Table 10. +-: shows borderline activity. 69: N-trans-feruloyl-3-O-methyldopamine 70: N-trans-feruloyl-4-O-methyldopamine
Quantification of Cochliophilin Rhizosphere of Spinach
A (6) in the Roots and the
Attempts to directly measure the levels of cochliophilin A (6) in crude extracts of plant material, or in the exudates from spinach roots, using gas chromatography or high performance liquid chromatography, were unsuccessful mainly because the compound occurred at a very low concentration. However, preliminary quantitative analysis using 500 MHz NMR, revealed that scanning samples about 1100 times was sufficient to detect the methylenedioxy signal (at ~i 6.11 in CDCI3) attributable to a few micrograms of cochliophilin A (6) in a few milligrams of crude plant extract. The successful micro-quantification of 6 by NMR was facilitated by using synthetic 2,3-methylenedioxybenzoic acid (76)as an internal standard, as this gave a recognizable methylenedioxy signal at ~i 5.73 in
CDC13. The amount of 6 in spinach roots was quantified as follows [75]: Spinach roots (48 g, fresh weight) at the harvest stage, were crushed and extracted with methanol. The concentrated extract was re-dissolved in 100 ml of water, adjusted to pH 2.7, and exhaustively extracted with ether.
SIMPLE FLAVONES
495
The ether extract was concentrated to yield a residue of 25.2 mg. 2,3Methylenedioxybenzoic acid (76) (10.3 Ixg) in ether was added to onefourth of this concentrate, and the mixture was then reduced to dryness. The residue was dissolved in CDCI3 and subjected to IH NMR analysis. From the ratio of the integrated methylenedioxy signal of 6 to that given by the known amount of internal standard, it was possible to calculate that the crude extract corresponding to 12 g of the fresh roots contained 64 ~tg of 6, with fresh roots containing 5.3 ~tg/g. The amount of cochliophilin A (6) in the rhizosphere of spinach [75] was also determined. One hundred spinach seeds were sown in a box containing a 32x24x7 cm layer of moist vermiculite. The resulting plants were supplied hydroponically with a nutrient solution of inorganic salts every 7 days. After 59 days growth, 3 litres of deionized water was carefully passed through the vermiculite layer, and the process was repeated with 1.5 litres of water after a further 2 days cultivation. The latter washings (ca 1000 ml), were collected and extracted with ethyl acetate. The extract was reduced to dryness to yield a residue (ca 7 mg) which was partitioned between hexane and 90 % aqueous methanol. The methanol layer was concentrated to dryness and the residue, which exhibited zoospore attractant activity at 30 ppm (particle method), was dissolved in CDCI3 containing the internal standard 2,3methylenedioxybenzoic acid (76), prior to NMR analysis. The amount of 6 that had accumulated over 2 days in the rhizosphere of spinach, was calculated to be 6.7 ~tg, or about 34 ng/plant/day. The above results show that apart from its presence in healthy spinach roots (ca 5.4 ppm), the flavone 6 also exudes into the rhizosphere where its level appears to be high enough to naturally attract pathogenic zoospores of A. cochlioides. Thus, the concentration of 6 in spinach root washings (6.7 ~tg/litre) was calculated to be ca 2xl 0 "s M, a level at which strong attractant activity would be expected on the basis of the data shown in Table 10. As with strigol [76, 77] and glycinoeclepin [78], both of which are well-known examples of host-recognition substances in root exudates, cochliophilin A must be wholly or partially responsible for the eco-chemical signalling between spinach (host) and the fungal pathogen ,4. cochlioides.
Structure-activity Relationships of Zoospore Attractants Twelve flavones (1, 6, 10, 53-55, 57-59, 77-79) including cochliophilin A (6), and a chromone (64), were chemically prepared in order to compare their attractant effects on zoospores ofAphanomyces coehlioides. Together with two isoflavones, genistein (8) and prunetin (9), the relative activity of each compound was determined using the particle method. The results for the various flavones lacking B-ring substitution are
TAHARA and INGHAM
496
T a b l e 12.
A t t r a c t a n t Effect of Flavones with an U n s u b s t i t u t e d B-ring on Z o o s p o r e s o f
Aphanomyces cochlioides J
Substituent
Comp. a
RI
Attractingacti vity (M)b
R2
R3
L
..
.
.
.
L,
, _,
6
OH
-O-C~12-O-
3.0x 10"9
100
58
H
"O'CH2"O"
3.0 x 10-8
10
10
OCH 3
1
H
H
59
OH
H
H
1.0 x 10-7
3.0
53
H
H
OH
1.0 x 10-7
3.0
57
OH
H
OH
2.0 x 10's......
15
55
OH
H
OCH3
1.0 x 10-8
30
79' .
Relative activity
54 . .
' OH .
.
.
.
77 "'a."
'
"O'CH2"O"
.....
H
1.7 x 10-7
1.8
1.0 x 10-7
3.0
H
OC2H5
6.5 x i0 '8
4.6
OH
OH
OH
1.0x l0 "7
3.0
OH
OCH 3
OCH 3
2.0 x 10.9
150
'
b: Minimum method. No concentration
concentration by the particle attracting actlvity was observed of one-third of the indicated
at a value.
given in Table 12. Data for other compounds (chromone, 4'-oxygenated flavones and isoflavones) are summarized in Table 13. T a b l e 13.
A t t r a c t a n t Effect of a C h r o m o n e , Two 7-O-Methylflavones, and Two Isoflavones on Z o o s p o r e s o f A p h a n o m y c e s cochlioides Substituent
C0mpd. a
ii
64
RI
R2
H
H
phenyl
H
78
p-OMe-phenyl
H
8
H
9
H
Relative activityc
>1.0 x 10-6
0.3>
R3 ....
55 ....
Attracting activity (M)b
OCH 3 OCH 3
1.0 x 10"8
30
OCH 3
>1.0 x 10"6
0~.3>
p-OH-phenyl
OH
>1.0 x 10"~
0.3>
p-OH-phenyl
OCH 3
>1.0 x I0 "6
0.3>
9
. . . .
b: The m i n i m u m active c o n c e n t r a t i o n of cochliophllin A (6} in this a s s a y w a s 3.0 x 10-gM (Table 12). c:
Based
on
the
activity
of
6,
see
Table
12.
SIMPLE FLAVONES
497
The flavones and related compounds used in these experiments were obtained as follows: Cochliophilin A (6) and 5-hydroxy-7methoxychromone (64) were prepared by the method of Horio et al. [ 10] and Jain [79], respectively. 6,7-Methylenedioxyflavone (58), 7hydroxyflavone (53), 5,7-dihydroxyflavone (57), and 5,6,7trihydroxyflavone (54) were synthesized according to the methods of Agasimundin and Siddapa [32]. Flavone (1) and 5-hydroxyflavone (59) were prepared by the method of Jain et al. [80]. Compounds (57), (54) and 5,7,4'-trihydroxyisoflavone (genistein, 8) were methylated to give 5hydroxy-7-methoxyflavone (55), 5-hydroxy-6,7-dimethoxyflavone (77), and 5,4'-dihydroxy-7-methoxyisoflavone (prunetin, 9) using diazomethane [81 ]. 5-Methoxy-6,7-methylenedioxy-flavone (10) and 5-hydroxy-7,4'dimethoxyflavone (78) were obtained from (6) and apigenin respectively by methylation with dimethyl sulphate in a mixture of K2CO3 and acetone. Ethylation of 57 with diethyl sulphate gave 5-hydroxy-7ethoxyflavone (79). Genistein (8) was isolated from the leaves of white lupin [82]. Although only a limited number of compounds were investigated, the structure-activity data indicate that 5,7-dihydroxyflavone (57)has stronger attractant properties than flavone (1; 2-phenylchromone) or 5and 7-monohydroxyflavone (62 and 61) (Table 12). These results strongly suggest that 5,7-dioxygenation is important for zoospore attracting activity. Furthermore, the fact that the potent chemo-attractant effect of 6 was markedly decreased by 5-deoxygenation (58), and by 5-0methylation (10), indicates that the free 5-hydroxyl group makes a strong contribution towards the observed activity (Table 12). The 5-hydroxyl group in flavones is known to form a strong hydrogen bond with certain metal ions [83] but it is not known at present if this property is linked with the ability of 6 and 77 to attract zoospores. A comparison of the attractant activity of 55 (5-OH, 7-OMe, tectochrysin), with that of 57 (5,7-di-OH, chrysin), and that of 77 (5-OH, 6,7-diOMe) with that of 54 (5,6,7-triOH) revealed that 7-O-methylation significantly increased activity. However, the 7-O-ethyl derivative (79) was of considerably lower activity when compared with the 7-O-methyl derivative (55), probably as a result of steric effects. Although 6hydroxylation had a negative effect on activity (compare 54 with 57), it was found that 6-methoxylation markedly increased activity (compare 55 with 77). Based on the high activity of the 6,7-methylenedioxysubstituted flavone (6) and the 6,7-dimethoxy derivative (77), the presence of small alkoxy groups at C-6 and C-7 would seem to be effective in enhancing the zoospore attractant properties (Table 12) [83]. Flavone 55 (tectochrysin) with a 5-hydroxy-7-methoxylated A-ring, and an unsubstituted B-ring, exhibited stronger activity than chromone 64 which lacked the B-ring, or 78 (apigenin-7,4'-dimethyl ether) with a pmethoxylated B-ring (Table 13). These results suggest that amongst
TAHARA and INGHAM
498
flavones, strong activity may be related to the presence of an unsubstituted B-ring. Although 5,7,4'-trihydroxyisoflavone (genistein, 8) and 5,4'-dihydroxy-7-methoxyisoflavone (prunetin, 9) have respectively been reported as zoospore attractants of Phytophthora sojae [11 ] and A. euteiches [12], these isoflavones were essentially inactive towards A. cochlioides at a concentration of 10.6 M (Table 13). The latter result is consistent with the fact that A. cochlioides is pathogenic on a species (Spinacia oleracea) belonging to a plant family (Chenopodiaceae) in which 5,7,4'-trioxygenated isoflavones do not appear to occur. As the coevolution of,4. cochlioides with its host plant appears to be closely linked with the ability of fungal zoospores to recognize a specific, and structurally unusual flavone (cochliophilin A, 6), the lack of a response to compounds such as genistein (8) and prunetin (9) is not entirely unexpected. Attractant
T a b l e 14.
Effect o f P r u n e t i n
and O t h e r . l s o f l a v o n e s
on Z o o s p o r e s
of
Aphanomyces euteiches, the C a u s e of a Root Rot of Garden Pea* .
.
.
.
Relative activity (M)
C-7
C-5 ,,
.
L.
i
Compd
.
Isoflavone substituent a
,
' '
C-4'
10 "6
10-7
10-8
='I
8
OH
OH
OH
++
9
OH
OCH3
OH
+++
++
+
8o
OH
OH
OCH3
++
+
-
81
OH
9OCH3
OCH3
++
+
-
82
OH
OCH3
H
+++
+
-
OH
OH
++
+
-
OH
H
OH
++
+
-
OH
H
OCH3
++
+
-
-
-
83 84 85
,,,
86
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
H .
.
OH
H
H
H
OH
OH
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+ .
.
.
.
.
.
The following isoflavones were only rated + o r - at 10"5 M. un'substituted. . . . . . . . . . monosubstituted: 5-OCH3; 7-OH; 7-OCH3; 4'-OH; 4'-OCH3.
.
disubstituted: 5-OCH3,7-OH; 5-OCH3,7-OCH3; 5-OCH3,4'-OH; 5-OCH3,4'-OCH 3" 7-OH,4'-OCH3" 7OCH3,4'-OH; 7-OCH3,4'-OCH3. trisubstituted: 5-OCH3,7-OH,4'-OH; 5-OCH3,7-OH,4'-OCH3; 5-OCH3,7-OCH3,4'-OH; 5-OCH3,7-OCH3,4'OCH3. ,
a:
,
,
,
.,,~,~ ~ 0 7[~ ~'~
0
,,,
,
,
,
=,
*The d a t a from two p a p e r s [84, 85] b y Seklzaki et al. were combined and rearranged.
4'
SIMPLE FLAVONES
499
Although structure-activity studies have not been carried out using compounds related to the ferulamide attractant, it is clear from Table 11 that exchanging the hydroxyl and methoxyl groups in the dopamine moiety (69, 3-O-methyl; 70, 4-O-methyl) has a significant effect on activity. Exchanging the hydroxyl and methoxyl groups of the ferulic acid unit also affected activity, but to a lesser extent [83]. Thus, the substitution pattern, 3-hydroxy-4-methoxy, in the dopamine part of the ferulamide molecule seems to be of most significance in conferring attractant activity. It is also noteworthy that the zoospores of A. cochlioides were attracted most effectively by a host-derived isomer (70) possessing a naturally rare substitution pattern. The importance of structure in the attractant activity of prunetin (9) and related compounds (mono-, di- and trisubstituted isoflavones) towards zoospores of A. euteiches has been analyzed by the capillary method [84, 85]. The results are partly shown in Table 14. According to Sekizaki et al. [84, 85], a hydroxyl group (but not a methoxyl) at the C-5 position of an isoflavone is necessary to strongly attract A. euteiches zoospores. In addition, the presence of an additional hydroxyl group at C-7 or C-4' strengthened the attractant activity, which was further increased by 7-0methylation, but slightly decreased by 4'-O-methylation. The strongest activity amongst 26 non-, mono-, di- and tri-substituted isoflavones was associated with the natural attractant prunetin (9).
Concluding Notes For plant pathogens which spread by means of soil-borne zoospores, an ability to successfully recognize, and respond to, chemical signals (chemotaxis) released by the host plant is of major importance. In the present study, cochliophilin A (6) has been isolated from spinach roots and found to be a powerful attractant of the zoospores of A. cochlioides. During the isolation procedure, it was found that the level of attractant activity was approximately in proportion to the total weight of material obtained at each step (Table 9). This strongly suggests that the activity in crude ether extracts of spinach roots is largely due to compound 6. In addition, the behaviour of the zoospores towards pure 6 was similar to that of their response on the surface of spinach roots. It may be concluded therefore, that cochliophilin A (6) plays an essential role in attracting A. cochlioides zoospores, and that this initial chemotaxic phenomenon leads directly to infection and disease development in spinach roots. Cochliophilin A (6) has previously been isolated from sugar beet roots infected with the basidiomycete fungus Rhizoctonia solani (Thanatephorus cucumeris) [9]. However, it is not yet known if this compound occurs in healthy, uninfected roots of sugar beet, nor if it has any attractant effect on the hyphae of R. solani, a fungus that is only distantly related to the zoospore-producing species of Aphanomyces. As A. cochlioides may cause
500
TAHARA and INGHAM
a seedling "damping-off' in sugar beet, it would not, in fact, be unexpected to find that cochliophilin A occurs as an attractant in root exudates, since the same compound is present in the root/rhizosphere of spinach, and in the roots of pigweed (Chenopodium album), both of which are good hosts for A. cochlioides. On the basis of the particle test, it was found that 6 exhibited attractant activity at the extremely low level of 10.9 - 10-10 M (Table 10). However, these values only represent the concentration of 6 in the ether or ethyl acetate solution used to coat entire Chromosorb W particles for use in the bioassay. It follows, therefore, that after being placed in an aqueous suspension of A. cochlioides zoospores, the concentration of the attractant in the medium .around the particles must be lower than 10.9 or 10-~0 M. Yet the zoospores clearly respond to the attractant, and aggregate around the treated particles. Using indirect (NMR) methods, it has been possible to estimate that fresh spinach roots contain only 5.3 ppm (5.3 ~tg/g) of 6, whilst the amount of 6 exuding into the rhizosphere was found to be approximately 35 ng/root/day. When considered together, the above results strongly suggest that 6 is a major factor in determining the ability of A. cochlioides zoospores to successfully locate, and subsequently colonize, spinach roots. GENERAL CONCLUSION Although many interesting properties have now been attributed to flavonoid compounds, no evidence, prior to the research described in this review, appears to have been obtained to suggest that certain flavones may act as antidotes against some of the benzimidazole (benomyl-type) fungicides now being used commercially to control plant diseases. As shown in Fig. 1 (left), the antidote effect of Polygonum-derived compounds against the benomyl metabolite MBC, can be easily and clearly demonstrated by means of a simple thin-layer plate bioassay using Cladosporium herbarum as the test fungus. Under normal circumstances MBC is active against C. herbarum, and inhibits fungal growth on much of the chromatogram (white areas). However, extracts of all three Polygonum species contained material which nullified the fungitoxic effects of MBC, and on corresponding areas of the chromatogram, a considerable amount of fungal growth was observed (black areas). Apart from the flavone eochliophilin A methyl ether (10), it has been found that other, non-flavonoid, natural products (e.g. the anthraquinone, emodin) also exhibit potent antidote activity. Whilst the significance of these plant-produced chemicals, in reducing the efficiency of several widely used benomyl-type fungicides, has still to be evaluated, the fact that flavones occur widely in the Plant Kingdom may suggest that crop plants should be routinely screened for potential flavone (and non-flavone)
SIMPLE FLAVONES
501
antidotes when control programmes are being devised to combat fungal pathogens. Monitoring plants for the presence of antidotes would seem to be particularly necessary when disease control involves the use of systemic fungicides (rather than those which act principally as protectants on the surface of the plant), since compounds of this type, which are now widely employed in agriculture, pass into the tissues of the plant where they are more likely to encounter any antidotes that may be present in the cells. If antidotes are detected in crop varieties, and if their presence appears to interfere with the efficiency of certain fungicides, the knowledge may allow different chemical treatments to be selected at an early stage. Alternatively, plant breeders may be able to develop varieties which lack, or contain only low levels of, the antidote compound(s) as, for instance, has already been done for different commercial reasons with crops such as lupin (low-alkaloid content), and sweetclover (low-coumarin content). Screening techniques such as the TLC plate bioassay also allow extracts to be quickly examined for the presence of fungitoxins occurring naturally within the plant, or on its surface. Referring back to Fig. 1., the same extract from P. thunbergii contained both a benzimidazole antidote and a fungitoxin (Fig. 1. fight), which could easily be located (white zone due to inhibition of fungal growth) against the dark, fungus-covered background. The ability to produce fungitoxins, either pre-infectionally in healthy tissues, or post-infectionally (e.g. as phytoalexins), is probably widespread in the Plant Kingdom, and may in part explain why all plants are resistant to most potentially pathogenic fungi. It is now generally accepted that fungi which are pathogenic on a given plant species are capable of withstanding the effects of any fungitoxins that may be present in (or produced by) the host plant. However, information on the presence (or absence) of fungitoxins may enable geneticists to select or develop varieties with a greatly enhanced ability to produce natural antifungal chemicals, thereby increasing their potential to resist the harmful effects of normally pathogenic fungi. There is now also the possibility that advances in genetic research may allow genes for chemical resistance to be transferred from one plant species to another, thus increasing the likelihood that a pathogenic fungus will be confronted with a novel compound (or compounds) exhibiting toxic effects which it is unable to overcome. The ability of fungal pathogens to tolerate, or in some way detoxify, the defensive chemicals of their host plants, is undoubtedly a major coevolutionary mechanism closely linked with the phenomenon of plantpathogen specificity. Other factors may, however, also be involved in determining specificity, including the ability of soil-borne pathogens to successfully locate their normal host. In contrast to compound 10, which occurs in the aerial parts of Polygonum species, the second section of this review deals with an almost
502
TAHARA and INGtlAM
identical compound (cochliophilin A, 6) found in the roots and rhizosphere of spinach, and which is a potent attractant of zoospores of the phytopathogenic fungus Aphanomyces cochlioides. From the limited available data, it seems possible that cochliophilin A may occur exclusively in the roots/rhizosphere of spinach and related Chenopodiaceae, where its presence allows the zoospores of A. cochlioides to detect, and then be guided to, their host species. In order to investigate the attractant properties of cochliophilin A, and related natural and synthetic compounds, a new technique (the "particle method") was developed which permitted the effects of chemo-attractants to be observed microscopically. Using this procedure, it was found that whilst cochliophilin A was a very strong zoospore attractant, the almost identical flavone 10 (from Polygonum leaves) was essentially inactive, whilst a third, synthetic, flavone had considerably greater activity. As A. cochlioides zoospores clearly have a very specific receptor for cochliophilin A, it is possible that knowledge derived from a combination of ecological chemistry and structure-activity studies may offer the prospect of developing new control measures, perhaps based on receptorblocking molecules, for soil-borne plant pathogens which often are difficult to eradicate successfully. Apart from its value in the study of natural and synthetic chemo-attractants, the particle method has also been used to investigate compounds which have an ability to repel zoospores, or cause them to lose motility, or burst (lyse) [87]. Again, these observations may ultimately be of importance in the development of more effective chemical control methods. Finally, as mentioned earlier, it is possible that geneticists may be able to increase the resistance of plants by incorporating into them a greater ability to produce defensive chemicals. Wider application of the particle method in the study of plant chemo-attractants, should allow further active compounds to be identified. With the knowledge thus gained, it may be feasible to decrease the susceptibility of plant species by phytochemical and genetic manipulation aimed at reducing or eliminating specific chemo-attractants from root tissues, and hence from the rhizosphere. ACKNOWLEDGEMENTS We thank Professors R. Yokosawa, F. Tomita, and M. Fujimura for kindly supplying the fungal strains, Aphanomyces cochlioides, Cladosporium herbarum, and Neurospora crassa, respectively. Much of the research on which this review is based, has been carried out by the following students, Masters Y. Matsukura, N. Toda, and H. Katsuta (benzimidazole antidotes), and by Dr. T. Horio; Masters T. Takayama, H. Kikuchi, K. Ohkawa, and M. Mizutani (zoospore attractants).
SIMPLE FLAVONES
503
Financial support (to S. T.) by CREST, Japan Science and Technology Corporation is also gratefully acknowledged. REFERENCES [l] [21
[3]
[4]
[5] [6] [7]
[8] [9] [~0] [11] [12] [131 [14]
[15] [16]
[17]
[18] [19] [20] [2~] [22] [23] [24] [25] [26]
Harborne, J. B.; Ingham, J. L.; King, L.; Payne, M. Phytochemistry, 1976, 15, 1485. Ingham, J. L. In Phytoalexins; Bailey, J. A.; Mansfield, J. W., Eds.; Blackie: Glasgow and London, 1982; pp. 21-80. Ingham, J. L. Phytopath. Z., 1973, 78, 314. Coxon, D. T.; O'Neill, T. M.; Mansfield, J. W.; Porter, A. E. A. Phytochemistry, 1980, 19, 889. Ingham, J. L.; Tahara, S.; Dziedzic, S. Z. J. Nat. Prod., 1986, 49, 63 I. Olah, A. F.; Sherwood, R. T. Phytopathology, 1971, 61, 65. Geigert, J.; Stermitz, F. R.; Johnson, G.; Maag, D. D.; Johnson, D. K. Tetrahedron, 1973, 29, 2703. Johnson, G.; Maag, D. D.; Johnson, D. K.; Thomas, R. D. Physiol. Plant Pathol., 1976, 8, 225. Takahashi, H.: Sasaki, T.; Ito, M. Bull. Chem. Soc. Japan, 1987, 60, 2261. Horio, T.;Kawabata, Y.; Takayama, T.; Tahara, S.; Kawabata, J,; Fukushi, Y.; Nishimura, H.; Mizutani, J. Experientia, 1992, 48, 410. Morris, P. F.; Ward, E. W. B. Physiol. MoL PL Pathol., 1992, 40, 17. Yokosawa, R.; Kuninaga, S.; Sekizaki, H. Ann. PhytopathoL Soc. Japan, 1986, 52, 809. Tahara, S.; Matsukura, Y.; Katsuta, H.; Mizutani, J. Z. Naturforsch., 1993, 48c, 757. Tahara, S.; Matsukura, Y.; Mizutani, J. J. Pesticide Sci., 1990, 15, 603. Millburn, P. In Biochemical Aspects of Plant and Animal Coevolution; Harborne, J. B., Ed.; Academic Press: London, 1978; pp. 35-73. Sijpesteijin, A. K. In Systemic Fungicides, 2nd Ed.; Marsh, R. W. Ed.; Longman: London, 1977; p.131.; Corbett, J. R.; Wright, K.; Baillie, A. C. The Biochemical Mode of Action of Pesticides, 2nd Ed.; Academic Press: London, 1984; p.202. Raeymaekers, A. H. M.; Van Gelder, J. L. H.; Roevens, L. F. C.; Janssen, P. A. J. Arzneim.-Forsch., 1978, 28, 586. Iwasaki, S. J. Syn. Org. Chem. Jpn., 1991, 49, 892. Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Hawel, E.; Blokhin, A.; Slate, D. L.J. Org. Chem., 1994, 59, 1243. Nicolaou, K. C.; Dai, W.-M.; Guy, R. K. Angew. Chem. Int. Ed. Engl., 1994, 33, 15. Cowden, C. J.; Paterson, I. Nature, 1997, 387, 238. Haar, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Longley, R. E.; Gunasekera, S. P.; Rosenkranz, H. S.; Day, B. W. Biochemistry, 1996, 35, 243. Homans, A. L.; Fuchs, A. J. Chromatogr., 1970, 51, 327. Hans, D. S.; Cho, H. J. Saengyak Hakhoe Chi., 1981, 12, 221; Chem. Abstr., 1982, 97, 20714s. Tamano, M.; Koketsu, J. Agric. Biol. Chem., 1982, 46, 1913. Kitanaka, S.; Takido, M. Yakugaku Zasshi, 1986, 106, 302.
504
[27]
[28] [29]
[30] [31] [32] [33]
[34]
[351 [36] [37]
[38] [39] [40] [41]
[42] [43] [44]
[45]
[46]
[47] [48]
[49]
[50] [511 [52]
[53] [54]
[551 [56]
[57]
TAHARA and INGHAM
Tschirk, A.; Pool, J. F. A. Arch. Pharm., 1908, 246, 315; Chem. Abstr., 1909, 3, 473. Turner, W. B.; Aldridge, D. C. Fungal Metabolites Hi Academic Press" New York, 1983, p. 143. Markham, K. R.; Mabry, T. J. In The Flavonoids; Harborne, J. B.; Mabry, T. J.; Mabry, H., Eds,; Chapman and Hall: London, 1975; p.65. Pelter, A.; Amenechi, P. I. J. Chem. Soc. (C), 1969, 887. Ser, N. A. Phytochemistry, 1988, 27, 3708. Agasimundin, Y. S.; Siddappa, S. ,1. Chem. Soc., Perkin Trans. I, 1973, 503. Iinuma, M.; Tanaka, T.; Matsuura, S. Chem. Pharm. Bull., 1984, 32, 1006. McPherson, D. D.; Cordell, G. A.; Soejarto, D. D.; Pezzuto, J. M.; Fong, H. H. S. Phytochemistry, 1983, 22, 2835. Handa, S. S.; Kinghorn, A. D.; Cordell, G. A.; Famsworth, N. R..1. Nat. Prod., 1983, 40, 248. Nishina, A.; Hasegawa, K.; Uchibori, T.; Seino, H.; Osawa, T. J. Agric. Food Chem., 1991, 39, 266. Nishina, A.; Uchibori, T. Agric. Biol. Chem., 1991, 55, 2395. Borisy, G. G.; Taylor, E. W. J. Cell Biol., 1967, 34, 525. Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J. M.; Lohavanijaya, P. Can. ,I. Chem., 1982, 60, 1374. Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.; Schmidt, J. M.; Pettit, G. R.; Hamel, E. Mol. Pharmacol., 1988, 34, 200. Luduena, R. F.; Roach, M. C. Biochemistry, 1981, 20, 4444. Kobayashi, A.; Ooe, K.; Kawazu, K. Agric. Biol. Chem., 1989, 53, 889. Matsukura, Y. M. S. Thesis; Graduate School of Agriculture, Hokkaido University: 1991, March. Katsuta, H. M. S. Thesis; Graduate School of Agriculture, Hokkaido University: 1994, March. Fujimura, M.; Oeda, K.; Inoue, H.; Kato, T. In Managing Resistance to Agrochemicals: From Fundamental Research to Practical Strategies; Green, M. B.; LeBaron, H. M.; Moberg, W. K., Eds.; Symp. Ser. Amer. Chem. Sot., 421: Washington D. C., 1990; p.224. Fujimura, M.; Kamakura, T.; Yamaguehi, I. J. Pestic. Sci., 1992, 17, 237. Fujimura, M.; Kamakura, T.; lnoue, H.; lnoue, S.; Yamaguchi, I. Pestic. Biochem. Physiol., 1992, 44, 165. Kilmartin, J. V. Biochemistry, 1981, 20, 3629. Davidse, L. C. In Microtubules and Microtubule lnhibitors; Borgers, M.; de Brabander, M., Eds.; North-Holland Publishing Co.; Amsterdam, 1975; p.483. Davidse, L. C.; Flash, W. J. Cell Biol., 1977, 72, 174. Jayasuriya, H.; Koonchanok, N. M.; Geahlen, R. L.; McLaughlin, J. L.; Chang, C.-J. J. Nat. Prod., 1992, 55, 696. Chang, C.-J.; Geahlen, R. L. J. Nat. Prod., 1992, 55, 1529. Toda, N. M. S. Thesis; Graduate School of Agriculture, Hokkaido University: 1995, March. Pallos, F. M.; Casida, J. E., Eds. Chemistry and Action of Herbicide Antidotes; Academic Press: New York; 1978. Banas, A.; Johansson, I.; Stenlid, G.; Stymne, S. Swedish J. Agric. Res., 1993, 23, 55. Yokosawa, R.; Ogoshi, A.; Sakai, R. Ann. Phytopathol. Soc. Jpn., 1974, 40, 46. Rai, P. V.; Strobel, G. A. Phytopathol., 1966, 56, 1365.
SIMPLE FLAVONES
[58] [59]
[60] [61] [62] [63]
[64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79]
[801 [81] [82] [83] [84]
[85] [86] [87]
505
Yokosawa, R.; Kuninaga, S. Ann. Phytopathol. Soc. Jpn., 1979, 45, 339. Ui, T.; Nakamura, S. Tensai-kenkyukai Kenkyu-hokoku. 1963, 3, 78. Yokosawa, R.; Sekizaki, H.; Kuninaga, S. Ann. Phytopathol. Soc. Jpn., 1988, 54, 133. Horio, T.; Yoshida, K.; Kikuchi, H.; Kawabata, J.; Mizutani, J. Phytochemistry, 1993, 33, 807. Donaldson, S. P.; Deacon, J. W. New Phytol., 1993, 123, 289. MUller, D. G. Z. Pflanzenphysiol., 1976, 80, 120. King, F. E.; King, T. J.; Manning, L. C. J. Chem. Soc., 1957, 536. Ohkawa, K. M. S. Thesis; Graduate School of Agriculture, Hokkaido University; 1996, March. Aritomi, M.; Komori, T.; Kawasaki, T. Phytochemistry, 1986, 25, 231. Suzuki, T.; Holden, I.; Casida, J. E. J. Agric. Food Chem., 1981, 29, 992. Agurr S.; Bruhn, J. G.; Lundst6m, J.; Svensson, U. Lloydia, 1971, 34, 183. Chiji, H.; Giga, T.; Izawa, M.; Kiriyama, S. Agric. Biol. Chem., 1984, 48, 1653. Tanaka, H.; Nakamura,; Ichino,K.; Ito, K. Phytochemistry, 1989, 28, 2516. Ramirez, F. A.; Burger, A. J. Am. Chem. Soc., 1950, 72, 2781. Hamilin, K. E.; Weston, A. W. J. Am. Chem. Soc., 1949, 71, 2210. Fuji, K.; Kawabata, T.; Fujita, E. Chem. Pharm. Bull., 1980, 28, 3662. Takayama, T.; Mizutani, J.; Tahara, S. Ann. Phytopathol. Soc. Jpn., 1998, 64, 175. Horio, T. Ph.D. Thesis; Graduate School of Agriculture, Hokkaido University; 1993, March. Cook, C. E.; Whichard, L. P.; Wall, M. E.; Egley, E. H.; Coggon, P.; Luhan, P. A.; McPhail, A. T. J. Am. Chem. Soc., 1972, 94, 6198. Hauck, C.; Mtiller, S.; Schildknecht, H. J. Plant Physiol., 1992, 139, 474. Masamune, T.; Fukuzawa, A.; Ikura, M.; Matsur H.; Kaneko, T.; Abiko, A.; Sakamoto, N.; Tanimoto, N.; Murai, A. Bull. Chem. Soc. Jpn., 1987, 60, 1001. Jain, A. C. Indian J. Chem., 1984, 23B, 1036. Jain, P. K.; Makrandi, J. K.; Grover, S. K. Synthesis, 1982, 221. Tahara, S.; lngham, J. L.; Mizutani, J. Phytochemistry, 1989, 28, 2079. Ingham, J. L.; Tahara, S.; Harborne, J. B. Z. Naturforsch., 1983, 38c, 194. Kikuchi, H.; Horio, T.; Kawabata, J.; Fukushi, Y.; Mizutani, J.; Tahara, S. Biosci. Biotech. Biochem., 1995, 59, 2033. Sekizaki, H.; Yokosawa, R. Chem. Pharm. Bull., 1988, 36, 4876. Sekizaki, H.; Yokosawa, R.; Chinen, C.; Adachi, H.; Yamane, Y. Biol. Pharm. Bull., 1993, 16, 698. Beutler, J. A.; Cardellina II, J. H.; Lin, C. M.; Hamel, E.; Cragg, G. M.; Boyd, M. R. Bioorg. Med. Chem. Lett., 1993, 13, 581. Mizutani, M.: Hashidoko, Y." Tahara, S. FEBS Letters, 1998, 438, 236.
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies #s Natural Products Chemistry, VoL 22
507
9 2000 Elsevier Science B.V. All rights reserved
MEDICINAL PLANTS WITH ANTICONVULSANT ACTIVITIES MOHSIN RAZA* M. IQBAL CHOUDHARY and ATTA-UR-RAHMAN
International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi- 752 70, Pakistan ABSTRACT: This review summarizes the literature on anticonvuisant activities of 334 medicinal plants used for the treatment of epilepsy and convulsive disorders in the indigenous system of medicine. Data on plants which have not yet been investigated for pharmacological activity are also presented. The details are presented in a tabular form. The data includes the plant part involved, the nature of the extract or fraction used and the names of active compounds and their structures wherever available. The terms used in ancient texts for various types of convulsive disorders are retained. The results of anticonvulsant activities of extracts, fractions and pure compounds isolated from the plants on various bioassays are also presented.
INTRODUCTION The use of medicinal plants for the treatment of epilepsy and convulsive disorders dates back to prehistoric times. Several plants that were reputed to possess antiepileptie properties in different folklore cultures have been found to contain active ingredients when tested with modem bioassays for detecting anticonvulsive activities. This provides justification for their use in many different indigenous medicinal systems. The activity of many other plants however remains to be scientifically established. Several such plants have been listed in order to promote further research with the hope that better medicines may be developed for treatment of epilepsy in the future. The term 'epilepsy' is derived from two Greek words meaning 'to seize upon' [ 1]. It is a chronic disorder characterized by recurrent seizures [2]. As it is a symptom complex and not a disease per se, several different factors contribute to the etiology of epilepsy. Epileptic disorders can be classified into four main groups; generalized, localized or focal, undetermined (either generalized or focal) and special syndromes; atleast 40 different types of epilepsies are known [3]. The incidence of epilepsy ranges between 40 and 200 per 100,000 population depending on the geographic location. The developing countries have a higher incidence rate [4]. All the currently available major antiepileptie drugs (AED) such as phenytoin, carbamazepine, phenobarbitone, valproie acid, lamotrigine,
508
MOHSIN eta/.
vigabatrin, gabapentin, felbamate and the benzodiazepines are synthetic molecules. About 50% of the patients can have complete control of seizures with AED. Problems associated with chronic therapy of epilepsy with AED are the incidence of side-effects which include CNS depression, psychiatric disturbances, osteomalacia, leucopenia, megaloblastic and aplastic anemia, hepatic failure, allergic reactions, teratogenic effects and withdrawal emergent disorders [5-9]. Ethnopharmacological research on natural products can contribute to the discovery of new active compounds with novel structures which may serve as leads to the development of new antiepileptic drugs. An example is the isolation of the active alkaloid piperine from Piper nigrum L. which is one of the component herbs of an ancient Chinese medicine used for the treatment of epilepsy. Its structural modifications resulted in the synthesis of seven derivatives including antiepilepserine which was found to be more potent than the parent compound with fewer side effects and it has been used as an antiepileptic drug [10-12]. A number of animal models are currently available for anticonvulsant screening [ 13,14]. Two basic models used are Maximal Electroshock Test (MEST) and Subcutaneous Pentylenetetrazole Seizure Threshold Test (ScMET). Anticonvulsant activity in MEST predicts the ability of the testing material or compound in preventing the spread of seizure discharge and effectiveness in the treatment of grandmal seizures, while activity in ScMET predicts the ability to elevate seizure threshold and effectiveness in myoclonic seizures. Further screening tests are directed to detect activity against bicuculline, picrotoxin and strychnine induced convulsions [15-17]. For completion, the data of anticonvulsant activities against various other chemoconvulsants eg. nicotine, theophylline which are not currently employed are also presented in this review. Several composite herbal preparations used for the treatment of epilepsy in different indigenous systems have been shown to contain plants with active principles. An example is TJ 960 (saiko-keishi-to), a kompo herbal formulation developed from ancient Chinese medicine comprising 9 medicinal plants has shown interesting antiepileptic activities in different in vivo, in vitro and clinical studies [18]. It exhibits inhibitory effects on audiogenic seizures in DBA mice, [19] PTZ induced convulsions in El epileptic mice [20] and PTZ induced power spectrum changes in EEG [21 ]. It has protective effect on hippocampal neuronal damage in cobalt-induced epileptic seizures in rats [22] and causes improvement of cognitive function in epileptic patients [23]. The aqueous extract as well as its albiflorin and pentagalloylglucose isolated from Paeonia albiflora (peony), a constituent of TJ 960, have strong inhibitory effect on PTZ induced EEG activity and Ca++ and K+ ion concentration changes related to seizures [24]. The extract has also shown to inhibit PTZ induced intracellular Ca++ release and inward Ca++ currrent [25].
ANTICONVULSANT ACTIVITIES
509
A number of pure compounds isolated from plants have been shown to possess several interesting activities related to the treatment of epilepsy. Methysticin, a kava pyrone isolated from the rhizomes of Piper methysticum (which is a shrub indigenous to south Pacific islands) and its dihydro derivative have neuroprotective effects in addition to anticonvulsant properties [26]. As progression of epileptogenesis is associated with neuronal loss, this activity may be useful in the treatment of epilepsy. Methysticin also inhibits seizure-like events in three different models of epileptiform activities in hippocampal and entorhinal cortex slices [27]. Linalool a monoterpene isolated from several species of aromatic plants including Aeolanthus suaveolens G. Dom., which is used for the treatment of convlusions in the Brazilian Amazon, has inhibitory effect on glutamate binding in rat cerebral cortex preparations [28]. As glutamate is the major excitatory amino acid in the brain and its role in pathophysiology of epilepsy is recognized, this activity provides a rational basis of the traditional use of linalool producing plants. Baiealein isolated from TJ-960 exhibited strong in vitro radical scavenging and antioxidative activity. In FeCl3-induced epilepsy model it significantly decreased the level of free radicals at the injection site in the rat brain [29]. Since FeCl3-induced epilepsy is an experimental model of posttraumatic seizures, this action of baicalein warrants further research on the probability of the role of anti-oxidants in the prophylaxis of post-traumatic epilepsy. It is a misconception, however, that all plants used for the treatment of epilepsy in folk medicine are safe to use and that their use is rational. A number of highly addictive and toxic compounds have been isolated from these plants; including LSD, A8 and A 9 tetrahydrocannabinols, cannabidiol and cocaine. A 8 and A9 tertrahydrocannabinols have anticonvulsant activity in various experimental models of epilepsy including kindling [30]. However their use in the treatment of epileptic disorders is obviously dangerous due to their addictive potential. Similarly a number of highly toxic alkaloids affecting the cardiovascular and central nervous system function have been isolated from several plants. This fact emphasizes the need for rational scientific investigations of the pharmacology and toxicology of the extracts as well as of the pure compounds isolated from them. However, the medicinal use of crude plants is usually safe since the amount of toxic components may be very minute. Convincing evidence that a particular herbal recipe is safe for human use must still be demonstrated by toxicological studies before their use can be recommended.
,,
S.No ,
I.
Family
N a m e
,
9
,
,
,
Abrus precatorius L.
Occu
rrance
Uses ,
trop
,,
,,
Part used
,=,
Papilionaceae
,
,,=
ep#epsy
decoction
leaves
convulsions in children
juice
trop Aft, lnd, Sri, Bur, Am
leaves flowering spike
3.
Achyranthes aspera L.
Amaranthaceae
lnd, Sri, Aus, Aft, E.S. Pac
4.
Aconitum japonicum Thunb.
Ranunculaceae
Chi, Indchi _.
Acorus calamus L.
Ara~,~
Ind, Pak, Sri, C. Asi, Sik, Him, Ksh
I ]
Ref.
!
31
70 % ethanolic ext.
32
epilepsy
decoction
33
hysteria
-
34
convulsions
decoction or alcoholic ext.
35
+PTZ -STN
plant
Euphorbiaceae
Nature of extract/active principle ,, ,"-',
roots
.... ,
Acalypha fruticosa Forsk.
Activity
ii|,l
,,,
,
rhizome
34, 3 5 , 37, 38
hysteria, convul-sions & epilepsy +EST -~Z +~Z -S~ -MES +~Z
essential oil AC-I ethanolic ext.
38 39
-~Z
asarone (1)
40, 41
+~Z -MES
3-asarone
40, 41
aqueous and 95% ethanolic ext.
42
= 2
6.
Aeollanthus suaveolens Mart. ex Spreng
Bra
Lamiacear
leaves
convulsions +PTZ +EST - PTZ
- EST + EST
+ PTZ
stem bark
W. Afr
anticonvulsant
Afraegle paniculata (Schum & Thorn) Engl. ,
Rutaceae
8.
Aglaia odorata Lour.
Meliaceae
S.E. Asi
roots & leaves
convulsions
9.
Albizzia harveyi Fourn.
Mimosaceae
E.S. Afr
roots
epilepsy
10.
Albi~ia lebbeck L.
-6
+PTZ
leaves
essential oil linalool (2)
43
&decanolactone &decen-2-1actone ~decanolactone 5-decen-2-1actone 7-decanolactone (3-5) .....
44
methanolic ext. chloroform sol. ext.
45 35
decoction
31
+PIC +PTZ +MES
chloroform fraction
46
+ PTZ
70% ethanolic ext.
> 2 ima
:Z 11.
Allium ascalonicum L.
Liliaceae
AfL I.nd
plant
12.
Allium cepa L.
Liliaceae
Ind, Pak, M.E., E.S. Afr, S.E. Asi, costa
bulb
36,47
epilepsy & infantile convulsions +PTZ
,,
13.
Liliaceae
Ailium sativum L.
70% ethanolic ext.
-STN
I 4 I
C. Asi, Ind, Pak, Sri, i E.N.S. Aft', costa, Bri, N. Eur, Egy, Mex, U S A
bulb
32
....
complex preparation
convulsive affections +PTZ
32 ,
I 37
70% ethanolic ext.
32
14.
Ailophylus africanus Beouv.
Sapindaceae
trop Aft"
leaves
convulsions
decoction with leaves of Ocimum basilicum L.
48
15.
Aloe vera L.
Liliaeeae
pant
sap ofleaves
epilepsy & convulsions in children
-
35
A. vera var chinensis Hain. Berger. ,
Tai, Indchi, Chi
....
> Z ,H >
,,q m
16.
i Alstonia boonei De Wilid.
Apocynaceae
Afr
17.
Alstonia venenata R.Br.
Apocynaceae
W.Pen
18.
Alstonia scholaris R.Br.
Apocynaceae
W. trop Aft', trop Aus, Sri, S. Chi, Ind, Ids, Indochi, Pak, Phil, Mal, Bur, Jav
! 9.
Ampelopsis japonica Thunb. Mak.
Vitacese
Kor, Chi, Jap
Anagallis arvensis L.
Primulaceae
Ind, Him, Pak
20.
.
i21. )
.
.
.
.
.
Commelinaceae ....
Annona muricata L.
Annonaceae
W. Pen, temp and trop Him, Ind Gha, Ind
!_
23.
Annonidium monni (Oliv.) Eng]. et. Diels
70% ethanolic ext.
32
leaves
+PTZ +EST
aqueous ext.
49
epilepsy.
,5o,51
epilepsy
35
roots
anticonvulsive
35
herb (whole plant)
epilepsy & hysteria
34, 50, 52
root bark
infantile convulsions
o
epilepsy
leaves
convulsive seizures
bark
epilepsy
ripe fruit
.
.
Aneilema scapiflorum Wight.
+PTZ
,l
L i
22.
.
plant
Annonaceae
C a m
.
.
.
.
34, 50 53 +PTZ
ethanolic ext.
54 55
l
24.
Apium graveolens L.
Umbelliferae
Chi, Ind, N.W. Him
seeds +IVIES -PTZ -STN
25.
Aralia continentalis L.
Araliaeeae
Chi
NM
26.
Arisaema consanguineum Schott.
Aracear
S.W. Chi
tuber
27.
A. amurense var serratum Maxim Nak.
Araceae
Kor
28.
A. heterophyllum.B.
Araceae
W.C. Chi
,
+
3-n-butylphthalide (6) 3-nbutyl 4-5-dihydrophthalide (7) alkaloid fraction
56 57
volatile oil
58
convulsions in children
35
c o m a
anticonvulsant
35
tuber
epilepsy
J,
i
35
29.
A. amurense Maxim
Araceae
N. Chi
tuber
convulsions in children
30.
A. japonicum Maxim.
Aracear
Chi, Tai, Jap
tuber
sedative for convulsions
Armillarta mellea Vahl. ex Fr. Quei.
31.
Tricholomataceae
Chi
dried mycelia
Arrabidaea platyphylla Bur.
Bignoniaceae
Bra
roots & leaves
33.
Artemisia verlotorum
Compositae
Bra, Spa
whole plant
34.
Artocarpus heterophyllus Lame
Momcear
Bur, Chi, Phil, Mal, Indchi, Ind, Bng
wood
Asarum macranthum Hook.
Aristolochiaceae
Tai
36.
Asparagus officlnalis L.
Liliaceae
Bulg, Ind
37.
Asparagus racemosus Willd.
Liliaceae
Ind, Him, Ksh
35 35
..
+IVIES +PTZ +STN
..
32.
I
epilepsy +MES +PTZ +3-mercapta -propionic acid +pilocarpine
aqueous ext.
59
decoction
60
95% ethanolic ext.
61
35
sedative for convulsions
. .
35.
Bacopa monnieri L.
38.
Scrophulariaceae
Ind, Pak, Mad, Phi, Sri
Bramia monnieri L. Herpestis monniera H. B. et K.
+EST -PTZ -STN
NM
Balsamodendron sp. vat. Commiphora myrrha (Nees) ..... E n s l .
Burseraceae
J Aft, Asi, Ara
ethanolic ext.
62
epilepsy
36
anticonvulsant
63
whole plant
epilepsy
50, 52, 64
root stalk & leaves
epilepsy & nocturnal
34, 36, 37
epilepsy
whole plant
39.
35
hysteria
gum resin from stem
epilepsy
+AUD +PTZ -IVIES
fraction aqueous ext. & 95% ethanolic ext.
65 66
37
Ot lint r
epilepsy
Barringtonia racemosa L. Roxb
Lecythidaceae
Ind, Sub Him
Basilicum polystachyon L. Moench
Labiatae
Jav, Aus
herb
sedative in convulsions & epilepsy
Benincasa hisplda (Thunb.) Cong.
Cucurbitaceae
lnd, Pak, Jay, trop, Chi, Mal, Ids
fruit
epilepsy & hysteroepilepsy
juice/decoction
fresh fruit
epilepsy
juice
B. cerifera Savi Bersama abyss!nica Fresen.
Melianthaceae
trop .S..Afr
roots
epilepsy
Boerhavia diffusa L.
Nyctogynaceae
Aft', lnd, Pak
root bark
anticonvulsive
B. repens L.
roots
Bombax malabaricum DC.
Malvaceae
Ind
seeds
Brassica nigra L. Koch
Cruciferae
Pak, Ksh, Ind
~
Buchnera cruciata Ham.
Scrophulariaceae
Butea monosperma (Lam.) Kuntze
33 +PTZ +PTZ +PTZ -EST
epilepsy
methanolic ext. 70% ethanolic ext. aqueous ext.
53 32 49
-
34
epilepsy
47
Chi
epilepsy
35
Papilionoideae
Pak, Ind
epitepsy
47
Caesaipinia puicherrima L. Sw.
Leguminosae
pant
flowers
convulsions m children
C bonduc L. Roxb
Caesalpiniaceae
Pak, Ind, S. Afr Nig
seeds plant
convulsions
+PTZ +STN
in a mixture
35
oil
53, 67 32
70% ethanolic ext.
i
51.
Mimosaceae
Calliandra portoricensis Jacq.
Afr
roots
32
anticonvulsant +PTZ +STN
70% ethanolic ext.
32
roots
+PTZ +EST
aqueous ext.
49, 68
stem
+PTZ +EST
aqueous ext.
68
-PTZ -EST
alkaloidal ext.
68
A8 and A9 tetrahydrocannabinols (8 and 9)
69, 70
plant
roots & stem ......
-6AtrO Cannabinaceae
Cannabis sativa L.
52.
N1d
Ind, Pak, Sub Him
+EST
-PTZ
SP-175 (I0)
71
i
2-q
cannabidiol (11) 72, 73, 74
2
-q
alcoholic ext.
34, 36, 47, 50, 64 75
epitep.sy
juice
48
convulsions in children
complex preparation
48
alkaloidal ext.
76
+PTZ +dexamphetami ne +EST 53.
Gentianaceae
i Canscora decussata Sehult. Roem. Roxb.
fresh plant
Ind, Sri, Bur, trop Aft
+EST
whole plant i
Rubiaceae
Canthium guein:ii Sond.
54.
Rubiaceae
Canthium bibracteatum Baker. Hiern.
55.
.
.
.
.
.
.
.
trop Aft"
.....
,
i
trop Aft
leaves
. . . . . . . .
roots
...........
.
56.
Capparis baducca
Capparidaceae
Jap
57.
Cardamine pratensis L.
Cruciferae
Ksh, W. Tib
flowering t o p s .
epilepsy
Umbeilifereae
Ksh, S. Ara, N. Him
seeds
anticon vuisant
Rutaceae
Mex
58.
59. ~,,
.
Carum carvi L. .
.
.
.
,,
Casimiroa edulis Llave et Lex. ~
i
,,
leaves .....
leaves
juice
epilepsy
u
,..
-6
50
+IVIES +PTZ
.
77
aqueous ext.
78
I !
i
. .
60.
Cassia accidentalis L.
Leguminosae
trop
seeds
convulsions in children
complex preparation
37
61.
Cassia sophera L.
Leguminosae
trop, Ind
seeds
convulsions in children
complex preparation
37
62.
Catunaregam nilotica Stapf. Tirvengadum
Rubiaceae
trop Aft
roots
convulsions in children
decoction
48
stem bark
convulsions
infusion
wood
epilepsy
,,,
63.
Cedeus libani Barrel var. Deodara Hook. f.
Coniferae
N.W. Him, Ksh
64.
Celastrus paniculta Willd.
Celastraceae
Sub Him, lnd
65.
Celtis cinnamomea Lindi.
Ulmaeear
Thi, Ids, Sik, Him, Bng, Ind
wood scrappings
66.
Centeila asiatica L.
Umbeiliferae
lnd
leaves plant
.
67.
Centipeda orbicularis Lour.
Compositae
Pak, Ind
.
.
.
a,nll,iepi/eptic
68.
Cerbera odollam Gaertn.
Apocynaceae
S.C. Vie, Cam, Ind
Cimicifuga dahurica
Ranunculaceae
USSR
70.
Cinchona spp.
Rubiaceae
S. Ame, Ind, Jay, Sri
bark
71.
Cinnamomum camphora L. F. Nces Eberm.
Lauraceae
Pak, Chi, Tai, Jap, Ind, Chi, Kor, lnd, Sri, Bra, Jam, Tai, Mau, Med regions
stearoptene from trunk root and branch camphor obtained by sublimation of chipped wood.
Cissampelos pareira L.
mucronata)
(C.
Menispermaceae
Aft, Ind
,,J
convulsions +PTZ +MES
leaves
plant
63 35
epilepsy
69.
72.
48
37
aqueous ext. 50% ethanolic ext.
79 80
.
47
+PTZ
95% ethanolic ext.
81
+STN +CAM
70% ethanolie tincture
82
+E
quinine (12)
anticonvulsant
83
35
epilepsy & hysteria
+PTZ +STN
camphor with olive oil
64
70% ethanolic ext.
32
73.
Cissus integrifolia (Bak) Planch.
Vitaceae
Tan
roots
epilepsy
74.
Citrullus colocynthis Schrad.
Cucurbitaceae
Mor
fruit
antiepileptic
75.
Citrus aurantium L.
Rutaceae
76.
Citrus maxima (Burro.) C. grandis L. C. decumana L.
Rutaceae
Ciausena anisata (Willd) Oiiv.
Rutaceae
77.
Aft, Ind, Chi, Ecu, Egy, S. Fra, W. Ind, Ira, Isr, Med, Mor, Pak, Por, .Spa, Tur, USA lnd, Pal<
W. Aft
+PTZ
plant
leaves
roots
decoction with root of 84 Azima tetracantha Lamb. Asparagus setaceus (Kunth) Jessop. Combretum xanthothyrsum i En~el. & Diels.
Nig
79.
Cnestis ferruginea
80.
Cocculus sermentosus Lour. Diels. (Nephroia sermentosa Lout.)
Menispermaeeae
Chi, Vie, Mal
81.
Colchicum luteum Baker
Liliaceae
Ksh, W.temp Him
82.
Colebrookea oppositifolia Sm.
Labiatae
lnd
83.
Coleus aromaticus Benth. C. amboinicus Lour.
Labiatae
Ind, Sri, cultivated
84.
Conium maculatum L.
Umbelliferae
Eur, temp Asi
Capparidaceae
70% ethanolic ext.
32
34, 50, 52
anticon vulsant +PTZ +PTZ
Cleome cileata Schum.et Thorn.
85
epilepsy & convulsive cough
+
78.
~
+PTZ +EST
leaves
with other ingredients methanolic ext. chloroform ext. heliettin (13)
45
aqueous ext.
49
Fm
roots
epilepsy & nocturnal epilepsy
decoction
;> 2 m,,l
r :Z ,<
61
;> 2 ,q ;>.
35, 36
N
epilepsy
47
roots
epilepsy
47, 50, 86
leaves
epileptic & convulsive af.fections
37, 64
epilepsy
87
,.q ,< ,.q
85.
Convolvulus arvensis L.
Convolvulaceae
Bulg, Ind, Him
NM
ethanolic ext. ,
Convolvulus pluricaulis
Convolvulaceae
Ind
dried plant
+EST +tremo-rine
95% ethanolic ext.
88
87.
Corydalis sp.
Fumariaccae
Chi
tuber
+PIC +PTZ
chloroform A ext. chloroform B ext.
89
88.
Crocus sativus L.
Iridaceae
Pak, Ksh, Irn, Spa, F.E., Chi
dried stigma
sedative in convulsions
89.
Crossostephium chinese L.
Compositae
Jap, Chi, Tai, Phi
leaves
infantile convulsions
90.
Cucumis sp. L.
Cucurbitaceae
Pak, Kor, Chi, Indchi, Ids, trop Aft, Med, W. Asi
stalks of unripe fruit
epilepsy
,
35 decoction
seeds
epileFsy
rhizome
convulsions
35
/~ts
37
Cucumis co!ocynthis L.
[92.
Curcuma aromatica Salisb.
Zingiberaceae ,
Ind, Chi, Indchi
Cuscuta europea L.
Convolvulaceae
Eur, W.C. Asi
.94.
Cynanchum saccatum
Asclepiadaceae
roots
+AUD
95.
Cynanchum decipiens
Asclepiadaceae
roots
+KND
96.
Cynanchum otophyllum
Asclepiadaceae
roots
-EST +AUD
97.
Cynodon dactylon L. Pers.
Graminae (Poaceae)
93.
Ind, Pak, Indigenous to tropics and subtropics of both hemispheres
35 35
91.
_
62
86.
,,
i
+EST -PTZ -STN
plant
oil
epilepsy & hysteria
3 alkaloids (names NM) . , 90
otophylloside A and B (14 and 15)
91
juice
34, 50, 51, 52,
leaves
+PTZ +EST
aqueous & alcoholic ext.
grass
-PTZ +IVIES
aqueous ethanolic ext.
98.
Cyperus esculentus L.
Gramineae
Mex, Ind
rhizome
99.
Cyperus rotundus L.
Cyperaceae
pant
rhizome roots
epilepsy epilepsy
+PTZ +STN
64
64
49 92
infusion
93
complex preparation 70% ethanolic ext.
35 32
100.
Cystoseira unsenoides L. Roberts
Cystoseiraceae
Spa
whole plant (alga)
101.
Datura fastuosa L.
Solanaceae
Ind, Pak
seeds
102.
Datura metel L.
Solanacear
.
103.
Daucus carota W.
Apiaceae (Umbelliferae)
Asi, Eur, costa, Ind, Pak
104. Deinbollia borbonica Scheff.
105.
106. 107.
Sopindaceae
Delphinium consolida var consolida
Ranunculaceae
Delphinium denudatum Wall.
Ranunculaceae
Desmodium adscendens Sw.
trap E. Afr
Bulg
+PTZ
34
epilepsy
47
red flowers in the centre of umbel roots
epilepsy
67
anticonvulsant
95
roots
convulsions
decoction
leaves
epilepsy & convulsions
concoction
NM
roots
Papillionaceae
Ghana
plant leaves
epilepsy
+F.ST
ethanolic
+PTZ
95% ethanolic ext.
+PTZ +KA
Desmodium polycarpum DC.
Leguminosae
Ind
plant
convulsions
109.
Desmodium tr~orum DC.
Leguminosae
Ind, Him, Pak, Chi, Phi, Med, Ids, Phi
leaves
convulsions & convulsions m children ..
110.
Desmodium pulchellum Benth.ex Baker
Leguminosae
Chi, Mal, Ids, Phi
1 1 1.
Dichrostachys cmerea L. Wight & Am.
Mimosaceae (Leguminosae)
Afr, Pak, Ind, Sri, N. Aus, Mal
Dictamnus albus L.
48
ext.
-PTZ -STN Ksh, W. temp Him
Rutaceae
Ind, Ksh, W. Him, Indochin Mal
94
epilepsy
108.
112.
methanolic ext.
decoction ethanolic ext.
root bark
epilepsy
47 96 97
50
complex preparation
37, 50, 51, 52 35
convulsions in infants undefined parts plant
62
decoction
98
epilepsy
64
hysteria
34, 50
lilt
113.
Diospyros usmabarensis F. White
114.
115.
.
.
.
.
E. & S.E.Aft"
Ebenaceae
epilepsy
roots
.
decoction
99
ethanolic ext.
62
,
Echium vulgare
Boraginaceae
Bulg
NM
Ekebergia senegalensis A.
Meliaceae
Sen
bark
+EST -PTZ -S'IN
epilepsy
53
Juss. .......
Elaeocarpus ganitrus Roxb.
116.
Elaeocarpaceae
Ind, Nep
....
epileptic fits
fruit .....
1 ! 7. [ Elaeocarpus sphaericus K.
Tiliaceae
Aft, lnd, Nep
fruit
Elaeocarpaceae
Ind
nuts
.
+IVIES
I
90% ethanolic ext.
epilepsy
53
epilepsy
5O
Schum.
118,
Elaeocarpus tuberculantus Roxb.
50, 85 I00
.....
119.
Emilia coccmea Don.
Nig
Compositae
.
.
.
.
+p'rz +EST
leaves .
.
.
.
120.
Erythrina stricta Roxb.
Fabaceae (Leguminosae)
Ind, Nep
bark
! 2 I.
Erythrina variegata L.
Leguminosae
Ind
bark
122.
Erythroxylum spp.
Erythroxylaceae
epilepsy
+STN +PTZ
~
,
123. ! 24.
Euphorbia hirta L.
Euphorbiaceae ......
Aus, Ind, Pak, pant
plant NM
Euphorbia fisheriana
Euphorbiaceae
Tai
125.
Euphorbia nyikae Pax.
Euphorbiaceae
Ken, Tan, S. Aft
126.
Euphorbta tirucalli L.
Euphorbiaceae
i
,,,
i Aft, Ind, B.ng
convulsions +E
,
roots
epilepsy
fresh milky latex
epilepsy +PTZ
aerial parts
aqueous ext.
49
.......
powder
50, 51
alkaloidal fraction
I01
cocaine (16)
102, 103
decoction
35
alkaline ext.
I04
decoction
33
with other ingredients
33
50% ethanolic ext.
I05
decoction
35, 50, 64
J~
I
127.
Excoecaria agallocha L.
Euphorbiaceae
! Ind, N. Aus, New Cal, ...... from S.E. Asi to poly
leaves
epilepsy
= ,
128.
Ferula alliacea Boiss.
Umbelliferae
Ira, Afg, Pak,
129.
Ferula foetida Re•el.
Umbelliferae
USSR
130.
Ferula galbaniflua Boiss. et.
Umbeiliferae
Per
gum resin
epilepsy
Moraceae
trop & S. Afr
root bark
covulsions
gum resin
epilepsy, hysteria & infantile
36, 37, 50, 52, 85
convulsions 47
Buhse 131.
Ficus capensis Thunb.
,,
132.
Flemingia strobilifera R.Br.
Leguminosae
31
Ind, Pak, Bng
roots
epilepsy, hysteria & nocturnal epilepsy
-
50, 87
trop S. Afr, trop & subtrop Asi, Aus, Mas
roots
epilepsy
decoction
33
......
,,
133.
Flueggea virosa Willd. Voight
Euphorbiaceae
134.
Galeopsis ladanum L.
Labiatae
Poi
overground parts
+PTZ -EST
aqueous ext. (lyophilized)
106
2-q
135.
Galium sylvaticum
Rubiaceae
Bulg
NM
+EST -~Z -STN
ethanolic ext.
62
2 ,< C
136.
Galium verum L.
Rubiaceae
W. Him, Ksh
plant
epilepsy & hysteria
juice/decoction
50
137.
Galphimia glauca Cav.
Malpighiaceae
Mex
2 ,q
shrub aerial parts
epilepsy
.,
....
decoction with Strychnos madagascariensis
.,
+PTZ +STN
107 methanolic ext.
138.
Gentiana compestris
Gentianacear
Ind, temp N.W. Him, Ksh
NM
139.
G. crassicaulis Duthie
Gentianaceae
Chi
roots
convulsions
35
140.
G. dahurica Fisch.
Gentianaceae
Chi
roots
convulsions
35
141.
G. decumbens L. f.
Gentianaceae
Pak
roots
convulsions
35
142.
G. fetisowii Reget Wink.
Gentianaceae
Chi
roots
convulsions
35
,
. . . . . .
-q ,< ,q N
N
swertianoline (45)
108
143.
Gentiana macrophylla Pallas
Gentianaceae
Mon
roots
convulsions
35
144.
G. tibetica King
Gentianaceae
Chi
roots
convulsions
35
145.
G. wutaiensis Marqund
Gentianaceae
146.
Gossypium herbaceum L.
Malvaceae
Gynandropsis pentaphylla Wiild.
roots
convulsions
35
Ind, Pak, Bang, E.S.
seeds
epilepsy
34, 47
Pak, Ind
seeds
convulsions
67
F i)
leaves
epilepsy
Afr, Egy
,
147.
Tib
Capparidaceae , ,
148.
Gyrocarpus americanus Jacq.
Gyrocarpaceae
149.
Haplophyllum dubium
Rutaceae
150.
Haplophyllum perforatum
151.
H. ~labrinu m
152.
Hedeoma pulegioides L. Pets.
+EST +PTZ
dubinine (17)
109
Rutaceae
seeds
+PTZ +CAM
haplophylidin (18)
110
Rutaceae
roots
infusion
! 11
decoction
99
Ame
~ ....
+STN L
convulsions & spasms
whole plant
Helichrysum setosum Ha rv.
Composi,tae
Tan
leaves
154.
Hemidesmus mdicus R. Br.
Asclepiadaceae
Bng, Ind
~
,
, epilepsy
155.
Heracleum sibiricum
Umbelliferae
156.
Heracleum verticillatum
Umbelliferae
157.
Hesperethusa crenulata Roxb. Roem.
Rutaceae
158.
Hibiscus abelmoschus L.
Malvaceae
Ind
seeds
159.
Himanthalia elongata L.S.F. Gray
Himanthaliaceae
Spa
whole plant (alga)
160.
Hippeastrum v i t t a t u m .
Amaryllidaceae
USSR
NM
+EST +PTZ +NIC -STN
roots
Bur ,
furocoumadns (sphondin
112, 113
pimpinellin, bergapten, isopimpineilin, angelicin & isoberl~apten) (19 to 24)
epilepsy
leaves
35
. . . . .
,
,
Malpighiaceae
47 36
epilepsy & nocturnal epilepsy
,,
Hiptage benghalensis Kurz.
63
leaves
~
153.
161.
decoction
,,
Mau, Ksh, Rod, Ind, Nep, Bng
tincture
34
+PTZ
chloroform ext.
114
+PTZ -STN
hippeastrine (25)
115
decoction
116
hysteria
epileptic fits
plant .
162.
Holarrhena floribunda 9 (G.Don) Dur et Schinz.
,,
Hoslundia opposita Vahl.
163.
Nig
Apocynaceac .....
Labiatar (Lamiaccae)
,
trop & S. Afr, Mad, Nig
aqueous ext.
49
epilepsy & convulsions
decoction with roots of Cassia petersiana Bolle.
33
convulsions
decoction with Grewia stuhlmannii Schum K. aqueous ext.
+EST +PTZ
leaves ,,
roots whole plant leaves
33 49
+PTZ +EST 164., Hoya australis R. Br.
, Asclepiadaceae
F~
leaves
convulsions
165., Humboldtia vahliana Wight
, Legumin0sae
Ind
bark
epilepsy
50
166.
Hyoscyamus niger L.
Solanaceae
Ksh, lnd
epilepsy & nocturnal epilepsy
47 36
167.
Hyptis suaveolens Poit.
Labiatae
Nig, Bra, Ind, Bng
leaves
+PTZ +EST
volatile oil
49
Icacinaceae
Nig
tuber
+PTZ -STN
50% mcthanolir ext.
117
1 6 9 . llex aqu!folium L.
Aquifoliaceae
Gre, Eur, Ind, temp
epilepsy
170. [ Impatiens repens Moon
Balsaminaceae
Sir
epilepsy
Leguminoseae (Papilionaceae)
Ind, Sri, Sen, W. trop Afr, Phi, Egy, Ids, Ame, Chi, Bra, Mal, Bur
lcacina trichantha Pflamzenfam
168. ,
,
63
decoction
)
87 64
i
171.
Indigofera tinctoria L.
roots
epilepsy
leaves
epilepsy
unspecified parts 172., Ipomoea hederacea L. Jacq 173.
lpomoea hispida Roem & Schult
,
Convolvulaceae
I Convolvulaceae
I !
epilepsy
Ind, Him Ind
powdered ext. with other ingredients juice juice with honey decoction
plant
epilepsy
with other ingredients
34, 67 64 98 ! 98 I !47 50
~
-l
r,
74
Jatropha curcas L.
Aft', Ind, tropAme
Euphorbiaceae
i
I roots & fresh leaves
convulsions & fits +PTZ -STN
roots
17 5 .
datropha gossypifolia L.
Euphorbiaceae ,,,
Air, lnd, Bra
roots & leaves
anticonvulsant
176.
J. multi~ida L.
Euphorbiaceae
S. Ame, Ind, (cultivated)
roots & leaves
anticon vulsant
177.
Juniperus macropoda Boiss.
Cupressaceae
Ind, Nep, Him, Pak
berries
178.
Kochia prostrata
Chenopodiaae
...
infusion
,
~
,
+PTZ
32
70% ethanolic ext. 70% ethanolic ext.
32
.
32
+EST
essential oil
118
+STN -PTZ
70% ethanolic ext.
119
(tincture)
Khaya ivorensis A. Chem.
Meliaceae
Aft"
stem bark
febrile convulsions in children
+PTZ
70% ethanolic ext.
120
180.
Khaya senegalensis A. Juss
Meliaeeae
Afr
stem bark
febrile convulsions in children
+PTZ
70% ethanolic ext.
120
181.
Lagochilus sp.
Labiatae
ext.
121
182.
Lammaria ochroleuca de la Pylaie
Laminariaceae
methanolic ext.
122
183.
Lantana camara L.
Verbenaceae
infusion 70% ethanolic ext.
53
decoction and concoction
99
i 179.
NM
+STN +CAF +PIC +CAM
deep seas
whole plant (alga)
-PTZ
Air, Gui, Ind, trop Ame
roots leaves
trop Aft
leaves
convulsions in children epilepsy
i i
.
.
.
.
.
.
.
.
.
.
anticonvulsant
,,
.
184.
Launaea cornuta Oliv. & Hiern. C. Jeffrey
185.
Lavandula stoechas L.
Labiatae
Por, Can, Med, As. min
-
186.
Lavandula sp.
Labiatae
Eur, Med, Asi
-
187.
Ledebouriella seseloides Wolff.
Umbelliferae
Chi, Man
roots
Compositae
+PTZ +STN
47 +PTZ +NIC +EST
convulsions & spasms .
oil vapour
123
~
35
188.
Leonurus cardiaca L.
Labiatae
Gre, Pak
189.
Leucas lavandulifolia J.Sm.
Labiatae
Irop Asi & Aft"
epilepsy leaves
Leucas zeylanica R.Br.
87
convulsions, epileptic seizures & coughing spasms
35
decoction or infusion
190.
191.
Licaria puchury major.
Lauraceae
192.
Limnophila chinensis Osb.
Scrophulariaceae
lndchi
branchlets & leaves
antispasmadic in convulsions
Rutaceae
Ind, Pak, Nep, Him, Bnl~,
leaves
epilepsy
seeds
Men'. 193.
Limonia crenulata Roxb.
+E~T
essential oil fraction
124
decoction with other ingredients
35 50
,
194,
Limonia acidissima L.
Rutaceae
Ind, Ids
i ! leaves
epilepsy
195.
Linum usitatissimum L.
Lineae
Ind
I seeds & flowers
hysteria
196.
Lobelia inflata L.
Campanulaceae
Ame, Ind
whole plant
epilepsy, hysteria & convulsions
197.
Maerua angolensis DC
Capparidaceae
Aft"
roots & leaves
epilepsy
34, 37 seed oil
111 decoction with Ricinus
epilepsy Magnolia obovata Thunberg
Magnoliaceae
Jap, Chi
Wilson
Magnoliaceae
Jap, Chi
199.
Mangifera odorata L.
Anacardiaceae
200.
Maprounea africana Muell.
Euphorbiaceae
201.
Marsilea sp.
bark
bark
Marsileaceae
Con Ind
plant leaves NM
mm
2
84
decoction +STN +Pie +PTZ +intra-cerebrovent-ricular inj of penicillin G-K
Magnolia officinalis Rehder et
....
Z ,.q
communis L.
roots 198.
34
hysteroepilepsy +PTZ
99
;r,,
ether ext.
125
,.q
magnolol (26)
126
compound mixture
35
ethanolic ext.
127
N
,.q
marsiline (27)
i
128
tirol
,'
,
,J
Marsilea minuta
202.
Marsileaceae
. . . .
Ind ,,
_ 203.
M. rajasthanensis
Marsileacear
204.
Martynia annua L.
Pedaliaceae (Martyniaeeae)
205.
M. diandra Glox.
206.
Matricaria aurea L.
207.
,,
+PTZ -EST
leaves ,,
Ind
,,
129
marsiline (27)
.....
129
leaves
50, 51, 86, 130
Mex, Ind, Nep, Pak
leaves
epitepsy
decoction
i Martyniaceae
Ind, Pak
fruit
epileptic attacks
decoction
I Coml:msitae
Pak
plant & flowers
hysteria
52
M. chamomilla L.
Compositae
lnd
dried flower heads
hysteria
34
208.
Melia azedarach L.
Meliacear
lnd, Pak, Bng, Sub Him
plant
209.
Melissa oflqcinalis L.
Labiatae
N.W. Gre, Pak
210
Meilittia usaramensis Taub.
Pa ilionaceae
Ken, Tan, Moz
Rutaceae
Phi
"
211.
~,,
Micromelum compressum Blco. ! Men'.
,
P
+PTZ +STN
,,
70% ethanolic ext.
131
32
epilepsy &. hysteria
~
roots
convulsions
decoction
31
young shoots
infantile con vulsions
with other ingredients
35
132
o
,
212.
Moghania strobilifera L. St. Hil. ex Jacks.
Leguminosae
Pak, Ind, Mal, Phi, Chi, Bur, from Ind to Phi
roots
epilepsy
35
Moringa oleifera Lamk.
Moringaceae
trop, Ind, PaL Him, E. Air, Sri, Mal, Bur, Phi
roots, root bark, leaves, gum, flowers & seeds
epilepsy & hysteria
roots
nocturnal epilepsy
34, 37, 50, 64,
.....
213.
70% ethanolic ext. ....
Moringa concunensis Nimmo
Moringaceae
used as a substitute ! f o r M. oleifera
.
.
.
.
.
215. [ Muntingia .calabura L. 216. ! Musa paradisiaca L. var sapientum Kumtzc Musa sapientum L. ,
,
Elaeoearpacee Musaceae
trop Ame, Indchi
flowers
Pak, lnd, Chi, Phi, trop
stem sap
antihysteric i epilepsy & hysteria
32 67
4
.
35, 47, 52, 67
36
+PTZ +STN 214.
~t
i
L
,,,
I
:infusion .
sap
.
.
.
.
.
.
35
35, 50, 52
217.
50
epilepsy
Ind, Chi
Mylitta lapidescens Horan. (Fungus)
I i
,
,
decoction
31
218.
Myrica salicifolia Hochst. ex. A.Rieh.
Moraceae
trop Afr
roots
convulsions
219.
Myrtus communis L.
Myrtaceae
Ind, from Med to Him
leaves
epitepsy
34, 50
220.
Nardostachys jatamansi DC.
Valerianaceae
Alp Him, Bhu, Nep, Sik
roots & rhizome
epilepsy, hysteria, convulsive affections & hysteroepilepsy
34, 37, 50, 67, +MES
221.
Nerium oleander L.
222.
223.
Apocynaceae
Newbouldia leavis (Beauv.) Seem. ex Bureau
Bignoniaceae
Nicotiana tabacum L.
Solanaceae
As min, Med
Lag, Gha, Nig
leaves & bark leaves bark & root bark leaves
epilepsy + PIC + BIC
convulsions in children
convulsions
Ind, Pak
jatamansone (isolated from essential oil) (28) fraction B-I fraction B-3
36, 47, 64, 86
133 64 134
infusion
53
+PTZ +EST
aqueous ext.
49
+PTZ
70% ethanolic ext.
32
+PTZ
70% ethanolic ext.
32
+~Z
juice 70% ethanolic ext.
33 32
+~Z -STN
70% ethanolic ext.
32
+EST +~Z
70% ethanolic ext.
135
decoction
33
oil
136
+STN
!
224. ....
Ocimum amencanum L.
Labiatae
Ind
225.
Ocimum basilicum L.
Labiatae
Asi, trop
226.
leaves
Ocimum gratissimum L.
Labiatae
Ind, (cultivated)
Ocimum sanctum L.
Labiatae
Ind, Pak, Phi
leaves
Ocimum suave Willd.
Labiatae
E.W. Aft, trop Asi
roots & leaves
Oleam miilefolii
Oleaceae
Pol
plant
epilepsy
i i 227. t
228. 229.
,
epilepsy & convulsions +PTZ
"..dl
230.
Origanum vulgare L.
Labiatae
Pak, Chi, Ksh, Indchi, Mal
-
epilepsy
23 !.
Ormocarpum kirkii S. Moore
Papilonaceae
E. & S. Aft"
roots
epilepsy
232.
paeonia albiflora (Pallas)
Paeoniaceae
Chi, Jap
233.
Paeonia emodi Wall.
Ranunculaceae
Him, Ksh, Pak
tubers
234.
Paeonia lactiflora Pall. vat trichocarpa Bunge.
Paeoniaceae
Jap
roots
.
35
-PTZ
roots
decoction
31
paeoniflorin (29)
137 34, 36, 47, 50, 52
epilepsy, convulsions, hysteria & hysteroepilepsy . pentagalloyiglucose (30)
+PTZ EEG
138
paeonifiorin (29)
-PTZ EEG albiflorin (31) methanolic ext. aqueous/acetone ext. aqueous ext.
+PTZ EEG +PTZ EEG +PTZ EEG +PTZ EEG 235.
Paeonia officmalis L.
Paeoniaceae
S. Eur, W. Asia
tubers
epilepsy, hysteria & convulsions
236.
Paeon!a ' suffruticosa Andr.
Paeoniaceae
Chi, Jap
dried.root bark
convulsions
237.
Palisota ambigua CB. Clarke.
Commelinaceae
Con, C. Air
leaves
epilepsy
238.
Panax ginseng C. A. Meyer
Araliaeear
Ame, E. & S.E. Asi, Bulg, Cana, Chi, F.E., Jap, Kor, Tib
roots
.
.
.
.
.
37, 50
~
35
99.9% ethanolic ext.
139
neutral saponins
140
~
111
.
-IviES +PTZ +NIC +STN ,,
,,
239.
Panax quinquefolius L.
Araliaeeae
Ame
roots
convulsions
240.
Paris polyphylla Sin.
Liliaeeae
Pak, Chi, Tai, lndchi
roots
epilep.~y
Passiflora incarnata
Passifloraceae
Jap
NM
241.
+PTZ -MES -PIC
~
.
+PTZ
_
.
concoction .
.
.
35
.
maltol and ethyl maltol (32 & 33). ,
141
acidic and neutral triterpene glycosides ..
142
.....
242.
Patrinia intermedia .
.
.
.
.
.
Valerianaceae
USSR
roots
+STN
243.
Rutaceae
Peganum harmala L. .
.
.
.
.
.
.
.
.
,
.
,
Ind, Pak, N. Aft, Eur, Mon, Rus, Tib, Tur
seeds
,
34
hysteria
i
decoction with Helichrysum setosum Harv.
31
244
Phoenix reclinata Jacq.
Orchidaceae
E. & S. Aft
roots
epilepsy
245.
Phyllanthus emblica L. (Syn. Emblica officinalis Gaertn ).
Euphorbiaceae
Ind, Ira, N. Ame, Nep, Cub
fruit
epilepsy & convulsions
246.
Phyllostachys nigra Lodd. var henonis Mitford
Graminae
Kor, Chi
stem
convulsions in children
complex preparation with raw ginger
35
247.
Phyllanthus urinaria L.
Euphorbiaceae
Nig, Moz, N. Ame, S. Ame, Ind, Sri, Nep, Chi, Mal, S. Kor, Indchi, Ids, Aus, Fij, Fra, Neth
roots
epilepsy & convulsions in children
used with dog's ear
144
used with Marrubium album
144
blossoms
+
alkaloids
145
dried unripe fruit
+AUD +MES -PTZ -PIC
75% ethanolic ext.
146
,
248.
Picnomon acarna L. Cass.
Compositae
Gre
249.
Piper iongum L.
Piperaceae
Gre, Chi, Jav, lnd, Sum, Tai, Bor, Sri, MaLl
250.
,
,
Piper methysticum
Piperaceae
143
leaves
,
,
,
NM
+EST
+PTZ +EST
2 tam
r-, kawain (34) dihydrokawain (35) methysticin (36) I dihydromethysticin (37) demethoxy yangonin (38) yangonin (39)
147 r
m
am
251.
Piper nigrum L.
Piperaceae
Piper retrofractum
Ind, Pak, Sri, Bra, Jav, Indochi, Ids, trop Asi, Bor, E.S. Aft, Phi, Mad, Nig, Chi
+STN +PTZ +IVIES -IVIES
fruit
seeds
piperine (40)
150 151 152 150
+
+PIC +AUD +I/C Fe + 1/V tub +l/V g!u 252.
Pithecolobium saman Benth.
253.
Plumbago :eylanica L.
254.
Polygonum japonicum Meisn. P. bistorta Sensu Chinese auct. Non L.
255.
Pongamia glabra Vent.
Leguminosae
: Plumbaginaceae Polygonaceae I
Leguminosae
Jam, trop, Ame
leaves
Ind, W. Pen, Bng
~
Chi, Tai, Jap, lndchi, Kor, Pak, S.E. Asi
rhizome
lnd, Pak
+PIC +NIC -PTZ -STN
alkaloidal fraction
153
+PTZ
70% ethanolic ext.
32
convulsions
epilepsy
,. . . . . . .
148, 149, 150
35
several complex prescript!ons
37
256.
Pothos scandens L.
Araceae
Chi, Mal, Ind
leaves
convulsions
257.
Psidium guyava L. P. guajava L.
Myrtaceae
Ame, Ind, Pak, subtrop, trop, Chi, Phi, S.E. Afr, Egy
leaves
ext. & tincture
37, 64
fresh leaves
epilepsy & convulsions in children convulsions
concoction
31
.
.
.
.
.
35
258.
Psychotria curviflora Wall.
Rubiaceae
Indchi, Mal
leaves
convulsions
poultice
35
259.
Punica granatum L.
Punicaceae
Ksh, Him, Ind, Pak
~
epilepsy.
~
47
260.
Quercus infectoria Oily.
Fagaceae
Randia esculenta Lour. Men'.
Rubiaceae
....
,,
261.
Gre, Syr, As.min, Ind, . . . . Ira,' Irq . . . . Indchi
47
epilepsy wood
convulsions
decoction with other plant drugs
35
1
Rauwolfia serpentina L. Benth.
262.
Rauwolfia vomitoria L.
263. 9
Apocynaceae
Apocynaceae
|
I
, Rosaeeae
,.266. ' Ruta chalespensis L.
, Rutaceae
267.
t
Ruta graveolens L. (Ruta graveolens var augustifoliea Pers W.)
....
f
epilepsy
stem bark 1
.
.
.
.
.
.
,,
!
leaves
i
,,,
|
Rutaceae
..,
S. Eur, Ind, Sri, (cultivated)
herb leaves seeds
35 51 130 154
+PTZ +STN
70% ethanolic ext.
32
+PTZ
70% ethanolic ext.
32
alcoholic ext.
155
~
Air, N. Ame, Arg, C.Asi, Bre, Chi, S. Eur, Sub Him, Ind, Ira, Mex, Pak, Parag lnd
with rose water decoction reserpine (41 )
.
+PTZ
.
epilepsy 1
Euphorbiaceae
.265:,, Rubus ellipticus
epilepsy
herb
Aft"
i
Ricinus communis L.
264.
=
Pak, Ind, trop Afr, C. Ame, Bur, Chi, Sri, Indchi, Thi, Sum, N. Bor, W. Lao, trop Asi
i
infantile conyu.!sions
+EST
L
Ruta tuberculata Forsk. Boiss.
269.
,,
|
Salvia nemorosa sub sp. i amplexicanlis
.
epilepsy hysteria convulsions & fits in children
juice used with other ingredients
and Z!ngiber purpuream
:
.
.
.
.
,
270. ] Salvia sclarea
I Sapindus emarginatus L. Roxb.
+EST -PTZ -S'IN
ethanolic ext.
62
Bulg
NM
+EST -PTZ -S'IN
ethanolic ext.
62
Pak, Ind
Sapindaceae t
i 272. ~ Sapindus . . . mukorossi . . Gaertn.
seed capsule .
i Sapindaceae
. . . . . . . . . .
Ind, Pak, Nep, Him, Bng, Chi
epileptic fits
Sapindus trifoliatus L. b
Ind, Bng, Sri
Sapindacear ,,
274. j Saussurea. lappa C.B. Clarke
|
.
.
.
Ksh
fruits & seeds
epilepsy
fruit & pulp of fruit
epilepsy, hysteria & hysteroepilepsy
.
'roots 9
.
9
|
2
9
epilepsy
r :Z ,<
F
:Z ,.q
,< N
.........
errhine
67
t
,
~
34, 35, 50, 52, 86
J
ra~
i
i errhine
,
Compositae
.
......
~.,
" 273.
.....
.
....
NM
_
271.
.
34, 37, 64 67, Ill 35
Bulg
. . . . . . .
i Labiatae
.
67
. . . .
Labiatae
|
36
,|
same as R. graveolens
Rutaceae
,, 9
,.
,,,
268.
,
. in preparations
36, 37, 47, 50, 64 47
|
275.
Scopolia japonica Maxim.
Solanaceae
Chi, Jap
stem & leaves
sedative for convulsions
used with other ingredients
35
plant
ep!leesv
decoction
52
,,
276.
Scutellaria 8a(ericu(ata L.
Labiatae
277.
Securidaca longepedunculata Fres.
Polygalaceae
Afr
roots
convulsions
decoction
53
Semecarpus anacardium L .
Anacardiaceae
Ind, Pak
fruit
epilepsy & hysteroepilepsy
juice of pericarp with other ingredients
34, 36, 47 37
lnd, Pak
roots
fever with fits
decoction with ginl~er
34
70% ethanolic ext. scopoletin (42)
32
.
278.
.
.
.
.Pal(, Ksh, W. tern pHim
.
279.
Sida cordifofia L.
Malvaceae
280.
Solanum americanum Jacq.
Solanaceae
281.
Solanum carolinense L.
Solanaceae
Ame
whole plant
epilepsy & convulsions
282.
Solanum dasyphyllum Schum. et Thonn.
Solanaceae
Nig
fruit
anticonvulsant
fruit
epilepsy
.
.
.
.
.
I|
ethanolic ext. scopoletin and scoparone (42 & 43)
156
283.
Solanum incanum L .
Solanaceae
crushed
157
284.
Solanum indicum L,
Solanaceae
trop lnd
+PTZ
70% ethanolic ext.
32
285.
Solanum melongena L.
Solanaceae
Ind
+PTZ
70% ethanolic ext. scopoletin and scoparone (42 & 43) ....
32
286.
Solanum nigrum L.s Lat.
Solanaceae
Aft, Pak
leaves
decoction 70% ethanolic ext.
157 32
287.
Solanum sodomaeum L.
Solanaceae
Mot
fruit
288.
Solanum torvum Swartz.
Solanaceae
lnd
289.
Solanum xanthocarpum Schrad. & Wendl.
Solanaceae
Ind
290,
Spilanthes mauritiana DC.
Compositae
trop
,,
,
anticonvulsant +PTZ +STN
antiepileptic
,,,
+PTZ
....
i
111
leaves
70~ ethanolic ext. coumarins
85 32
epilepsy
47
convulsions in children
99
epilepsy
35
291.
Stephania cepharantha Hay.
292.
Streptomyces primprina
pimprinine (metabolite)
293.
Streptoverticill.ium olivoreticuli
(44)
294.
Strychnos cinnamomifolia Thw.
Menispermaceae
Loganiaceae
S. nux vomica L. 295.
Swertia japonica
2 9 6 . . Swertia perennis L.
Tai
tuberous root
Ind
S . E . Asi, Bur, Chi, Tim, Sri, Vie ,,
epilepsy
roots seeds bark
158
decoction infusion infusion
50 34, 35 64
swertianoline (45) isoswertianoline (46)
108
Jay,
Gentianaceae
N. temp regions
NM
Gentianaceae
N. temp regions
, NM
+
swertianoline (45)
108
i
297. 298.
Swertia purpurascens Wall.
j Gentianaceae
Ind, Ksh, temp N.W. Him
NM
+
norswertianoline (47)
108
Swertia randaiensis
i Gentianaceae
-
NM
+
norswertianoline (47)
108
infusion
159
. 2 9 9 . ' Synaptolepis kirkii Oliv.
Tabernanthe iboga
300.
9
Thymel.eaceae
Zim
, roots
emleosv , epilepsy +
Apocynaceae
160
, Tabernaemontana .spp. ii
301. ~ Tamarix articulata Vahl. 302.
i,
Tamarix ~allica L.
303. i Taxus baccata L. T. wallichiana 304.
J
. ibogaine (48)
305.
Tamaricaceae
Pak, Ind
Tamarieaceae
Pak, Ind
Taxaceae (Coniferae)
Him, Pak
-
-
leaves
, ,
epilepsy
-
~| 4 7
epilepsy
-
47
90% ethanolic ext.
34, 37, 50, 52 161
epilepsy -PTZ
Terminalia chebula Retz.
Combretaceae
Ind, Bng, Bur, Sri, Ire, Mal, Pak
Tetrameles nudiflora R.Br.
Datiscaceae
S.E. Asi
epilepsy young plant
convulsions
47 infusion
35
I
306.
"Tetrapleura tetraptera (Schum.
& Thorn)
Aft"
Mimosaceae
fruit with other parts fruit
drink
53
+lrfZ +PTZ +EST
alcoholic ext. volatile oil
162 49
+PTZ +STN
talizopine (thalisopine) (49)
163
anticonvulsant
....
seeds, roots & subterrenian parts .....
307.
Thalictrum isopyroides T. rugosum T. foliolosum T. minus vat microphyllum
Ranunculaceae
308.
Thuja orientalis L.
Pinaceae ....
309.
Trema gumeensis Schum & Thorn.
Ulmaceae
310.
Trema orientalis Blume
Ulmaceae
Ind ..
plant
31 !.
Trichosanthes anguina L.
Cucurbitaceae
Ksh, Ind, Phi
~
312.
Uncaria rhyncophylla Miq.
Rubiaceae ....
Chi, Jap
dried vine ~..
convulsions in children
Valeriana hardwickii Wall. ....
Valerianaceae _ .....
Him, Ksh, Bhu
roots
epilepsy, hysteria & hysteroepileps.y
314..
Valeriana ~atamansi DC..
Valerianaceae....
Pak, Nep, Ind
roots
52
315.
Valeriana leshchenaultii DC.
Valerianaceae
Ind
roots............
50
316.
Valeriana officinalis L.
Valerianaceae
Ksh, Pak, Eur, Asi, Ind, N. Ame
roots
Pak,
Chi, Indchi
C. Aft
..
313. .,
[
35
convulsive disorders o f children
seeds ............
+PTZ -EST -PIC
epilepsy
...
epilepsy
ethanolic ext.
47
..........
47, 50, 86
infusion or tincture
+STN
roots & rhizome
crude fractionsA 7, A 8,
All
valepotriatefractions ext. Valerianaceae ....
Him, Ksh, Nep, Bhu
roots
epilepsy & hysteria
318.
Veratrum ni.g.rum L.
Liliaceae
.Chi, Kor, Tai
roots & rhizome
convulsions
, .
35
epilepsy, hysteria & hysteroepilepsy
Valeriana wallichii DC.
164 50
.....
epilepsy
317.
d~
34, 35, 36, 50, 87 165, 166 167 168
I
37, 50, 86 ~. ..
~
35
....
319.
Verbasum thapsus L.
Scrophulanaceae
temp Him, Bri, Ksh, Bhu
seeds
infantile convulsions
37
320.
Vernonia chinensis Less. V. patul a Men'.
Compositae
Mal
leaves
convulsions in children
35
321.
Vernonia hildebrandtii Vatke.
Compositae
trop Aft-
roots
convulsions in children
decoction
99
1322.
Vitex negundo var heterophylla French Rehd.
Verbenaceae
Chi, Pak
twigs
convulsions in children
infusion
35
323.
Voacanga thouarsii
Apocynaceae
+
ibogaine (48)
160
324.
Withania ashwagandha
Solanaceae
Ind
roots
+IVIES -PTZ
acetone soluble alkaloidal fraction
169
325.
Withania somnifera L. Duna
Solanaceae
Afr, Ind, Him, Pak
roots
-IVlEST +PTZ
95% ethanolic ext. (after 8 days administration)
170
antiepileptic & as sedative in epilepsy
acetone soluble fraction
leaves +SME 326.
Xanthoxyion hostile Kunth.
Xanthoxylaceae
Ind, Pak
gum, fruit & bark
debility after epilepsy
327.
Ximenia americana L.
Olacaceae
pant
roots
convulsions in children
328.
Ximenia caffra Sond. vat natalensis
Olacaceae
Ken, Tan, S.Afr
roots
epilepsy
329.
Xylotheca tettensis (Klotzsch) Gilg varfissistyla (Warb.) Steumer
Flacourtiaceae
Tan
roots
convulsions
Zanthoxylum holtzianum Engl. Waterm.
Rutaceae
Ken, Tam, Som, Moz
root bark
convulsions
Zanthoxylum chalybeum Engl.
i Rutaceae
f
53 67 ii decoction
31
I
decoction
31
i decoction
33
i
i330. 331
used wtih other ingredients
48 i
stem bark
skimmianine (50)
157
332
Zanthoxylum simulans
Rutaceae
Chi
roots
+MES +PTZ -PIC
edulinine (5 i )
171
in a lotion
35
calophyllolide and friedelan-3b-ol (52 & 53)
172
+thiosemcarbazide + Na gluta-mate
333
Zingiber ottensi Val.
334
Unidentified and un mentioned species
Zinl~iberaceae
Mal, Sum, Jav
rhizome
convulsions
decoction +
i|
i
173
ANTICONVULSANT ACTIVITIES
~ MeO-
537
O
~H= CHCH 3 OMe
H
T OMe
8-Decen-2-1actone (3)
Linalool (2)
AsEtroll~ (1}
~
O
y-Decanolactone (5}
8-Decanolactone (4}
O
Bu H
Bu 3-n-Butyl-4,5-dihydrophthalide (7}
3-n-Butylphthalide {6)
OH 0 Me
(CH2)4Me
(CH2)4Me ~ 0 Me
(A8 ) Tetrahydroeannabinol (8)
(A9 )
Tetrahydrocannabinol (9)
538
MOHSIN et aL
2)3~" HCI
Me
HO {CH4)4Me
....
M
Me
)"
~
~,ao~
""CHMeCHMe(CH2)4Me
Me~CH a
Cannabidiol
SP-175
(11)
(10}
zo HO MeO~N-~~
~
\
\
~/"
/ Heliettin (la)
Quinine {12)
OR
~ Me
I
e
Me
COMe H
l'~176
Cocaine (16}
OtophyHoside A, R = C ~ O H (14) and (15)
Otophylloside B, R = COCH ~CMeCHMe2
ANTICONVULSANT ACTIVITIES
539
Me
jCH2OCOCH 3
~,oFr
HO"" OMe
Dubinine (17)
----~
HaplophyHdin
(18)
H3C
~ CH3
)CH 3
)CH 3 Sphondin (19)
Pimpinellin (20)
Isopimpinellin (21)
)CH 3
Isobergapten
Bergapten (22}
(23}
0
Angelicin (24)
Hippeastrine (25)
540
MOHSIN et
OI-DH o
Ill
CH3--(C H2}2A.---C-O(C H2) 29" CH3
f
Marsiline (27} Jatamansone
(28)
ll~agnolol
(26)
O m.,m,m
ooo
;~~o~"
o
Q--
Paeoniflorin (29)
~>---0
OH
(~=--
PhCOOCH 2 Albiflorin
OH
(31}
Pentagalloylglucose OH (ao} Me
0
CH2CH 3
OH
Me
OH P
Me
0 P
0 H
Maltol (32)
Ethyl Maltol (aa}
Kawain
(34}
Dihydrokawain (35} Me
OMe
0
Methysticin
(36)
Dihydromethysticin (37)
ANTICONVULSANT ACTIVITIES
541
Mc
Me
0 Yangonin
(ag)
Demethyoxyyangonin
{a8)
) Piperine
{40)
Mea3. Mooo~- y o~ Reserpine (41)
-ooc~ //--o~o \ OMe
MeO HO ~
O
Scoparone {43)
Scopoletin (42)
OR I
H Pimprinlne (44)
Swertlanollne R = glucopyranoslde, R i = Me, R 2 = H I s o s w e r t l a n o U n e R -- H, R ! = Me. R 2 - glucopyranoslde N~176 R = glucopyranoslde, R I = R 2 = H (45), (48) a n d (47)
542
MOHSINet
MeO.%,~"~.
~
E.
[1
I[
~N
OMe 0
CH2CH3 Me~
Ibogaine (48) Mq
Talizopine(thalisopine) (49)
)Me
)Me
~
Me(
Me
)Me Skimmianine (50)
CH2CHIOH)CMe20H O
Edulinine (51)
Me
MemO M
e
m
O
H(
eOr M -Me
Me
CalophylloUde (52)
ABBREVIATIONS Afg Aft Alp
= = =
Friedelan-3~-ol (s3)
OF COUNTRY NAMES AND PLACES
Afghanistan Africa Alpine
ANTICONVULSANT ACTIVITIES
Ame
Ara Arg Asi As.min Aus Bng Bhu Bor Bra Bri Bulg Bur Cal Cam Cana Can Con cosm Cub Ecu Egy Eur F.E.
m
m m
u n
D
Fij
USSR Fra Gha Gre Gui Him Ind Indchi Ids Irn Irq Ire Isr Jam Jap Jav Ksh Ken Kor Lag
m m
America Arabia Argentina Asia Asia minor Australia Bangladesh Bhutan Borneo Brazil Britain Bulgaria Burma Caledomia Cameron Canada Canaries Congo cosmapolitan Cuba Ecuador Egypt Europe Far East Fiji Former Soviet Union France Ghana Greece Guiana Himalaya India Indo China Indonesia Iran Iraq Ireland Israel Jamaica Japan Java Kashmir Kenya Korea Lagos
543
544
Lao Mad Mal Mas Mau Med Mex M.E. Mon Mor Moz N.Guin Nep Neth Nig Pae Pak pant Parag W. P e n Per Phi Pol Poly Por Rod Rus Sen Sik Som Spa Sri Sub Him Sum Syr Tai Tan temp Thi Yib Tim trop Tur USA Vie
MOHSIN et aL
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
Laos Madagascar Malaya Peninsula Masearene Island Mauritius Mediterranean Mexico Middle East Mongolia Morocco Mozambique New Guinea Nepal Netherlands Nigeria Pacific Pakistan pantropie Paraguay Western Peninsula Persia Philippines Poland Polynesia Portugal Rodrigues Russia Senegal Sikkim Somalia Spain Sri L a n k a Sub H i m a l i y a n tract Sumatra Syria Taiwan Tanzania temperate Thailand Tibet Timor tropical/tropics Turkey U n i t e d States o f A m e r i c a Vietnam
ANTICONVULSANT ACTIVITIES
W. Ind
=
Zim
=
545
West Indes Zimbabwe
LIST OF ABBREVIATIONS
NM +
= Details not mentioned in Chemical Abstracts = No anticonvulsant activity in animals = Anticonvulsant activity in animals
+PTZ
= Anticonvulsant activity against pentylenetetrazole-induced seizures in animals
-PTZ
= No significant anticonvulsant activity/no activity against pentylenetetrazole-induced seizures in animals.
+EST
= Anticonvulsant activity against eletroschock-induced seizures in animals
-EST
= No anticonvulsant activity against eletroschock-induced seizures in animals
+SME
= Anticonvulsant activity against supramaximal electroshock-induced seizures in animals
-SME
- No anticonvulsant activity against supramaximal electroshock-induced seizures in animals
+MES
= Anticonvulsant activity against maximal electroshockinduced seizures in animals = No anticonvulsant activity against maximal electroshockinduced seizures in animals = Anticonvulsant activity against strychnine-induced seizures in animals = No significant anticonvulsant activity/no activity against strychnine-induced seizures in animals - Anticonvulsant activity against picrotoxin-induced seizures in animals
-MES +STN -STN +PIC +AUD
= Anticonvulsant activity against audiogenic seizures in animals
+KND
- Anticonvulsant activity against kindling-induced seizures in animals
+NIC
= Anticonvulsant activity against nicotine-induced seizures in animals
546
MOHSIN eta/.
+CAF
=
+CAM
=
+PTZ EEG -PTZ EEG = + 1/C Fe
=
+l/V tub
-
+l/V glu
=
+E
=
Anticonvulsant activity against caffeine-induced seizures in animals Anticonvulsant activity against camphor-induced seizures in animals Significant inhibition of EEG power spectrum changes after pentylenetetrazole administration in animals No/insignificant inhibition of EEG power spectrum changes after pentylenetetrazole administration in animals Anticonvulsant activity against clonic seizures produced by intracerebral injection of ferrous sulphate in animals Anticonvulsant activity against seizures produced by intraventricular injection of d-tubocurarine in animals Anticonvulsant activity against seizures produced by intraventricular injection of L-glutamate in animals Antiepileptic activity in epileptic palients
ACKNOWLEDGEMENT Dr. M. Raza was a recipient of research grant from F.E.M.T. The authors wish to thank Mr. Syed Tauseef Hussain Naqvi for typing the manuscript. REFERENCES [l] [2] [31
[4]
[51 [61 [7]
IS] [9]
[10] [11]
Dorlands illustrated medical dictionary, 28th edition. W. B. Saunders, 1994. Gastaut, H. Dictionary of epilepsy. World Health Organization, Geneva, 1973. Commission on classification and terminology of the international league against epilepsy. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 1989, 30, 389-399. Sander, J. W. A. S. and Shorvon, S. D. J. Neurol. Neurosurg. Psychiat., 1996, 61, 433-43. French, J. Ann. Intern. Med., 1994, 120, 411-422. Mastroiacovo, P., Botto, L., Serafini, M. and Zampino, G. Ann. Inst. Super. Sanit. Rom., 1993, 29, 77-82. Holmes, G. L. Am. J. Hosp. Pharm., 1993, 50, (Suppl 5), 85-116. McNamara, J. O. In, Goodman and Gilman's The Pharmacological Basis of Therapeutics;. Joel G. Hardman et.al. (Ed.) McGraw Hill. 1996; 9th edition, pp. 461-486. Ketter, T. A., Malow, B. A., Flamini, R., White, S. R., Post, R. M. and Theodore, W. H. Neurology, 1994, 44, 55-6 I. Chow, C. J. Beijing Med. Coll., 1974, 4, 214-216. Pei, Y. Q. Epilepsia, 1983, 24, 177-182.
ANTICONVULSANT ACTIVITIES
[12] [131 [14]
[16] [17]
[18] [19]
[20] [211 [221 [23]
[24] [25] [26]
[27] [28] [29] [30] [311 [32] [33] [34]
[35] [36]
547
Chow, C.. `l. Beijing Med. Coll., 1981, 13, 1-8. Loscher, W. and Schmidt, D. Epilepsy Res., 1988, 2, 145-181. Purpura, D. P., Penry, J. K., Tower, D., Woodbury, D. M. and Walter, R. Experimental models of epilepsy- A manual for the laboratory worker Raven Press, 1972. Porter, R. J., Cereghino, J. J., Gladding, G. D., Hessie, B. J., Kupferberg, H. J., Scoville, B. and White, B. J. Clev. Clin. Quart., 1984, 51,293-305. Krali, R. L., Penry, J. K., White, B. G., Kupferberg, H. J. and Swinyard, E. A. Epilepsia, 1978, 19, 409-428. Swinyard, E. A. In: Anticonvulsant Drugs, International Encyclopedia of Pharmacology and Therapeutics, J. Mercier Ed.; Pergamon Press, 1972; Section 19, Vol 1, pp 47-65. Sugaya, E. SK (TJ-960). Drugs of the Future, 1987, 12, 360-363. Takato, K., Takamura, K., Sugaya, A., Tsuda, T. and Sugaya, E. IRCS Med. Science, 1982, 10, 86-87. Sugaya, E., lshige, A., Sekiguchi, K., Iizuka, S., Sugimoto, A., Yuzurihara, M. and Hosoya, E. Epilepsy Res., 1988, 2, 337-339. Sugaya, E., Ishige, A., Sekiguchi, K., Iizuka, S., Ito, K., Sugimoto, A., Aburada, M. and Hosoya, E. Epilepsy Res., 1988, 2, 27-31. Sugaya, E., Ishige, A., Sekiguchi, K., Yuzurihara, T., lizuka, s., Sugimoto, A., Takeda, S., Wakui, Y., lshihara, K. and Aburada, M. J. Ethnopharmacol., 1991, 34, 13-19. Nagakubo, S., Niwa, S., Kumagai, N., Fukuda, M., Anzai, N., Yamauchi, T., Aikawa, H., Toyoshima, R., Kojima, T., Matsuura., M., Ookubo, Y. and Ootaka, T..lap. .l. Psychiat. Neurol., 1993, 47, 609-620. Sugaya, A., Suzuki, T., Sugaya, E., Yuyama, N., Yasuda, K. and Tauda, T. J. Ethnopharmacol., 1991, 33, 159-167. Sugaya, A., Tasuda, T., Kubota, K., Motoki, M. and Sugaya, E. Bioimages, 1993, 1, 115-123. Backhaub, C. and Krieglstein, J. European `l. Pharmacol., 1992, 215, 365-269. Schmitz, D., Zhang, C. L., Chatterjee, S. S. and Heinemann, U. Nauynschmiedeberg's Arch. Pharmacol., 1995, 351, 348-355. Elisabetsky, E., Marschner, J. and Souza, D. O. Neurochem. Res., 1995, 20, 462465. Hamada, H., Hiramatsu, M., Edamatsu, R. and Mori, A. Arch. Biochem. Biophys., 1993, 306, 261-266. Wada, J. A., Wake, A., Sato, M. and Corcoran, M. E. Epilepsia, 1975, 16, 503501. Chhabra, S. C., Mahunnah, R. L. A. and Mshiu, E. N. J. Ethnopharmacol., 1990, 29,295-323. Adesina, S. K. Fitoterapia, 1982, 53, 147-162. Chhabra, S. C., Mahunnah, R. L. A. and Mshiu, E. N. `l. Ethlopharmacol., 1990, 28, 255-283. Kirtikar, K. R. and Basu, B. M. Indian medicinal plants. Apura Krishna Bose Indian Press Allahabad, India, 1918. Perry, L. M. Medicinal plants of east and southeast Asia. The MIT Press USA, 1980. Nadkarni, K. M. Indian materia medica. Popular Prakhshan Pvt. Limited Bombay, India, 1976.
548
[37]
[381 [39] [40]
[41] [42] [43]
[44] [45] [46] [47] [48] [49]
[50] [51] [52]
[53] [541 [55] [561 [57] [58] [59l [601 [611 [62] [63]
MOIISIN eta/.
Dymock, W. Pharmacographia Indica. (Ed.) Said, H. M. (reprinted 1972) The institute of health and tibbi research Hamdard National Foundation, Pakistan, 1890. Madan, B. R., Arora, R. B. and Kapila, K. Arch. Intern. Pharmacodynamie., 1960, 124, 201-211. (cited from Chemical Abstracts 54: 12388e). Vohora, S. B., Shah, S. A. and Dandiya, P. C. J. Ethnopharmacol., 1990, 28, 5362. Dandiya, P. C. and Sharma, J. D. Ind. J. Med. Res., 1962, 50, 46-60. (cited from Chemical Abstracts 57: 1495i). Dandiya, P. C. Menon, M. K. Brit. J. Pharmacol., 1963, 20, 436-442. (cited from Chemical Abstracts 59: 3235h). Martis, G., Rao, A. and Karanth, K. S. Fitoterapia, 1991, 62, 331-337. Elisabetsky, E., Coelho De Souza, G. P., Dos Santos, M. A. C., Siquieira, I. R., Amador, T. A. and Nunes, D. S. Fitoterapia, 1995, 66, 407-414. Coelho De Souza, G. P., Elisabetsky, E., Nunes, D. S., Rabelo, S. K. L. and Nascimento da Silva, M. J. Ethnopharmacol., 1996, 58, 175-181. Adesina, S. K. and Ette, E. I. Fitoterapia, 1982, 53, 63-66. Kasture, V. S., Kasture, S. B. and Pal, S. C. Ind J. Exp. Biol., 1996, 34, 78-80. Said, H. M. Hamdard pharmacopoeia of eastern medicine. Hamdard National Foundation, Times Press, Sadar Karachi, Pakistan, 1970. Chhabra, S. C., Mahunnah, R. L. A. and Mshiu, E. N. J. Ethnopharmacol., 1991, 33, 143-157. Akah, P. A. and Nwambie, A. I. Fitoterapia, 1993, 64, 42-44. Chopra, R. N., Chopra, I. C. and Nayar, S. L. Glossary oflndian medicinal plants. Council of Scientific and Industrial Research New Delhi, India, 1956. Singh, V. K. and Khan, A. M. Medicinal plants andfolkores. J. N. Govil. (Ed.) Today and Tomorrow Printers and Publishers New Delhi, India, 1990. Baquar, S. R. Medicinal and poisonous plants of Pakistan. Printas Karachi, Pakistan, 1989. Oliver, B. B. J. Ethnopharmacol., 1983, 7, 1-93. N'gouemo, P., Koudogbo, B., Pambou-Tchivounda, H., Akono-Nguema, C. and Etoua, M. M. Phytother. Res., 1997, 2, 243-245. Perera, P., Kumlin, E., Sandberg, F., Dutschewska, H., Christov, V. and Philipov, S. Fitoterapia, 1991, 62, 139-143. Junshan, Y. and Yuwu, C. Yaoxue Tongbao, 1984, 19, 670-671. (cited from Chemical Abstracts 103: 2687a). Kulshrestha, V. K., Singh, N., Saxena, R. C. and Kohli, R. P. Ind. J. Med. Res., 1970, 58, 99-102. (cited from Chemical Abstracts, 72: 109572a). Guizhi, W., Jingxia, L., Hongbo, C., Wei, L., Guangshu, W. and Jingda, X. Zhongcaoyao, 1988, 19, 77-79. (cited from Chemical Abstracts 108:180057d). Junhua, H., Dechao, Y., Xianyu, C., Zenin, H., Xiaozhang, F., Araki, P., Tsuchida, K., Asami, Y., Aihara, H., Tamai, M., Watanabe, N., Obuchi, I., Omura, S. and Araki, H. Fitoterapia, 1990, 61, 207-214. Hirschmann, G. S. and de Arias, A. R. J. Ethnopharmacol., 1990, 29, 159-172. De Lima, T. C. M., Morato, G. S. and Takahashi, R. N. Planta Med., 1993, 59, 326-329. Atanasova, S. V., Rusinov, K. and Markova, M. Bulg. Akad. Nauk., 1969, 12, 205216. (cited from Chemical Abstracts 72: 98942t). Steiner, R. P. Folk medicine, The art and the science. American Chemical Society, Washington DC. Maple Press Company, York, PA, USA, 1986.
ANTICONVULSANT ACTIVITIES
549
Jayaweera, D. M. A. Medicinal plants used in Ceylon. The National Science Council of Sri Lanka, Columbo, 1981. [65] Ganguly, D. K. and Malhotra, C. L. Ind. J. Med. Res., 1967, 55, 473-482. (cited from Chemical Abstracts 67: 42392t). Rao, G. M. A. and Karanth, K. S. Fitoterapia, 1992, 63, 399-404. [66] [67] Murray, J. A. The plants and drugs of Sind. (reprinted 1984) Periodical Expert Book Agency Delhi, India, 1881. [68] Akah, P. A. and Nwaiwu, J. I. J. Ethnopharmacol., 1988, 22, 205-210. [69] Consroe, P. F., Man, D. P., Chin, L. and Picchioni, A. L. J. Pharm. Pharmacol., 1973, 25, 764-765. (cited from Chemical Abstracts 80: 440000. [70] Consroe, P. E. and Man, D. P. Life Sci., 1973, 13, 429-39. (cited from Chemical Abstracts 80: 44003j). [71] Carasso, R. L., Yehuda, S. and Frommer, R. Int. J. Neurosci., 1978, 8, 65-9. (cited from Chemical Abstracts 89: 140507h). [72] Karler, R. and Turkanis, S. A. Adv. Biosci. 22-23, Marihuana: Biol. Effl, 1978, 619-641. (cited from Chemical Abstracts 92:52119q). [73] Karler, R. and Turkanis, S. A. J. Clin. Pharmacol., 1981, 21, Suppl. 437-448 (cited from Chemical Abstracts 95: 37089e). [74] Karler, R. and Turkanis, S. A. Br. J. Pharmacol., 1980, 68, 479-484. (cited from Chemical Abstracts 93: 37089h). [751 Dikshit, S. K., Tewari, P. V. and Dixit, S. P. Ind. J. Physiol. Pharmacol., 1972, 16, 81-83. (cited from Chemical Abstracts 77: 29159j). [761 Hashimoto, Y. Japan 1967, 10, 922 (CI.30A31) June 16, Appl, Dec. 19, 1964. (cited from Chemical Abstracts 67: 42392t). [77] AI-Yahya, M. A. Fitoterapia, 1986, 57, 179-182. [78] Ruiz, A. N., Ramirez, B. E. B., Estrada, J. G., Lopez, P. G. ad Garzon, P. J. Ethnopharmacol., 1995, 45, 199-206. [79] Diwan, P. V., Karwande, I. and Singh, A. K. Fitoterapia, 1991, 62, 253-257. [80] Sakina, M. R. and Daniya, P. C. Fitoterapia, 1990, 61, 291-296. [81] Hien, T. T., Navarro-Delmasure, C. and Train, V. J. Ethnopharmacol., 1991, 34, 201-206. [821 Nikorskaya, B. S. and Shreter, A. I. Meal Prom. SSSR, 1961, 15, 47-8. (cited from Chemical Abstracts 56: 9375d). [83] Lesany, I., Dittrich, J. and Odvarkova, J. Casopis L~karu Ceskych, 1957, 96, 707713. (cited from Chemical Abstracts 51:18348d). [84] Chhabra, S. C. and Uiso, F. C. Fitoterapia, 1989, 59, 307-316. [85] Beilakhdar, J., Claisse, R., Fleurentin, J. and Younus, C. J. Ethnopharmacol., 1991, 35, 123-143. [86] Suwal, P. N. Medicinal plants of Nepal. His Majesty's Govt. Press, Sinha Darbar Khatmandu, Nepal, 1982. [87] Vokou, D., Katradi, K. and Kokkini, S. J. Ethnopharmacol., 1991, 39, 187-196. [88] Sharma, V. N., Barar, F. S. K., Khanna, N. K. and Mahawar, M. M. Ind. J. Med. Res., 1965, 53, 871-876. (cited from Chemical Abstracts 77: 29159j). [89] Tseun, H. M., Jung, H. H., Yann, T. H., Yuan, C. N., Chan, C. H., Feng, H. K., Shiow, L. J., Chen, C. H. and Shung, W. T. Chung-hua Yao Hsueh Tsa Chih, 1989, 41,227-237. (cited from Chemical Abstracts 111' 187306e). [90] Viquan, P., Jin, D., Winxiang, C., Zhiqin, L. and Quanzhang, M. Beijing Yike Daxue Xuebao, 1987, 19, 29-32. (cited from Chemical Abstracts 110: 69348b). [911 Quanzhang, M., Jianrong, L. and Qialan, Z. Sci. Sin., Ser. B., 1986, 29, 295-301. (cited from Chemical Abstracts 106:81545p). [641
550
[92] [93] [94] [95] [96] [97] [98] [99] [ 100] [101] [ 102] [103] [ 104] [ 105] [106] [107] [108] [ 109] [110] [111] [112] [113] [114] [115] [116] [117]
MOHSIN et aL
Odenigbo, G. O. and Awachie, P. I. Fitoterapia, 1993, 64, 447-449. Zamora-Martinez, M. C. and Pascal Pola, C. N. J. Ethnopharmacol., 1992, 35, 229-257. V[izquez-Freire, M. J., Castro, E., Lamela, M. and Cajella, J. M. Phytother. Res., 1995, 9, 207-210. Lokar, L. C. and Poldini, L. J. Ethnopharmacol., 1988, 22, 231-278. Authors, unpublished data, 1995. N'gouemo, P., Baldy-Moulinier, M. and Nguemby-Bina, C J. Ethnopharmacol., 1996, 52, 77-83. Samuelsson, G., Farah, M. H., Claeson, P., Hagos, M., Thulin, M., Hedberg, O., Warfa, A. M., Hassan, A. Q., Elmi, A. H., Abdurahman, A. D., Elmi, A. S., Abdi, Y. A. and Alin, M. H. J. Ethnopharmacol., 1992, 37, 93-112. Chhabra, S. C., Mahunnah, R. L. A. and Mshiu, E. N. J. Ethnopharmacol., 1989, 25, 339-359. Bhattacharya, S. K., Debnath, P. K., Pandey, V. B. and Sanyal, A. K. Planta Med., 1975, 28, 174-175. Ghosal, S., Dutta, S. K. and Bhattacharya, S. K. J. Pharm. Sci., 1972, 61, 12741277. (cited from Chemical Abstracts 77: 101963m). Guiti~rrez-Noriega, C. and Ortiz, V. Z. Rev. Med. Exptl., 1947, 4, 249-267. (cited from Chemical Abstracts 42: 980c). Guti6rrez-Noriega, C. and Ortiz, V. Z. Rev. Med. Exptl., 1945, 4, 59-70. (cited from Chemical Abstracts 41:2173c). Liu, Y. X., Wang, M. Z. and Sun, X. F. Chung Kuo Chung Hsi I Chieh, 1994, 14, 282-284. Samuelsson, G., Farah, M. H., Claeson, P., Hagos, M., Thulin, M., Hedberg, O., Warfa, A. M., Hassan, A. O., Elmi, A. H., Abdurahman, A. D., Elmi, A. S., Abdi, Y. A. and Alin, M. H. d. Ethnopharmacol., 1992, 37, 47-70. Czarnecki, R., Librowski, T., Zebala, K. and Kohlmunzer, S. Phytother. Res., 1993, 7, 9-12. Tortoriello, J. and Lozoya, X. Planta Med., 1992, 58, 234-236. Ghosal, S., Sharma, P. V. and Chaudhuri, R. K. J. Pharm. Sci., 1974, 63, 12861290. (cited from Chemical Abstracts 81" 176084k). Polievtsev, N. P. Voprosy Biol. i. Kraevoi Med., Akad. Nauk. Uzbek. SSR. Otdel. Biol. Nauk., 1960, 231-237. (cited from Chemical Abstracts 56: 10874e). Magrupova, M. A., Kamilov, I. K. and Polietsev, N. P. Farmakol. Alkaloidov, Akad. Nauk. Uz. SSSR., Inst. Khim Rast. Veshchestv, 1962, 1, 169-173. (cited from Chemical Abstracts 61" 112120. Krochmal, A. and Krochmal, C. A guide to the medicinal plants of the United States. Quadrangle, The New York Times Book Co., USA, 1980. Rusinov, K., C. R. Acad Bulg Sci., 1966, 19, 985-987. (cited from Chemical Abstracts 66: 27550d). Rusinov, K. S. Izv. Inst. Fiziol., Bulg. Akad. Nauk., 1968, 11, 141-150. (cited from Chemical Abstracts 69: 852820. Anca, J. M., Lamela, M. and Cajella, J. M. Planta Med., 1993, 59, 218-220. Zakirov, U. B., Abdumaliova, N. V., Sadritdinov, F. and Kamilov. I. K. FarmakoL Alkaloidov, Akad. Nauk. Uzb. SSR. Inst. Khim. Rast. Veshcheslv., 1965, 2, 243252. (cited from Chemical Abstracts 66: 54109y). Fakim A. G., Sewraj, M., Gueho, J. and Dulloo, E. J. Ethnopharmacol., 1993, 39, 175-185. Asuzu, I. U. and Abubakar, I. U. Phytother. Res., 1995, 9, 21-25.
ANTICONVULSANTACTIVITIES
551
[118] Mishra, P. and Agrawal, R. K. Fitoterapia, 1989, 60, 339-345. [119] Alidzhanov, A., Magrupova, M. A. and Sadritdinov, F. S. (Ed.) Kamilov, I. K. Farmakol. Alkaloidov. Glikozidov. pp. 178-181, 1967. (cited from Chemical Abstracts 70: 56096y). [ 120] Adesina, S. K. Fitoterapia, 1983, 54, 1412-3. [121] Akopov, I. E. FarmakoL Toksikol., 1954, 17, 34-37. (cited from Chemical Abstracts 48: 13988d). [122] Vilzquez-Freire, M. J., Lamela, M. and Cajella, J. M. Phytother. Res., 1994, 8, 422-425. [123] Yamada, K., Mimaki, Y. and Sashida, Y. Biol. Pharmaceut. Bull., 1994, 17, 359360. [124] Carlini, E. A., De Oliveira, A. B. and De Oliveira, G. G. J. Ethnopharmacol., 1983, 8, 225-236. (cited from Chemical Abstracts 100: 32182k). [125] Kazuo, W., Yoshiaki, G. and Kyosuke, Y. Chem. Pharm. Bull., 1973, 21, 17001708. (cited from Chemical Abstracts 79: 121892g). [126] Watanabe, K., Watanabe, H., Goto, Y., Yamaguchi, M., Yamamoto, N. and Hagino, K. Planta Med, 1983, 49, 103-108. [127] N'gouemo, P., Nguemby-Bina, C. and Baldy-Moulinier, M. J. Ethnopharmacol., 1994, 43, 161-166. [128] Chatterjee, K. A., Kumar, D. P. and Ranjan, M. S. Indian 90, 813 (CI. A61K27/14), 30 Aug, 1975. Appl. 14 Nov. 1963. (cited from Chemical Abstracts 92: 82422a). [ 129] Chatterjee, A., Dutta, C. P., Choudhary, B., Dey, P. K., Dey, C. D., Chatterjee, C. and Mukherjee, S. R. Sci. Cult, 1963, 29, 619-620. (cited from Chemical Abstracts 61: 3587h). [ 130] Nagaraju, N. and Rao, K. N. J. Ethnopharmacol., 1990, 29, 137-158. [131] Tiwari, V. J. and Padhye, M. D. Fitoterapia, 1993, 64, 58-61. [ 132] Malamas, M. and Marselos, M. J. Ethnopharmacol., 1992, 37, 197-203. [133] Arora, R. B., Sharma, P. L. and Kapila, K. Ind. J. Med. Res., 1958, 46, 782-91. (cited from Chemical Abstracts 53: 5515g). [ 134] Zia, A., Siddiqui, B. S., Begum S., Siddiqui, S. and Suria, A. ,1. Ethnopharmacol., 1995, 49, 33-39. [135] Sakina, M. R., Dandiya, P. C., Hamdard, M. E. and Hameed, A. J. Ethnopharmacol., 1990, 28, 143-150. [136] Kudrzycka-Bieloszabska, F. W. and Glowniak, K. Diss. Pharm. Pharmacol., 1966, 18, 449-454. (cited from Chemical Abstracts 67: 62837r). [137] Takagi, K. and Harada, M. Yakugaku ZasshL 1969, 89, 879-886 (cited from Chemical Abstracts 71:100282k). [138] Sugaya, A., Suzuki, T., Sugaya, E., Yuyama, N., Yasuda, K. and Tsuda, T. J. Ethnopharmacol., 1991, 33, 159-167. [ 139] N'gouemo, P., Baldy-Moulinier, M. and Nguemby-Bina, C. Phytother. Res., 1994, 8, 426-429. [140] Nabata, H., Saito, H. and Takagi, K. Japan J. Pharmacol., 1973, 23, 29-41. [141] Nobuo, A., Ryohei, K. and Toshiro, M. Chem. Pharm. Bull., 1974,22, 1008-1013. (cited from Chemical Abstracts 81:86167k). [142] Lapik, A. S., Bukharov, V. G. Talan, V. A. and Karlin, V. V. Farmakol. ToksikoL, 1968, 31,650-652. (cited from Chemical Abstracts 70:56100v). [143] Unander, D. W., Webster, G. L. and Blumberg, B. S. J. Ethnopharmacol., 1990, 30, 233-264.
552
MOHSIN et aL
[ 144] Unander, D. W., Webster, G. L. and Blumberg, B. S. J. Ethnopharmacol., 1991, 34, 97-133. [145] Gonzalez, B. J., Peinado, I. I. and Garcia, Z. F. An. Bromatol., 1983, 35, 177-182. (cited from Chemical Abstracts 102: 57377g). [146] Pei, Y. Q.J. Traditional Chinese Med., 1983, 3, 17-22. [147] Kretzschmar, R. M. and Joachim, H. Arch. Int. Pharmacodyn. Ther., 1969, 177, 261-277. (cited from Chemical Abstracts 72: 30057c). [148] Lee, E. B., Shin, K. H. and Woo, W. S. Arch. Pharm. Res., 1984, 7, 127-132. [149] Woo, W. S., Lee, E. H. and Shin, K. H. Arch. Pharm. Res., 1979, 2, 121. (cited from Chemical Abstracts 93:61624b). [ 150] Pei, Y. Q. and Tao, C. J. Beijing Med. Coll., 1974, 4, 217-2. [15 l] Woo, W. S., Lee, E. B. and Shin, K. H. Soul Taehakkyo Saengyak Yonguso Opjukjip, 1979, 18, 66-70. (cited from Chemical Abstracts 93: 215482j). [ 152] Shin, K. H., Yun, H. S., Woo, W., S. and Lee, C. K. Soul Taehakkyo Saengyoak Yonguso Opjukjip, 1979, 18, 87-89. (cited from Chemical Abstracts 93:215484s). [153] Leonard, B. E. Arch. Int. Pharmacodyn. Ther., 1967, 166, 435-438. (cited from Chemical Abstracts 67: 1928d). [154] Chowhan, J. S. Antiseptic, 1956, 53, 858-860. (cited from Chemical Abstracts 52: 14971g). [155] Rana, Santani, D. D. and Saluja, A. K. Ind. J. Pharm. Sci., 1990, 52, 174-177. (cited from Chemical Abstracts 115:4181 lb). [156] Adesina, S. K. J. Nat. Prod., 1985, 48, 147. [157] Samuelsson, G., Farah, M. H., Claeson, P., Hagos, M., Thulin, M., Hedberg, O., Warfa, A. M., Hassan, A. O., EImi, A. H., Abdurahman, A. D., Elmi, A. S., Abdi, Y. A. and Alin, M. H. J. Ethnopharmacol., 1993, 38, 1-29. [158] Narasimhan, M. J. Jr. and Gangla, V. G. Hindust. ,4ntibiot. Bull., 1967, 9, 138142. (cited from Chemical Abstracts 67: 20358j). [ 159] Borris, R. P., Blasko, G. and Cordell, G. A. J. Ethnopharmacol., 1988, 24, 41-9 I. [ 160] Dictionary of natural products Vol. 3. Chapman & Hall, U. K. pp. 3263, 1994. [161] Vohora, S. B. and Kumar, I. Planta Med., 1971, 20, 100-107. [162] Adesina, S. K. and Sofowora, E. A. Planta Med., 1979, 36, 270-271. [ 163] Tashbaev, K. I. and Sultanov, M. B. Farmakil. ,41kaloidov, ,4kad. Nauk. Uz. SSR, Inst. Khim. Rast. Veshchesti, 1962, 1, 210-219. (cited from Chemical Abstracts 61: 8790a). [ 164] N'gouemo, P., Pamb0u-Tchivounda, H., Baldy-Moulinioer, M., Koudogbo, H. and Guemby-Bina, C. N. Planta Ailed., 1994, 60, 305-307. [165] Cionga, E. Pharmazie, 1961, 16, 43-44. (cited from Chemical Abstracts 56: 14396d). [ 166] Muresan, V., Simionovici, M. and Cucu,V. Farmacia, 1958, 6, 43-52. (cited from Chemical Abstracts 52:14971 g). [167] Manolov, P. and Petkov, V. Farmatsiya, 1976, 26, 24-34. (cited from Chemical Abstracts 85: 137513u). [168] Mueller, J. and Rudmann, M. Arzneim Forsch, 1993, 43, 638-641. (cited from Chemical Abstracts 119: 85975v). [169] Prasad, S. and Malhotra, C. L. Ind. J. Physiol. Pharmacol., 1968, 12, 175-81. (cited from Chemical Abstracts 7 l: 89860s). [ 170] Prabhu, M. Y., Rao, A. and Karanth, K. S. Fitoterapia, 1990, 61, 237-240. [171] Zhiqing, C., Gengxin, H., Zhong, T., Ligang, Q., Gengsheng, L., Kunjan, G., Ruyun, J., Wencai, J. and Bin, C. Yaolixue Yu Dulixue Zazhi., 1988, 2, 109-112. (cited from Chemical Abstracts 109: 48274h).
ANTICONVULSANTACTIVITIES
553
[172] Chaturvedi, A. K., Parmar, S. S., Bhatnagar, S. C., Mistra, G. and Nigam, S. K. Res. Commun. Chem. Pathol. Pharmacol., 1974, 9, I 1-22. (cited from Chemical Abstracts 82:38600b). [173] Gill, R. C. (1949) Brit. 619, 235, Mar. 7, 1949. (cited from Chemical Abstracts 43: 5155g).
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22
555
9 2000 Elsevier Science B.V. All rights reserved
CHEMISTRY, STRUCTURE AND BIOLOGICAL ACTIVITY OF ANTHRACENONES OF THE KARWINSKIA GENUS P I N E Y R O - L O P E Z , A.* and W A K S M A N , N.
Departmento de Farmacologia, Y Toxicologia, Apdo. Postal 146, Col. del Valle, 66220, Garza Garcia, N.L. M~xico ABSTRACT: The genus Karwinskia is included in the order Rhamnaceae and comprises 15 different species of trees and shrubs whose habitat goes from the south part of the U.S.A., all Mexico, Central America, North of Colombia, Cuba, Haiti and the Dominican Republic. So far in Mexico 11 of these species have been reported; most of them, as toxic plants. Karwinskia humboldtiana is the most widespread species. The ingestion of its fruits in humans produces a flaccid paralysis similar to the Guillain-Barr6 syndrome and poliomyelitis. From the fruits of these plants, besides some hydroxyanthraquinones already reported for other Rhamnaceae, newly dimeric reported hydroxyanthracenones have been shown to be responsible for the aforementioned neuromotor toxic effects. Structure and chemical properties of hydroxyanthracenones were determined along with their biological activity, focusing on animal toxicity, cytotoxicity and their potential effects on celular function. One of these compounds, T 514 (peroxisomicine A l) has demonstrated a selective in vitro cytotoxicity and therefore a patent for its use as an antineoplasic agent was requested and obtained. Roots of Karwinskia sp. have been also studied on the basis of the popular belief that they act as antidote for the intoxication produced by the ingestion of the fruits. In roots, identical compounds as those obtained from the fruits were isolated, as well as other anthracenones not previously described in Karwinskia sp. Dimeric hydroxyanthracenones have been isolated from Cassia sp and the fungi Dermocibes sp and Cortinarius sp by other researchers. Although there are many papers describing different types of pigments isolated from fungi, such descriptions have been for taxonomic aims and not for investigating their biological activity. INTRODUCTION In M e x i c o , there is a total o f 11 different species o f Karwinskia (K. calderonii, K. humboldtiana, K. johnstoniL K. latifolia, K. mollis, K. parvifolia, K. rzedowskiL K. subcordata, K. tehuacana, K. umbellata, and K. venturae). The m o s t c o m m o n one is K. h u m b o l d t i a n a (Fig. 1), w h i c h can be found t h r o u g h o u t the country and even b e y o n d its borders: as far north as the states o f T e x a s , A r i z o n a , N e w M e x i c o and C a l i f o r n i a in U . S . A . a n d as far south as Central A m e r i c a [1,2,3]. The other species o f
556
Pll~IEYRO-LOPEZ and WAKSMAN
Karwinskia described below are normally restricted to geographically smaller habitats [2, 3, 4, 5, 6,].
Fig. (1). Photo of Karwinskia humboldtiana.
The epidemiology of poisoning from Karwinskia is linked to the geographical distribution ofK. humboldtiana although there is also a report on poisoning from a Karwinskia species other than humboldtiana [7]. Karwinskia humboldtiana was most certainly known as a poisonous plant to various indigenous tribes as well as to the Spanish and Mestizo settlers. It was not until the end of the 18th century, however, that the Jesuit Francisco Xavier Clavijero rendered the first description of the plant in his book Historia de la Antigua o Baja California, which was published in Venice in 1789, two years after the priest's death [8]. He describes a plant whose fruit ( the size of a chick-pea and black when ripe) paralyzes whoever eats it. Clavijero also mentions that the members of one tribe (the Pericfis) protect themselves by not eating the seed, which is where the poison is located. Subsequent clinical studies have emphasized the toxicity of these seeds or pits. This fact has been established by experiments proving that the neurotoxins are present in the endocarp and definitely not
ANTHRACENONES OF THE KARWINSKIA GENUS
557
in the pericarp and mesocarp [9, 10]. The leaves do not contain any dimeric anthracenones but rather some anthraquinones that are known for their purgative effects [ 11 ]. The roots have been considered as an antidote for poisoning from eating the fruits although their use could be dangerous because of their high content of tullidinol (T544) [2]. Since then, numerous reports on poisoning from K. humboldtiana have been documented, where the prevailing pathology has been the flaccid paralysis described by Clavijero, which bears some resemblance to poliomyelitis and the Guillain-Barr6 syndrome. This kind of poisoning occurs chiefly in children although some cases of poisoning of adults have also been reported. There have also been occasional reports of death through respiratory arrest without signs of paralysis [12]. It has been estimated that the annual figures amount to between 30 and 40 human deaths caused by K. humboldtiana in Mexico [ 13]. It is also known that during dry seasons, goats and sheep feed from the plant and die [14], causing considerable losses to farmers and ranchers [I0]. Those who have studied the plants have had different intentions: Some wanted to described an event they had witnessed or experienced themselves, such as in the case of Castillo N~ijera, an army surgeon who wrote down his experiences in 1920. During the Mexican revolution, in 1910, he lived through the poisoning of 106 soldiers lost in the Altar desert in the State of Sonora. Out of hunger and thirst, they ate from the fruit of the plant, which led to a mortality rate of over 20%; some of the soldiers died without presenting signs of paralysis [ 15] Living in the homeopathic delusion of the 19th century, others studied the roots of K. humboldtiana in their search for an antidote or a cure for tetanus and/or poliomyelitis [16]. Pathologists, neurophysiologists and others, wanted to find out what the lesion was and what caused the symptoms; from nerve biopsies, they reported a segmental loss of myelin, wallerian degeneration and disorders in neural conduction [ 16, 17, 18]. But it was the veterinarians who used a more pragmatic approach: By searching systematically for the substances contained in the plant, they isolated and characterized four dimeric anthracenones and used their molecular weights as names: T496, T514, T516, and T544 [2]. On the other hand, the medical literature contains excellent reviews of the subject, as for example the one written by Weller, Mitchell, and Daves [ 10]. We as toxicologists are not only interested in finding the effects of the different substances contained in the plant but also in the dose-response correlation. Our attempts in that direction failed, however, until we started working together with some organic chemists. From then on, we have been able to make steady progress, for we were certain that we had chemically pure and correctly Characterized substances, that we were isolating new compounds, and that we could contrast the effects with other anthracenones of different botanical origins. When we found a distinct
558
PII~IEYRO-LOPEZ and WAKSMAN
cytotoxic effect, particularly from the compound named T514, we decided to determine the median lethal dose (LDs0) in various animal species. We found a particularly abrupt slope, suggesting that if the toxic effect had a certain selectivity between cells of normal origin and cells of neoplastic origin, the substance could potentially have antineoplastic effects [ 19]. ETHNOBOTANY OF THE KARWINSKIA GENUS The genus Karwinskia (Rhamnaceae) is a taxon that consists basically of shrubs or small trees growing in the southern USA, in Mexico, Central America, Colombia, Cuba, Haiti and the Dominican Republic. Of its 15 species, 11 are found in Mexico; most of them have been reported to be poisonous [4]. The name Karwinskia was given in honor of Wilhelm Friedrich von Karwinski, a Bavarian botanist, who in 1826 collected plants in Mexico [20]. In the literature, this genus appears frequently as restricted to arid regions, but in more recent systematic studies, Fernandez found samples in deciduous tropical forests and pastures. The plants grow in altitudes ranging from sea level to about 2200 m.. The species of this genus have a reproductive and vegetative development restricted to the rainy season; in general, fructification takes place between August and October. Among all Karwinskia species, the most widely distributed and most resistant from an ecological point of view is K. humboldtiana. This species is found in almost all of Mexico and the southern USA, but the most extended populations are located in arid regions. The major concentration is in northern Mexico, where it can be found from sea level up to an altitude of 2200 m. In Mexico, K. humboldtiana has over 10 synonyms, the most common of which are capulincillo, coyotillo and tullidora. The name coyotillo goes back to the popular knowledge that coyotes eat the fruit and disseminate the seeds over considerable distances without showing signs of poisoning; the name tullidora refers to its paralyzing effect. The plant is a spineless shrub or small tree which grows to a height of from 1 to 7 meters. It may be in flower at any time of the year depending upon weather conditions. Its fruit is drupaceous, dark purple when ripe, measures between 1.0 and 1.5 cm in diameter, and has a ligneous endocarp and four seeds, two fertile and two abortive. The pulp of the fruit is sweet and edible, but the pits are toxic; when eaten they produce paralysis, particularly of the lower limbs. The seeds are used in Mexico as an anti convulsive, particularly for cases of tetanus. In popular tradition, a decoction of the roots is used as an antidote for the poisoning produced by ingestion of the seeds, which is effective as long as it is administered immediately after ingestion of the poison [10]. The bark is used as a laxative in some arid regions. In some places, an infusion made from leaves and roots is used for treating fevers and also as a remedy against neuralgia and toothache. Taxonomic characterization is not easy
ANTHRACENONES OF THE KARWINSKIA GENUS
559
due to the polymorphism of the leaves, flowers and fruits. According to Aguilar [21 ], tullidora is one of the best-known poisonous plants growing in Mexico. The rest of the Karwinskia species known in Mexico grow in deciduous tropical forests in altitudes of between 50 and 1700 m above sea level. Measured in biomass, most of the plants are found in arid regions; most of the species, however, grow in deciduous tropical forests. For K. parvifolia, known as cacachila in the Mexican states of Sonora and S inaloa, there is no use reported. In northwest Mexico its toxicity is well known; livestock dies after feeding on this plant, with death by asphyxia normally occurring a few minutes after ingestion. In the reference by Standely, K. parvifolia is considered as a synonym for K. h u m b o l d t i a n a although both species can be well differentiated morphologically as well as phytochemically. In these days K. parvifolia is the source ofperoxisomicine A1 [22]. K. mollis, known as capulin and cualzorra, grows in central Mexico in the states of Quer6taro, Guanajuato, Hidalgo and San Luis Potosi. No use is known, but its toxicity is common knowledge in the arid regions of Mexico, where domestic animals as well as children have been poisoned by ingestion of the fruits. K. venturae Fem~.ndez, known as diente de le6n, grows in the states of Mexico and Guerrero. No use is reported. K. rzedowskii FemS.ndez, known as margarita in Jalisco and Zacatecas, is used to lower fever and provoke vomiting by a decoction made from its leaves. K. mollis, K. venturae and K. rzedowskii are very similar, but Fernfindez has proposed some characteristics for distinguishing each one [5]. K. johnstonii FernS.ndez, known as cerezo, guanito, and guayabillo, grows in Michoac~in and Guerrero. In Michoac~in a decoction of leaves is used to lower fever caused by dengue. Its toxicity is popularly known although it had not been documented until recently [7]. K. latifolia Standl is known as margarita in state of Jalisco. For K. subcordata Schlecht., there are no reports of use or common names. It is found in the states of Hidalgo and Quer6taro. K. umbellata (Cav.) Schlecht., known as guayabillo, laurel de chile and 4rnica, grows in the state of Morelos, Guerrero and Puebla. Standley reports the presence of this species the state of in Michoac~in, but the only species that could be corroborated for this region is K. johnstoniL which is similar to K. umbellata. An infusion made from its leaves is used to reduce inflammations caused by contusions. K. tehuacana Fem~indez et Waksman has been found in the state of Puebla. It is related to K. humboldtiana Zucc. but can be differentiated by its leaves and fruits. Phytochemical survey also permits the differentiation of both species [6].
560
PII~IEYRO-LOPEZ and WAKSMAN
K. calderonii Standl., known as huiliste, grows in Chiapas (Mexico) and is better known in Central America (Guatemala, El Salvador, Nicaragua). It is described as a large tree, sometimes 12 m high or more. The wood is hard. and is used in Guatemala and El Salvador for railroad ties and wagon axles. Although the toxicity of its fruits is well known in Mexico, this feature does not seem to be common knowledge in Central America. Its pulp is sweet and edible and is eaten by birds and mammals [23].
H
a
C
O
~
CHa
CH3
OH OH O
H IH HaCO-
~
~
CH3
~CHa
OH
OH
OH OH
OH O
3
O
4
ANTHRACENONES OF THE KARWINSKIA GENUS
561
COUPLED HYDROXYANTHRACENONES Dimeric Hydroxyanthracenones from the Karwinskia Genus
From the air-dried grounded fruits of Karwinskia humboldtiana, Dreyer et al. isolated four major constituents in similar quantities, which were reported to be responsible for the neurotoxic properties of the plant [2]. These toxins were obtained from the semipolar toxic extract and were originally named according to their molecular weight as T544 (1), T496 (2), T516 (3) and T514 (4). They turned out to be coupled dimeric anthracenones possessing at least one subunit of 3,4-dihydroanthracenl(2H)-one. These compounds were reported as stable in solid form, but unstable in solution, yielding anhydrous and oxidized products, particularly under basic conditions [24]. Fractionation of the hexane extract (which is non-toxic) also yielded the anthraquinones 5, 6, and 7, as well as compound 8, which are probable precursors of the dimeric structures.
'CH3
CHa
O
O
5
6
CH30
CHa OH I
CH30
CH 3
0 7 I
8 III
I
The main spectroscopic characteristics of the coupled anthracenonic compounds are the following" in visible UV spectra, there is a weak maximum between 410 and 420 nm and intense absorption in the region of 220-270 nm; in IR, all isolated compounds show a strong absorption band at 3390 cm -l, due to hydroxyl groups, and a chelated carbonyl group absorption at 1600-1635 cm -~. Mass spectra show the loss of one or two molecules of H20 and a prominent ion representing the monomeric subunits. ~HNMR spectra reveal the presence of three or four chelated
562
PII~EYRO-LOPEZ and WAKSMAN
hydroxyl groups, two of which are highly deshielded (16 ppm approx.) con'.esponding to the OH hydrogen bonded to the carbonyl. The aliphatic region was not well resolved in spectra run at 100 MHz, but at 400 MHz these signals could be resolved in most cases, allowing each of the diastereotopic protons to be assigned. More recently, 13CNMR were reported, too. From the results obtained after further screening of other species of the Karwinskia genus growing in Mexico, we demonstrated that the presence of at least one of these dimeric anthracenones is characteristic for this genus (table 1) [25]. This finding agrees with the known fact that plants belonging to a restricted taxonomic group such as this one tend to biosynthesize similar classes of chemical compounds. Table 1.
% of Toxins in K a r w i n s k i a
Species Percentage
T-544
T-496
T-514
K. humboldtiana I
1.60
0.80
0.70
K. humboldtiana 2
0.70
0.58
0.36
K. mollis
1.33
1.08
0.95
K. umbellata
0.75
0.50
0.05
K. subcordata
1.51
0.98
1.07
Specie
K. latifolia
I.I0
K. parvifolia
1.58
i.04
2.00
0.70
K. calderonii
0.75
K. rzedowskff
1.60
K. venturae
0.11
K. tehuacana
0.83
0.40
K. johnstonii
2.10
0.70
0.33
i- Villa de Garcia, Nuevo Le6n, M6xico. 2- Linares, Nuevo Le6n, M6xico.
Some isomers of the toxin named T514 by Dryer were obtained from semipolar extracts of the Karwinskia parvifolia fruit. In order to avoid the confusion caused by compounds being called by their MW, T514 was renamed peroxisomicine. The name refers to the selective damage that this substance produces on peroxisomes, as will be explained later on. It is well known that these substances may exhibit hindered rotation around the
ANTHRACENONES OF THE KARWINSKIA GENUS
563
biaryl bond; atropisomers resulting from this restriction can be differentiated by characteristic Cotton effects in CD spectra [26]. A large negative Cotton effect to longer wavelengths and a large positive effect to shorter wavelengths at around 270 nm defines an A-type dimer; Cotton effects are inverted in the isomers of the B-type. According to this nomenclature, diastereoisomeric molecules described by the same letter possess the same axial chirality since it is this structural feature which dominates their CD spectra. CD curves for the T514 toxin originally isolated from Karwinskia humboldtiana, (Fig. 2), demonstrated that the substance belongs to the A-type; for being the first isomer isolated, it was named peroxisomicine A l (the lowercase number corresponds to the order of isolation). Besides peroxisomicine Al itself, an equal quantity of a stereoisomer was isolated from fruits of Karwinskia parvifolia which gave the same sign in the DC curve Fig. (2), and was named peroxisomicine A2 [27]. The stereochemistry of these compounds is complicated because of the presence of two chiral centers and one chiral axis. Peroxisomicine A~ could be crystallized from CHCI3 yielding prismatic yellow crystals that incorporate the solvent. Peroxisomicine A2 was crystallized from a mixture of toluene and mesitylene in the form of small irregularly shaped yellow crystals; ours is the first report about crystallization of dimeric
301
261 nm
20-
10" CD
428nm I
-10 I 218 nm
-20 200
278nm 300
400 Wavelength (nm)
Fig. (2). CD spectra for peroxisomicine A I ~ ,
A2 . . . . . . . and A 3 + + + + .
500
564
PllglEYRO-LOPEZ and WAKSMAN
Table 2.
Selected Torsion Angles (~ for Peroxisomicines A 1 and A 2 Corresponding to Equivalent Atoms in Both Molecules A1
,,, 9
,,,
,,,
II
.
I
.
.
.
A2 I
I
I
C(I ')-C(2')-C(3')-O(3' )
+72
+63
0(3')-C(3')-C(4')-C(4a')
-63
-69
C( 1')-C(2')-C(3')-C(Me-3 ')
-171
-177
C(1)-C(2)-C(3)-C(Me-3)
+177
+178
O(3)-C(3)-C(2)-C(!)
+65
-62
C(4a')-C(10')-C(7)-C(6)
-105
-106
C(4a')-C( I 0')-C(7)-C(8)
+69
+72
hydroxyanthracenones that appears in the literature. Through X-ray analysis of the crystals obtained, relative stereochemistry of these two compounds was established: peroxisomicine A I and A2 have the same axial configuration; chirality of C-3 and C-3' in peroxisomicine Al is the same; both compounds are epimers in C-3 [28]. The angle between the naphthalene frame rings (constrained to be planar) was 109.2 o in peroxisomicine Al and 107.4 o in peroxisomicine A2 (Fig. 3). The most
Fig. (3). X-ray structure for peroxisomicine A 1.
ANTHRACENONES OF THE KARWINSKIA GENUS
565
important angles for both compounds are given in table 2. All OH in peroxisomicine A l are involved in hydrogen bonds. In both epimers, Me groups adopt the more stable equatorial position in both chiral carbons. More recently we have isolated a third stereoisomer from the same extract. Because the CD spectra of these dimers are dominated by interactions between the naphthalene chromophores without any significant influence from substituents or chiral centers elsewhere in the molecule, the stereochemistry of the axis in this new compound had to be the same as in (9) which was deduced through comparison of the CD spectra. The new compound was named peroxisomicine A3, which is found in the plant in very small quantities [29]. Geometric optimization of the four possible peroxisomicine stereoisomers originated by the two chiral centers was performed by means of molecular modeling using semiempirical methods (AM1). Dihedral angles for local minima obtained from the R rotamers are summarized in table 3. The optimized structures found by AM1 Table 3.
Parameters for Peroxisomicine Isomers (R Rotamers) as Calculated by AM1 Mehod
* Peroxisomicine A i
**Peroxisomicine A 2
*** C4a'-CI 0'-C7-C8
calculations showed a difference of 7-9 kcal/mol for conformers with methyl groups in equatorial position and structures with the methyl groups in axial position, showing that the equatorial position is also the preferred one in the gas phase. The correlation between the NOE effect observed between H-4'ax and H-4'ec with H-6, and OH-8 and the internuclear distances calculated from optimized geometries is consistent with the proposed 3'R stereochemistry for both (9 and 10). Dihedral angles between the naphthalene subunits were calculated for peroxisomicines A1 and A2 and were found to be very close to those obtained from X-ray crystal analysis. The NOE values found for peroxisomicine A3 (11) are in accordance with those expected from the R rotamer with 3'S stereochemistry (table 3), confirming the results obtained through CD spectra analysis, although the stereochemistry of C-3 for this compound remains undefined. Some NOE correlations found for peroxisomicines are seen in Fig. (4). The rotation barrier obtained through calculations using the AM1 semiempirical method of more than 25
566
PII~IEYRO-LOPEZ and WAKSMAN
O i
7' ~ 6'
R 5'
l
lO'a T 4'a "~,
~..
"2
9
Rl
R2
Me
OH OH Me
10 Me 11
R 4 ~)
OH
Ra
R4
Me OH OH (Me}
Me
OH Me {OH}
Ix3
kcal/mol for peroxisomieines is high enough to allow the existence of two atropisomers with minimal energy for each compound, as seen in table 3. According to the AE calculated, interconversion between them should not take place at room temperature, Fig. (5). From the fruits of these plants, however, only one atropisomer has been isolated for each case up to now.
H~
H
H
H
H OH Ha H
OHfi
Fig. (4). Some NOE effects found for peroxisomicine A 1 and A 2 (left) and peroxisomicine A 3 (right).
The great number of quatemary aromatic carbons made it difficult to assign all of them, but careful examination of the ~H and ~3C spectra using HMQC and HMBC with different pulse lengths has led to an unambiguous assignment of all signals (Tables 4 and 5).
ANTHRACENONES OF THE KARWINSKIA GENUS
567
.230
Heat o#
form kcaVmol .240,
-250'
-260'
-270 -200
-100
0
100
dihedral angle
Fig. (5). Energy vs. dihedral angle for peroxisomicine A 1 obtained through AM I semiempirical calculation method.
From the semipolar extract obtained from fruits of Karwinskia parvifolia, two other compounds with the same MW (514) were isolated. UV-Vis spectra (DAD detector, HPLC) showed that in this case, the chromophores were similar although not identical to those present in peroxisomicines [29]. In NMR spectra, the isolated and purified compounds 12a and 12b showed the presence of half of the expected signals both in ~H and ~3C atoms. In this case, the simplicity of NMR spectra reflected a symmetrical structure for these compounds. The appearance of two doublets and one singlet in the aromatic region (IH spectrum) was consistent with a 7,7' linkage between the naphthalene OH
H3c..j
O H H0
~
O
H
\
12a and b
\
/ "~ CH3 HO
I
I
568
PINEYRO-LOPEZ
T a b l e 4.
I HNMR Spectral MHz, DMSO-d6)
Perox. A 1
Data
and W A K S M A N
for Peroxisomicines
Perox. A 2
I
Perox. A 3 ,
,
and
Isoperoxisomicines,
Isoperox.,, A I
(400
Isoperox.A 2 |
5 ppm
JHZ
8 ppm
3.01 (d)
17.4
3.01 (d)
I q5 ppm
[ JHZ ,,
H2
2.74 (d) H4
3.13 (d)
8 ppm
17.2
2.99 (d)
i.
16.8
iI 2.74 (d)
3.13 (d)
16.0
1 JHZ ,
,
3.01 (d)
2.75 (d) 16.0
i JHZ
.
8 ppm
,,.=,
i
17.2
15.8
i
3.00 (d)
17.3
2.73 (d)
2.73 (d)
3.14 (d)
JHZ
! 3.10 (d)
3.11 (d)
16.0
16.0
i
3.01 (d)
3.03 (d)
i
3.02 (d)
3.00 (d)
2.99 (d)
H5
7.41 (d)
8.2
i 7.42 (d)
8.3
7.40 (d)
8. I
7.30 (d)
8.3
7.30 (d)
8. I
H6
7.32 (d)
8.2
7.32 (d)
8.3
7.31 (d)
8.1
i 7.58 (d)
, 8.3
, 7.58 (d)
8.1
OH-8 ,9 9.74 (s) OH-9
9.73 (s)
15.93 (s)
15.94 (s) |
HIO
7.23 (s)
Me-3
1.34 (s)
! H2'
3.00 (d)
2.78 (d)
!
/
7.32 (s) 1.34 (s)
' 17.1
2.70 (d) H4'
,
2.63 (d)
9.92 (s)
15.95 (s)
16.04 (s)
7.23 (s)
7.15 (s)
1.34 (s)
i
1 2.71 (d)
16.5
2.93 (d)
2.78 (d)
16.3
H6'
7.7
7.15 (s) 1.32 (s)
i
|
17.0
2.74 (d)
2.65 (d)
,i 6.64 (d) 8.3 ' i 7.37 (t) 8.0
16.05 (s) i [
2.76 (d) 16.4
2.61 (d) ,
H5'
9.91 (s)
!.32 (s) |
3.01 (d) 16.5
9.96 (s)
6.62 (d) . . . 7.36 (t)
8.3 . 8.1
6.81 (d)
7.7
.,
.
i 6.66 (d) 8.4 . . . 7.37 (t) 8.0
H7'
6.81 (d)
OH-8'
9.98 (s)
9.97 (s)
OH-9'
16.13 (s)
16.13 (s)
i 16.10 (s)
Me-Y
I. 17 (s)
!. 17 (s)
1.15 (s)
]
i |
6.81 (d) 9.96 (s)
7.7
.
.
'
.
.
.
.
.
.
i
il
i
I
I
subunits, similar to the one observed in flavommanines isolated from
Macromycetes [30]. The other possibility, that of a 5,5' linkage, was
discarded because of the positions of the signals and the NOE enhancement observed between the signal of one of the aromatic doublets and the singlet. In this case, too, unambiguous assignation for all carbons was achieved by means of HMQC and HMBC experiments (tables 4 and 5). CD spectra were similar for 12a and 12b (Fig. 6) and showed the same Cotton effect as peroxisomicines. The substances were named isoperoxisomicines A x and A2. The crystallization of isoperoxisomicines has not been achieved yet, but through molecular modeling (AM1
ANTHRACENONES OF THE KARWINSKIA GENUS
T a b l e 5.
569
1 3 C N M R S p e c t r a l D a t a for P e r o x i s o m i c i n e s and I s o p e r o x i s o m i c i n e s (100.6 MHz, DMSO-d6) ,i
Perox A 1
#C ....
Perox A 3
ppm
ppm
ppm |
1
Perox A 2
,
,
,
,
205.41
'
,,
'205.34
|
lsoperox A 1
lsoperox A 2
ppm
ppm
'
""l
205.39
,
205.25
i
205.18
i
f i
2
50.99
50.98
50.96
50.96
50.91
3
69.23
69.68
69.60
69.71
69.61
4
42.36
43.27
42.33
42.33
42.30
4a
137.24
137.19
137.16
136.91
136.83
5
118.31
119.30
118.32
117.41
117.32
6
135.08
135.09
134.97
135.21
135.12
7
119.41
8
154.43
119.40
119.39
119.38
119.33
154.45
154.11
153.96
153.92
8a
112.10
111.98
I 11.99
111.90
!
111.88
9
163.76
163.78
163.81
164.08
i
164.05
9a
110.00
109.96
109.92
109.80
10
117.88
10a
138.80
J i i
28.99
J
Me-3 1' i
i
205.63
109.75
117.88
117.81
117.74
117.67
138.80
138.76
138.45
138.37
28.93
28.87
29.00
205.59
205.29
2'
50.81
50.80
50.90
3'
69.73
69.21
69.01
4'
40.57
41.52
40.65
4a'
135.08
135.01
134.83
'
.
I
.
.
,
.
.
i .
5'
116.48
116.49
116.55
6'
132.17
132.15
132.09
7'
110.47
110.45
110.36
8'
157.61
157.60
157.52
8a'
111.99
112.08
111.99
9'
163.91
163.95
163.89
9a'
109.47
109.46
109.43
10'
124.55
124.60
124.81
10a'
138.80
138.80
138.50
Me-3'
29.06
29.03
28.58
I
28.90 I
.
!
I
semiempirical method), internuclear distances in the optimized geometries were calculated. From NOE diff. enhancement (Fig. 7), it was concluded that biaryl linkage in both compounds should correspond to the S series. The absolute stereoehemistry of C3 and C3' remains undefined, but
570
PIIC/EYRO-LOPEZ and WAKSMAN
30
( - " 264 201 nm
nm
I |
I
|I
CD
-I0
218 nm
327nm
42e.~
24~ ~n
l
-20 200
,
I 300
I
Waveleng(h(nm)
Fig. (6). CD spectra for isoperoxisomicine A I ~
I 400
i 500
and A 2 ...... .
according to the symmetry of the NMR spectra for both compounds, the stereochemistry of C3 and C3' should be identical; one should be the 3R,3'R isomer and the other the 3S,3'S. Rivas et al. has also reported the isolation of compound 12a from fruits of Karwinskia humboldtiana and Karwinskia umbellata [31 ]. From semipolar extracts obtained from the fruit of Karwinskia tehuacana (previously known as Karwinskia affin humboldtiana), Rivas et al. obtained a compound (13) identified as anhydroperoxisomicinequinone-Al or T510 [33,5]. This substance may have been formed by oxidation of peroxisomicines ; however, when over 50 specimens of OH
H3~.J
(
o/i,
, HO
/ .o
/
OH
~
]-"~ CH 3 HO Fig. (7). Some NOE correlations found for isoperoxisomicines A l and A 2.
ANTHRACENONES OF THE KARWINSKIA GENUS
~
OH
571
OH
0 OH
la
[ CH 3
different Karwinskia species, especially those belonging to the species humboldtiana, were collected in different regions of Mexico at different times of the year and examined for the presence of compound 13, not even traces of that compound could be detected although it is closely related to peroxisomicine. This fact has been suggested for use as a taxonomic marker to help distinguish the new species tehuacana from humboldtiana. Karwinskia tehuacana is the only species for which the presence of compound 13 has been established up to now.
R"
v
v
v
CH3 OH 0
X"
y
v
14 15
y
~, CH 3
OH
X- H X= OMe
0
CH3 1 6 R=OMe 17 R=H I
I
572
Pll~IEYRO-LOPEZ and WAKSMAN
Table 6.
Ratio of Compound 1 to Compound 18 in Karwinskia Species Species
,
,|,
,
1:18
,
,,
K. humboldr
,,,
33:67
K. umbellata
17:83
K. subcordata
I:1
K. mollis
3:22
K. johnstonii
3:7
From the air-dried ground roots of Karwinskia humboldtiana, Dominguez et al. reported the isolation of T544 (tullidinol) and the flavones baicalein and quercetin [11]; three further constituents were isolated from semipolar extracts by application of fractionation techniques in conjunction with bioassays [32]" karwinaphtol A (14), karwinaphtol B (15) and 2-acetyl-6,8-dimethoxy-3-methyl-l-naphtol (8). More recently, Yussim et al. [33] conducted a phytochemical screening of Karwinskia subcordata, K. humboldtiana, K. mollis, K. umbellata, and K. johnstonii roots, isolating anthraquinones 16 and 17, as well as the new compound 18, the 7'desmethoxy analogue to tullidinol. The presence of atropisomerism in 18 was indicated by the presence of doublets in the resonance signals for H-7' and H-6'. Stereochemistry of C-I' and C-3' in the dihydrodimethylpyran ring was determined by a NOE diff test that rendered a correlation between H-I' and H-3' (6.4% enhancement), proving that both protons are pseudoaxial. The analysis by IHNMR in the methoxyl group region of the crude mixtures obtained by precipitation of
CH3 OH OH 0
18
2H 3
573
ANTHRACENONES OF THE KARWINSKIA GENUS
the methylene chloride extracts with cold hexane led to the determination of the relative proportion of I and 18 in these five species (table 6). The authors suggest that these data could be of chemotaxonomic relevance for the classification of the genus. Table 7.
I H N M R Spectral data for Tullidinol B 1 , B 2 and Desmethoxytullidinol (400 M H z , d values in CDCI3)
i
iii
=
1
Tullidinol B 1
~
Tumdinol B2
Desmethoxytullidinol ,
H
d ppm
JHz
d ppm
i
2
2.87 (d) 2.93 (d)
! 4
8.2
L
. 7.30 . .(d).
.
. 8.2 . .
.
.
8.1
7.42 (d)
8.1
8
9.84 (s)
9.84 (s)
9.83 (s)
9
16.06 (s)
16.05 (s)
l0
7.13 (s)
16.05 (s) t 1 7.14 (s)
1.52(s)
1.50(s)
Me-3
7.42 (d)
8.2
r
I 5.27 (c)
6.1
3.71 (m)
4'(ec)
2.29 (dd)
18 and 2
2.37 (dd)
18 and I0
,
8.2
7.13 (s)
'
1.51(s)
5.26 (c)
3'
6'
6.0
5.30 (c)
3.71 (m) 2.30 (dd) ,,,
6.27 (d)
19 and 2
2.38 (dd)
.
1.5
2.39 (m)
19 and I0
9
6.23 (d)
!.4
6.98 (d)
6.43 (d)
10' Me-I .
1.5
6.43 (d)
9.63 (s)
. . . . . .
.
8.4
7.13 (m)
8'
.
6.2
3.72 (m)
7'
,
!
. . . .
i
1.69 (d)
.
6.2
1.21 (d) .
OMe-9'
.
.
...........
i
6.1
1.71(d)
,
.
.
.
.
.
i J
i
1.22 (d)
6.0
,
6.2
1.23 (d)
3.58 (s) .
5.9
i .
.
4.05 (s) |
8.0
9.85 (s)
1.70 (d)
6.0
3.58 (s)
.
6.74 (d)
9.65 (s) l
OMe-7'
1.4
,
.
Me-3'
|
i
. I. 7.30 . . (d) .
7.42 (d)
4'(ax)
r
I
18.4 18.0
3.16(AB)
6
I'
i
! ,,,'; . . . .
2.88 (d) 2.93 (d)
1
/
l
JHz
'
18.1 18.1
2.89 (d) 2.91 (d) 3.17(AB)
~[ 7.30 (d)
5
|
d ppm
i
17.6 17.7
3.16(AB)
"
JHz
.
4.04 ii
.
~.
(s)
.
.
.
4.08 ,
ii
i
I
j
.
(s)
11
|
The air-dried ground roots of Karwinskia parvifolia were successively extracted with petroleum (bp 60-80), EtOAc and MeOH at room temperature through bioassay guide fractionation (34). Only the two first extracts showed biological activity (brine shrimp test) and were further
574
PI~EYRO-LOPEZ and WAKSMAN
13CNMR Spectral Data for Tullidinol B 1, B 2 and Desmethoxytullidinol
T a b l e 8.
(100.6 MHz, 8 values in CDCI3) ,, I
,
Tullidinol B2 5 (ppm)
Tuilidinol B 1 8 (ppm)
C
|
,
,,i
1
202.97
202.90
2
51.28
51.28
[ ,
Desmethoxytullidinol 5 (ppm)
L
,
F
'
.
,
., |
202.95 51.37
3
71.10
71.19
71.09
4
43.30
43.25
43.36
4a
134.50
134.49
134.52
5
118.51
118.50
! 18.41
6
136.58
136.61
136.42
7
121.56
121.60
121.44
8
154.86
154.88
! 54.97
l
|
112.95
I,
165.81
165.87
165.84
i
9a
109.38
109.39
109.54
I0
118.65
118.65
118.65
10a
139.17
139.21
29.20
29.26
.
8a
.
.
9
Me-3
;
i
9
J
9
.
.
.
112.92 .
.
.
.
.
.
.
112.93 .
.
.
.
.
139.21 ~
29.05
I'
71.20
71.22
71.29
3'
69.39
69.39
69.45
4'
36.71
4a'
135.19
i
36.78
46.70
135.11
134.69 124.00
5'
122.99
I
123.04
5a'
134.30
i
134.32
6'
97.68
7'
157.13
,
..
,
97.56
|
157.20 ..
,
..
133.67 .
,
l 19.73
i
125.07
8'
96.93
97. i I
103.44
9'
157.46
157.44
156.40
9a'
109.52
109.51
113.56
I O'
150.25
150.26
150.15
lOa'
119.71
119.69
121.71
Me-l'
21.84
21.87
21.75
Me-3'
21.72
21.74
21.73
MeO-7'
55.10
MeO-9'
56.28
i
55.16 56.28
|
.
.
.
.
.
,
-56.20
i ,,,,,,,
ANTHRACENONES OF THE KARWINSKIA GENUS
575
fractionated (LD50 310.73 and 272.36 ppm, respectively). From the petroleum extract and after repeated column and preparative TLC chromatography, two active anthraquinones were isolated (16 and 17); both had been reported previously for the roots of other plants of this genus. Biological tests afforded LDs0 of 49.99 and 4.44 ppm for 16 and 17, respectively (brine shrimp test). From the EtOAc extract, the main component was a compound with Rf and spectroscopic properties identical to those previously reported for tullidinol (1). Further HPLC analysis of this compound demonstrated the presence of two components with close Rt and identical spectra (DAD detector). By means of preparative HPLC, it was possible to isolate small quantities of each component of the mixture. Both compounds had the molecular formula C32H3208 as determined by mass spectrometry. Careful examination of the 1H and 13C spectra (including HMQC and HMBC) has allowed the unambiguous assignment of all signals (tables 7 and 8); both compounds are isomers with the same planar structure as the one previously proposed for T 544 or tullidinol. Tullidinol is optically active due to the combined effects of three centers and one axis of chirality; the axis of chirality must be the same for both compounds isolated by comparison of their CD spectra. CD curves for both isomers (Fig. 8) exhibit a strong positive Cotton effect to longer ~, and a large negative Cotton effect to shorter ~,, which indicates that the two long axes are twisted in the same sense in both compounds. CD curves are opposite to those obtained for peroxisomicine An (Fig. 2). From these experimental data, and assuming that the peroxisomicines are the R rotamers, it was concluded that the biaryl linkage in the two isomers from tullidinol belonged to the S series. Stereochemistry of the three chiral centers is not totally resolved up to 30 4§ +4 4+ 4 4 Ib 4 4
20
10 44 I ,+'l'
CD
o'
t
~
'
'
'
'
'
'
'
'
'
'
'
'
'
i, § 4.
.,o t " ! 20 2O0
§
9
9 300
Fig. (8). CD spectra for tullidinol B I ~
I
I 400
Wlvelength(nm)
and B 2 ++++.
I
I 500
CO0
576
PII~EYRO-LOPEZ and WAKSMAN
T a b l e 9.
Local M i m i m a for I s o m e r s of Tullidinol ( A M I m e t h o d )
Isomer
Comp. no. i
Heat of formation ll|
-260.46
i**
1'R3'S
Torsional angle C4a'-C5'-C7-C8
L
+94 ......
ii*
-254.70
+76
I'R3'S
iii*
-260.62
-80
1'R3'S
iv**
-254.40
-108
1 'S3'R
v*
-260.65
I'R3'S .
.
.
.
.
.
.
.
.
.
.
+83 ,
vi*
1 'S3'R
,
-260.48
-70
,,
*Conformation of A-ring as in fig 9a **Conformation of A-ring as in fig 9b
now; however, considering the stereochemistry of C-I' and C-3' in the A ring, NOE difference spectra demonstrated an interaction between H-I' and H-3' in both isomers (6% enhancement). I'R3'S as well as I'S3'R isomers have these hydrogens in pseudoaxial position and at a distance (2.5-2.7 A) that allows this effect to be observed. The structures for both isomers were optimized by means of semi-empirical molecular models (AM1 method); six local minima were obtained; the characteristic of each one is summarized in table 9. As can be observed, the A ring can adopt two low-energy conformations (Figs. 9a and 9b); the J found for the coupling between H-4' and H-3' (11 Hz) agree with those conformations in which H-4' is pseudoaxial to the plane formed by 02'- C I'-C 10a'-C4a'-C4' atoms (Fig. 9a), giving the presumed structures i, iii, v and vi (Table 9) for both compounds. Internuclear distances for each of these optimized 3'
3'
4'ax
4'ec
(a)
tb)
Fig. (9). Conformations of A-ring in tullidinol.
geometries were determined and quantitative NOE's were performed in order to correlate the results. The values obtained for both isomers were not significantly different; the effects observed were consistent with the internuclear distances calculated for the l'R3'Snegative rotamers (structure iii in Table 9) which displayed a counterclockwise twist between the naphthalene chromophores, which in tum resulted in an S configuration.
ANTHRACENONES OF THE KARWINSKIA GENUS
577
Although all other NOE's observed are in accordance with the calculated internuclear distances for the I'R3'S isomer, more experimental surveys must be done in order to assure stereochemistry on A ring. From these results we think that both isomers are epimers, the differences lying in the chirality of C-3, and that both belong to the B-type according to the classification proposed by Steglich, which is why they were named tullidinol BI and B2 (19). The lowercase numbers correspond to the order of Rt in the chromatographic system used, which is identical to the order of isolation. Compound 18, reported in roots of other Karwinskia species, was not found in Karwinskia parvifolia. However, after oxidation of the mixture of tullidinols to the corresponding anthraquinones, a small quantity of the anthraquinone 17 (less than 1%) was detected, showing that 18 has to be present in the original mixture. ......
H3CO
CH3H
7'
""stJ
6'
19 II
'-
H 0 / 3~CH3
II
It is important to point out that tullidinol B~ and B2 are not present in the fruits of Karwinskia parvifolia, and the anthracenones which are most abundant in the fruit (peroxisomicines Ai, A2, and T496) are absent in the roots. Establishment of the metabolic pathway in this species might be of interest because it appears to be different from the other species of this genus, where tullidinol is present in great quantities both in aerial and in subterranean parts (see tables 1 and 6). Tullidinol Bl and B2 were found to be biologically active with a LDs0 of 12.3 and 9.88 ppm, respectively (A. salina test). Using HPLC to examine several samples of tullidinol isolated previously from fruits of Karwinskia humboldtiana, it was possible to verify that all of them consisted of four compounds, as seen in Fig. (10): two of them correspond to the tullidinols Bl and B2 isolated from roots of Karwinskia parvifolia; the other two (Rt 4.37 and 4.86) are not pure
578
and
PilqEYRO-LOPEZ
WAKSMAN
enough at present to make structural assignments although we could conclude from preliminary data that both compounds have the same planar structure as tullidinol. Karwinskia johnstonii was recently reported to be highly toxic [7], which could be confirmed through experiments with laboratory animals. A bioassay guide fractionation of the fruits of this plant led to the isolation of tullidinol as responsible for the toxicity. In this case, HPLC analysis revealed that the tullidinol isolated from Karwinskia johnstonii consisted of the same four compounds found in humboldtiana, although in slightly different proportions. Tullidinol was reported to be the agent responsible for the neurotoxic effects of Karwinskia humboldtiana fruits, so it will be necessary to complete the isolation and purification of all isomers of tullidinol that have been found and repeat toxicological test with each of them, since all toxicological assays reported up to now were made with mixed substances.
r
I
,, o
.
w
I'-
9 ,4
C
c,i
,4
o
.o
"w.
d
t'o
Fig. (10). HPLC of tullidinol mixture obtained from a)K. humboldtiana (fruits), b)K. johnstonii (fruits), c) K. tehuacana ( fruits), d) K. humboldtiana (roots), e) K. parvifolia (roots), 0 K. tehuacana (roots).
As there are several reports about isolation of monomeric hydroxyanthracenones in glycosidic form from plants, we thought that dimeric anthracenones could also appear in the same form in methanolic
ANTHRACENONES OF THE KARWINSKIA GENUS
579
extracts. Up to now, however, we have not been able to sustain this hypothesis; for only ubiquitous flavonoid glycosides such as rutin have been isolated from methanolic extracts of the Karwinskia species.
Dimeric Hydroxyanthracenones from Macromycetes Dimeric hydroxyanthracenones similar to those obtained from the
Karwinskia genus were isolated from various toadstools of Macromycetes belonging to the genera Cortinarius, Dermocybe and Tricholoma [35]. The isolation, characterization and chemistry of these substances isolated from
Macromycetes have been reviewed previously by Steglich [36] and Gill [37]. The main difference with the compounds isolated from the
Karwinskia species is that in Macromycetes positions 6 and/or 6' are oxygenated. In general these compounds are used for taxonomy and are considered precursors of the anthraquinones; this is the reason that some authors call this type of compound dimeric preanthraquinones; no other biological properties of compounds isolated from Macromycetes were revised. The compounds were classified according to the position of the biaryl coupling [37]. It is noteworthy that, in many cases, both atropisomeric forms were isolated from the same species, but there is no report of the isolation of diastereoisomeric forms from the same species, as opposed to the substances obtained from the plants of the genus Karwinskia. This shows that whereas it has been suggested that in Macromycetes these dimers are formed by initial phenolic coupling of two dihydroanthracene units which have specific chirality and nonstereospecific bonding, in the case of plants of the genus Karwinskia, the monomeric units, if present, should be in racemic mixtures and the coupling could be stereospecific. The corresponding monomer unit has not yet been found in these plants, but the isolation of R-prechrysophanol (20) from the subterranean stem of A. gramnicola [38] was recently reported.
CH 3 OH 20
580
PII~EYRO-LOPEZ and WAKSMAN
Dimeric Hydroxyanthracenones from Plants Dimeric hydroxyanthracenones of this type are rare in plants. Monomeric hydroxyanthracenones have been isolated from a wide variety of plants, for example from the genera Aloe [39], Cassia [40], Vismia [41], Psorosporum [42], and Gasteria [43]. The only reports published to date on isolation of dimers of the latter type from plants, however, involve certain species of Cassia and Senna. These genera are known to possess important medicinal uses; they are a rich source of anthraquinones and flavonoids which account for most of their therapeutical properties that have been reported [44]. In some cases, however, there is no clear correlation between the biological activity and the isolated compounds; an example of this is Cassia occidentalis, a plant toxic to cattle [45]. From seedlings of Cassia torosa, a pair of atropisomeric dimers were isolated [46] 9phlegmacine A2 and B2 (21 and 21), enantiomerics from those isolated previously from the fungus Cortinarius odorifer [47], as well as a pair of anhydrophlegmacine-9,10-quinones A2 and B2 (22 and 22). From unripe seeds of Cassia torosa [48], anhydrophlegmacine B2 (23) and torosanin (24) were isolated. This last compound represents a new type of dimeric anthracene derivative. The finding of anthrones instead of quinones in the unripe seeds suggested that the anthrones could be oxidized to quinones as the seeds mature. Singueanol-I (25a) and singueanol II (26) were isolated from Cassia singueana, an East African medicinal plant [49]. The absolute configuration at 3 and 3' was presumably S, which was achieved by application of the chiral excitation coupling method" Torosachrysone (27a) was converted into its benzoate (27b), and the absolute configuration at C-3 was determined. By comparing the CD curve for 27b and CD curves for singueanol I and II, the configuration at the biaryl linkage was established. From roots of Cassia occidentalis, two new dimeric anthracenones were isolated" occidentalol-I (25b) and occidentalolII (25e). The plane structure of 25b was established by comparing spectral data with those reported for singueanol-I; the CD curve exhibited strong positive first and negative second Cotton effects, indicating that the two long axes of the naphthalene nuclei are twisted in a clockwise manner, since the C10-C10' configuration is S. Occidentalol-II had the same configuration [50]. From the fresh roots of Cassia torosa, together with the known phlegmacines A2 and B2 and singueanol I, two new dimeric hydroxyanthracenones were reported: toroasol I (28) and toroasol II (29) [51], the latter of which possesses a lactone group in ring A; no stereochemical assignation has yet been published. From Senna multiglandulosa, compound 30 was obtained; as 30 can be obtained by oxidation of torosanin, the question of whether it is a true natural product or an artifact arising by oxidation of torosanin remained unclear. The isolation of hydroxyanthracenones from some toxic plants, such as C.
ANTHRACENONESOFTHEKARWINSKIAGENUS
H
H
H
CHa
CHa OH
CHa~OH
H
O
CHaO O CH3
581
~ ~ "-CHa OH OH
~
OH
0
O
OH
3
CHa
t
21
H
22
H
H
CHa
CH3 O CH3
~
OH
OH
CH30 O CH3
H
CH3 ~
O
0 OH
OH
CHa 23
OH
OH
CH3 24
occidentalis, makes it necessary to carry out further analyses on extracts that were analyzed years ago. The hydroxyanthraquinones reported could have been formed through oxidation of the corresponding hydroxyanthracenones during the different steps of extraction and purification, so it is possible that these compounds are the real cause for the known toxicological properties of some of these plants.
582
PII~IEYRO-LOPEZ and WAKSMAN
H
H
H
H
H3 OH
,,,CH3 CH OH
CH
:
3
H3~
H3C~ OCH 3
HO ~'-
CH 3
R 0 O
OH
25a: 25b: 25c:
OH
26
R l= R2= Me Rl=Me; R2=H RI =R2=H
H
OH
OH
H
Rl
H3
CH 3
CH3 OH
OH
CH30
~],,.~ ~I,~ "r ~
~ V
HO
HO
~
OCH3
O
OH
OH
0
OH
27a: RI= Me: R~= H 27b: RI=R2--H
_
~...--CH3 \ OH OCH 3
OH
28
_ I
I
I
As we pointed out previously for the dimeric hydroxyanthracenones isolated from Macromycetes, all compounds obtained until now from Cassia and Senna species are oxygenated at positions 6,6' and/or 5,5'. Peroxisomicines and isoperoxisomicines from Karwinskia plants are the sole compounds of this type that are not oxygenated in any of these positions. This fact could possibly be of taxonomic relevance.
ANTHRACENONES OF THE KARWINSKIA GENUS
o.
o.
o
v, ~
o
c
.
~
IL 51 5L
583
?"?"~
]
o"
I tl
1
YY"
o
I
I
IN VIVO TOXICITY OF DIMERIC HYDROXYANTHRACENONES Poisoning in Humans The ingestion of the fruit of the different species of Karwinskia leads to different clinical pictures depending on the amount of fruit eaten and on the type and concentration of anthracenones it contains. On one hand, these substances have different effects [52]: T496 as well as the anthraquinonic compounds found in other Rhamnaceae (Rhamnus catartica and Rhamnus purshiana) are basically purgative; peroxisomicine A l (T514) is strongly cytotoxic; and T544 has a demyelinizing effect on the motor nerves. On the other hand, the concentration of these substances in the different species of the genus is not constant; in fact, we have found distinct variations related to precipitation and to the type of terrain where the plants are growing. Additionally, at least two species, K. parvifolia and K. latifolia, do not contain any T544 [25], so that no acute paralyzing effect can be observed after poisoning from these two species. Apart from these considerations, the neurological symptomatology appears after some days of latency, which frequently lasts two weeks. The shorter this latency period, the more severe the case of poisoning. Montoya Cabrera reports that during this period, the patient suffers from vomiting, diarrhea, and muscular fasciculations [53]. The prevailing clinical picture for poisoning by K a r w i n s k i a humboldtiana is a flaccid and ascending, bilateral and symmetrical paralysis with tendinous hyporeflexia that appears first in the lower limbs, then in the upper limbs, and finally in the respiratory muscles as
584
PINEYRO-LOPEZ and WAKSMAN
well, see Fig. (11). This symptomatology is preceded by weakness in the lower limbs (paresis), which perfectly fits the clinical description given by Padron Payou and Velazquez [16]. The histopathologic lesion consists of a segmental demyelinization accompanied by wallerian degeneration [3]. Occasionally, other symptoms appear" acute hepatic insufficiency, respiratory dysfunction, and a general attack of the central nervous system that is characterized by psychomotoric slowness, emotional tranquillity and affective indifference, similar to a neuroleptic syndrome that has been
Fig. (11). Female quadriplegic patient who later recovered completely.
described for the effects of phenothiazine derivatives; all this without producing notorious peripheral neurologic symptoms except for the characteristic position of the "clawhand" (see Fig. 12) [7]. This picture corresponds to the one observed in apes that had received T514 and also to the symptomatology described for goats and sheep, which normally is fatal [ 10]. Some authors, such as Puertolas [54], emphasize the possibility of a confusion that would lead to the erroneous diagnosis of poliomyelitis, Guillain-Barr6 syndrome or other types of polyradiculitis, because
ANTHRACENONES OF THE KARWINSKIA GENUS
585
especially in children it is not always possible to identify the causal agent. This is why it is important to develop analytic techniques that help to achieve diagnostic differentiation.
Fig. (12). Patient (brother of the former patient) who did not show any paralysis and died from respiratory damage. Observe the hand in "claw" position.
Although it is thought that most cases occur during the first three months of the year because that supposedly is the period when the fruit is available, accidents can happen at any time of the year, it all depends on climatic variables such as temperature and precipitation. There have been reports on epidemic cases in 1983 by Carrada et al. [54] and in 1984 by Puertolas [55], who revised 108 reports published in Mexico since 1918 on poisoning from ingestion of K. humboldtiana. More recently, Cervantes et al. [7] published a report on 12 cases of flaccid paralysis that were treated in the children's hospital of the city of Morelia, located in the Mexican state of MichoacS.n; in all cases, the cause was identified as ingestion of Karwinskia johnstonii. This is the first document that names this type of Karwinskia as a causal agent although the author considers it to be an ancestral health issue for that region. The botanical characterization of the causal agent was performed at the School of Biology belonging to the National Polytechnic Institute in Mexico City. Chromatographic analysis of most patients' blood showed the presence of tullidinol (T544). Bermudez et al. [ 12] have described the poisoning of 10
586
PIIC/EYRO-LOPEZ and WAKSMAN
members of a family of 13 who ate the ripe fruit of K. humboldtiana. Three of them, the father and two daughters, died; in these cases, peroxisomicine A1 (T514) was identified through TLC [12, 56] in their blood samples. It is important to note that Bermudez reports that death occurred without signs of quadriplegia or bulbar paralysis, which are normally identified as causes of death. This observation concurs with data obtained from cattle and laboratory animals [10, 57]. Additionally, Bermudez et al.[ 13] have analyzed 150 cases of acute flaccid paralysis that occurred between 1991 and 1993 in 18 Mexican states. The study referred to patients who had ingested the fruit and whose blood was found to contain toxins. The greatest number of cases appeared in the Northeastem states of Mexico (Nuevo Le6n, Tamaulipas and San Luis Potosi). For 36.4 % of the cases studied, the antecedent of ingestion was confirmed, and toxins where identified in 89.3 % of the cases, which was possible even seven weeks after the ingestion had taken place. For those patients for whom the antecedent of eating the fruit had not be established, toxin detection was negative in 96.9 % of the cases. This report situates most cases between the months of March and June; some isolated cases appeared during the rest of the year because, as mentioned before, the plants may bear fruits at other times of the year. Latency between ingestion of the fruit and the appearance of symptoms varies from one to six weeks. The sensitivity and the specificity of the method used were 89% and 96.9%, respectively, with a X2test probability of <0.001. The paralysis observed in those patients who exhibit it and do not die tends to remit slowly. In these cases, the disappearance of the neurological symptoms can be observed over a six-month period; early rehabilitation efforts favor this evolution and reduce secondary muscular atrophy [ 12]; without any physiopathologic support, thiamine has been used and has failed to give any positive results [10].
Poisoning in Livestock For lack of appropriate food during the dry season, goats and sheep eat leaves and fruits of the plants belonging to the Karwinskia genus. Some days later, they also present paresis, paralysis of the rear legs, and ataxia that impedes locomotion; they probably die due to the combination of toxic effects and inanition, as they are not able to move on to new pastures. Anatomopathologie studies have demonstrated lesions similar to those of peripheral neuropathy found in humans, where Schwann's cells show damage, segmental demyelinization and wallerian degeneration. Additionally, there have been reports of necropsies that found signs of damage in the liver, lungs and kidneys as well as a degeneration of the skeletal and cardiac muscles [58, 59, 60, 61, 62]. In goats, there was a strong increase of glutamie-oxalacetie transaminase (SGOT) and a lesser increase of glutamic-pyruvie transaminase (SGPT) [63].
ANTHRACENONES OF THE KARWINSKIA GENUS
587
Toxicity in Laboratory Animals Oral feeding of the dried and ground fruit has been the most commonly used method for experimental poisoning. Administered in this way, both the green and the ripe fruit cause neurological symptoms and lesions compatible with those described for humans, such as pronounced weakness, hyporeflexia, loss of spontaneous activity, loss of muscular T o x i n s F o u n d in Karwinskia humboldtiana
T a b l e 10.
Part of the plant
Collection site ,
g
Fruit
,,
Leaves
i,
General Ter,'tn*
November 1981
(Green) (Ripe) (Green) (Ripe)
1.06 0.46 I.I0 0.40
0.68 0.28 0.62 0.20
Garza Garcia
December 1984
(Green) (Ripe)
0.91 0.70
1.08 0.36
0.68 0.29
Linares*
October 1984
(Green) (Ripe)
0.70 0.50
0.58 0.08
0.36 0.20
Marin*
December 1985
(Green) (Ripe)
0.69 0.57
0.53 0.24
0.32 0.21
General Terfin*
November 1981
(Ripe)
0.01
Garza Garcia
December 1982
(Ripe)
0.02
General Ter,'tn*
November 1981
Garza Garcfa*
i
-
0.15
October 1984
0.15
Tamaulipas
August 1994 .
.
0.06
December 1982
Garza Garcia* Fruit
i
Percent Percent T-496 l T-514
,'
December 1981
Pulp
Percent T-544
Date of Collection
.
.
.
.
.
.
.
.
(Green)
0.20
0.30
0.40
1.10
0.35 1.20
.
Tamaulipas
October 1994
Garza Garcia*
October 1994
0.90
Quer6taro
August 1994
0.30
Hidalgo
September 1994
0.78
0.80
0.38
Hidalgo
October 1994
0.70
1.00
0.28
Baja California
October 1994
2.00
1.38
0.38
Yucatfin
November 1994
0.70
1.06
0.67
All collection sites are in Mdxico * Nuevo Le6n state
0.70 0.33
,|
588
Pll~IEYRO-LOPEZ and WAKSMAN
tone, bristling of the hair, dyspnea, paralysis of the rear legs, and death. The lungs of several species (mouse, rat, guinea pig, hamster) show interstitial congestion, edema, and massive hemorrhage; the liver occasionally shows centrilobular necrosis and sometimes massive necrosis; the kidneys present turbid swelling of the proximal convoluted tubuli; the brain exhibits necrosis of astrocytes. The toxic effects are more evident for the green fruit than for the ripe fruit, which can be explained by differences in toxin concentration (table 10). Surviving animals showed weight loss, reduction of muscular mass, palpebral ptosis, and conjunctival secretion. Independently of whether they survived or not, all animals presented a pronounced loss of weight without anorexia. Curiously, oral administration to the dog did not lead to any symptomatology, and its weight curve remained equal to that of the control animals, which makes us think about one of the common names for the plant, which is coyotillo" It suggests that the coyote (Canis lactrans) can eat the fruit without being poisoned [64]. In rats, intraperitoneal administration of T496 [52] produces intense diarrhea; that of peroxisomicine A1 (T514) is characterized by vague symptoms such as anorexia, weight loss, bristling of the hair, and death. Histopathologic lesions in this case consisted of the same pulmonary lesions described above for the entire fruit, but in the liver lesions included marked congestion, hemorrhage and massive necrosis. The pattern of lesions obtained with peroxisomicine A2 showed less damage in both liver and lungs as compared to the most extensive lesions observed with peroxisomicine A1; LD50 in mice also resulted three times lower with peroxisomicine A2 than with peroxisomicine A1, both administered orally as i.p. [65]. The administration of T544 was characterized by the classical neurologic symptoms of paralysis. For T516, however, no experiments on acute poisoning have been carried out due the fact that this substance is very rare in our extracts and does not appear in all species of the genus. It is paradoxical that when using this means of administration, dogs are just as sensitive as other species, which perhaps explains the contradictory reports given by Weller et al. [ 10] on toxicity in dogs. Mufioz-Martfnez and Chavez [66] as well as Aoki [67, 68] used a gastric probe for administering extracts from K. humboldtiana dissolved in peanut oil to adult cats. They were able to confirm segmental demyelinization and found some evidence of functional denervation of the rear legs due to the difficulty of propagating the electrical impulse through the whole nerve. They observed different effects for cutaneous and motor nerves and used electrophoresis for establishing the increased presence of a 68 KDalton polypeptide among the myelinic proteins, which is believed to be involved in normal neurofilaments. The same authors performed intraneuronal injections of tullidinol (T544) in the sciatic nerve and observed local demyelinization without signs of degeneration of the axons
ANTHRACENONES OF THE KARWINSKIA GENUS
589
[69], concluding that the toxin has a primary effect on the metabolism of Schwann's cells. Additional toxicological information on toxins from K. humboldtiana has been obtained through the research done by Bermudez et a1.[52], who reported that those animals that died within the first 48 hours because of the dose administered did not present any neurologic symptoms, which concurs with Weller [ 10], who observed that under those conditions, death occurs due to hepato-pulmonary insufficiency. For the first time in the medical literature, the histologic findings show direct toxic damage in the liver and the lungs. In other experiments that remain unpublished, we have found fatty infiltrations of rats' livers within 30 minutes after the animals have received an intraperitoneal administration of high doses of peroxisomicine A1 (T514). These observations could explain the human deaths originally attributed to a bronchopneumonic complication, while in truth it might have been a primary and direct effect on the organs involved. Similar to the efforts undertaken for K. humboldtiana, a systematic survey has been conducted in order to correlate the toxicity of other Karwinskia species to their toxin content [25]. The findings are shown in table 11. Both clinical and anatomopathologic results are similar to those reported for K. humboldtiana. Necropsies showed pulmonary congestion and hepatic necrosis, especially for K. umbellata and K. parvifolia. Table
11.
Acute Toxicity of Different K a r w i n s k i a Species on CD 1 Mice I
I
i
Dose 2.5 gr / Kg weight Species
I" . . . .
II
% Lethality Ill
i
,
Death-Days llu
,
I I
K. humboldtiana
100.0
2
K. humboldtiana
85.7
2 -6
K. mollis
71.4
2- 5
K. umbellata
85.7
2-6
K. subcordata
14.3
2 -4
K. latifolia
0.0
30
K. parvifolia
100.0
2-6
K. calderonii
21.4
4-11
28.5
8-14
K. rzedowskii . .
K. venturae
0.0
K. tehuacana
28.5
8-21
K. johnstonii
100.0
1-3
!- Villa de Garcia,Nuevo Le6n, M6xico. Lhnares. Nuevo Le6n, M~xlco.
2-
30
I
590
PIr~EYRO-LOPEZ and WAKSMAN
CYTOTOXICITY Selective Toxicity 72 hours after in-vitro administration, peroxisomicine A1 (T514) shows a toxicity that is selective between cells of benign origin and cells of neoplastic origin (hepatomas, different types of lung and of colon carcinomas), resulting in a particularly high "therapeutic index" (table 12). It can be observed that cells of benign origin are several times more resistant than those of neoplastic origin [70]. Fiebig found a similar invitro effect for 66% of 30 different cell lines [70]. Peroxisomicine AI has been patented for treatment of liver, lung and colon carcinomas [71 ]. Table 12.
C T 5 0 (~tg/ml) Obtained After 72 h i
Vinca ,,,,
i
i
,,ll
5-Fu
i
Doxo
Epidoxo
Mito
PA l
,,,,
PLC/PRF/5
0.023
70.7
0.05
0.10"*
1.0
5.0**
Hep 3B
8.000*
50.0
0.40*
0.56*
0.71
7.1 **
Hep G2
0.710*
100.0
0.40*
0.80*
0.71
7.1 **
Chang
0.016
70.7
0.05
0.14
1.00
0.360
25.0
0.10
0.10"*
!.00
20.0**
0.360
25.0
0.20
0.20**
0.71
20.0**
2.000*
70.7
0.40*
0.40
!.00
ChaGo K-I
113.0
I
Calu-3
i
SK-mes-I
28.3** I
NCI-H69
0.360
400.0*
Lung (normal)
0.500
70.7
0.28 0.20
i i
0.28
0.71
0.40
1.00
1.13
0.71"*
I0.0'* 160.0
I
Lovo Colon (normal) --
|
i|
0.360
25.0
0.720
565.0
0.56
1.13
0.56 i
i
5.0**
4.00 i
160.0 i
l
Vinca, vincristine; 5-Fu, 5-fluorouracil" Doxo, doxorubicin; Epidoxo, epidoxorubicin; Mito, mitomycin; PA l, peroxisomicine A 1 **Malignant cells were significantly more sensitive than benign cells. P < 0.025. *Benign cells were significantly more sensitive than malignant cells. P < 0.05.
Peroxisomicine A2, although cytotoxic, was found not to be selective. These results were corroborated by the National Cancer Institute's invitro anticancer screening system" Peroxisomicine A~ was selected for invivo studies, whereas peroxisomicine A2 was rejected due to its lack of selectivity.
ANTHRACENONES OF THE KARWINSKIA GENUS
591
On the other hand, the in-vitro hepatotoxicity of peroxisomicine Al and A2 showed similar results, as assessed by LDH leakage and MTT test in a primary culture system of rat hepatocytes [72]. In the same system, T544 (tullidinol) turned out to be less hepatotoxic than peroxisomicines A l and A2 [73]. There is only a small number of reports in the literature about the cytotoxicity of dimeric hydroxyanthracenones: Toroasol I (28) and II (29) inhibit an in-vitro KB cell line with an EDs0 of 1.7 and 5.5 lag/ml respectively; occidentalol I (25a), occidentalol II (25b) and phlegmacines (21) have been patented as cytotoxic agents by Taisho pharmaceutical Co.[74]; however, phlegmacines At and Bl did not show selective toxicity.
Genotoxicity Different types of tests for genotoxicity of peroxisomicine A~ (T514), such as inhibition of mitotic index, lymphocyte proliferation kinetics, frequency of sister chromatid interchange, and frequency of chromosomic aberrations, have yielded negative results [75].
Fetal Toxicity T544 and peroxisomicine Al (T514) were administered orally to female mice on the eighth day of pregnancy with a dose equivalent to half of LD50. T544 produced underweight embryos with encephalitic dilatation, exencephalia, abdominal eviscerations, deformities of the rear legs, inflammation and hemorrhage of the jaw, and alterations of the tail [76]. Animals treated with peroxisomicine Al (T514) did not exhibit any kind of malformation [77]. ANALYTICAL PROCEDURES On account of the biological activity of some of the dimeric hydroxyanthracenones isolated from the genus Karwinskia, analytical procedures have been developed over the past few years for determining concentration levels in plant material as well as in body fluids. The main purposes of these efforts were: 1) to evaluate the different extracts in order to optimize peroxisomicine A~ production; 2) to help in the diagnosis of poisoning from ingestion of fruits; 3) to assure the purity of the peroxisomicine used in preclinical trials; 4) to improve the production of the compound; and 5) to support research on the mechanism of action (especially pharmacokinetics and pharmacodynamics). The first laboratory test appearing in the literature was published by Flores Otero et al. [78]. The authors considered the detection of tullidinol by spectrophotometric and chromatographic methods. Sera of rats treated
592
PII~IEYRO-LOPEZ and WAKSMAN
with tullidora and/or pure tullidinol were deproteinized by tungstic acid; then absorption spectra of the supematant were taken. At 400 nm a peak attributed to tullidinol appeared, but less interference was found at 410 nm; serum absorbance at this wavelength was high immediately after tullidora treatment and diminished over time. A linear relationship was found between absorbance at 410 nm and tullidinol concentration. The findings suggested that tullidinol could be detected in the blood of tullidora-treated rats, with a potential use for the diagnosis of tullidora poisoning. Guerrero and Waksman [56] suggested the use of TLC and in situ densitometry in order to quantify all the hydroxyanthracenones known at that moment that could be present in Karwinskia extracts; this method also took advantage of the strong absorbance of these compounds in the Vis region. By this method, T514 (peroxisomicine Al), T 544 (tullidinol) and T496 were quantified in different plant extracts obtained from K. humboldtiana (table 10). The great variations in the toxin content of the fruits collected in different regions and times of the year made this method more specific than the previously described spectrophotometric method without separation of the compounds. The detection limit reported by this method was 0.2 nmol for each toxin. This method is easy to use and not too expensive for routine work. It was also used in biological samples: In deproteinized serum taken from rats, the presence of tullidinol and/or peroxisomicine Al could be detected even one month after the poison was administered. The method has since been applied in toxicological screening of human poisoning with Karwinskia fruits; there is a good correlation between clinical survey and toxin quantity detected in the serum [ 12,13]. Although convenient for toxicological screening, the method described resulted inadequate for studying the mechanism of action of peroxisomicine A1 as well as for the analysis of the minor components in the extracts; for these purposes we have proposed the use of HPLC in C-18 and C-8 reverse phase with DAD detection [79]. The quantification limit achieved until now by this method has been 0.01 nmol using Vis region; in the UV region, this limit could be improved by a factor of five; however, the wavelength of 410 nm presented less interference due to the different matrixes used. Deproteinization of biological samples was achieved by AcCN. Solid phase extraction did not lead to good recuperation and was discarded. Quantifications were made by means of external standard methods; the use of internal standard methods did not give good results. The analytical HPLC method was also scaled up for the isolation and purification of peroxisomicine At using preparative HPLC. One of the first problems we had to solve in order to obtain larger quantities of the potential antineoplastic agent peroxisomicine Al was to evaluate the best source for its extraction. We used the analytical methodology described here (TLC and/or HPLC) to conduct a phytochemical survey of the genus Karwinskia, which allowed us to choose the best source for peroxisomicine Al: the fruit of K. parvifolia
ANTHRACENONES OF THE KARWINSKIA GENUS
593
[22]. The absence of tullidinol in the fruit (table 1), as well as lesser variations in peroxisomicine content due to time of the year (table 13), made this species the most adequate for our purpose. K. parvifolia grows at a distance of one thousand Kilometers from our laboratory (in the states of S inaloa and Sonora), and a project was started for growing it experimentally in our region. A survey to find the specimen that produces principally peroxisomicine Al is now in progress; we are using the analytical methodology described above to compare the quantity of the compound of interest produced by the cultivated plants to the one that can be extracted from wild specimens. Quantification Sinaloa
T a b l e 13.
of T o x i n s
in K.
parvifolia
Samples
from
Sonora
and
Percentages of Sample
T514
T 496 !
i
green
0.50
1.20
green
1.03
1.33
ripe
0.78
0.90
ripe
1.00
1.25
3 .
.
.
.
3
ripe
0.88
1.15
4
green
0.75
0.90 0.80
,
4
ripe
0.70
5
green
0.83
! .25 .
5
ripe
0.55
1.08
T544 (tullidinoi) was not detected
After obtaining peroxisomicine A~ in our production laboratory, quality control was another analytical problem that had to be solved before initiating clinical trials. Some of the isomers in plant extracts, especially isoperoxisomicine A~, which has a Rt very close to that of peroxisomicine Al [80], are not easily separated (table 14). In all systems examined, this causes problems with purity and, above all, the biological activity of the final product, so the results are not always reproducible. Some analytical test methods were examined in order to establish the best ones for this specific case. Until now, melting point, TLC (silica and reverse phase), HPLC (in C-18 and C-8 reverse phase) and absorbance relationships at different wavelengths have been the physical tests in use, while percentage inhibition of catalase activity and selective cytotoxicity in cell lines are
I
F
594
PIIgIEYRO-LOPEZ
Table 14.
[
and WAKSMAN
Reti~ntion Times o f Dimeric Anthracenones from
Compound
C8 Column
I
Karwinskia parvifolia C18 Column
I ....
II
Peroxisomicine A2
1.66
!.64
Peroxisomicine A 1
2.65
2.43
T-516
2.85
2.42
Isoperoxisomicine A 1
2.55
2.85
Peroxisomicine A 3
3.00
3.17
T-496
6.92
6.99
used as biological tests. Only after evaluation of the results obtained by applying all the aforementioned tests can the purity of peroxisomicine Al be certified [80]. Using the aforementioned tests, we calculated the percentages for each of the possible natural contaminants (peroxisomicine A3, isoperoxisomicine Al and T516) that could be detected (table 15). To !mprove the results achieved, however, several analytical investigations are ~n process to determine the appropriateness of using derivative spectrophotometry, polarimetry and ~HNMR. Table 15
% of Impurities that can be Detected by Means of the Tests Currently in Use for Quality Control of Peroxisomicine A 1
Compound
UV-VIS
A~.l ( nm )/A~2(nm )
HPLC
% Catalase inhibition I
1.80*
Perox A 3 lsoperoxA 1
1.25 (280/437)
1.80"
1.80
T516
0.80 (270/222)-
2.50**
2.50
* in C-I 8 reverse phase ** in C-8 reverse phase
Spectrophotometry, TLC and HPLC were also used to evaluate some physicochemical parameters of the biologically active dimeric anthracenones that we had isolated. As these compounds are weak acids with specific absorption in the Vis region, spectrophotometry proved to be useful in the calculation of pK by using bathochromic shift effects in alkaline media. Solubility tests were also recorded by means of spectrophotometric techniques. Partition coefficients and half life were run under different conditions of temperature and pH and analyzed by means
ANTHRACENONES OF THE KARWINSKIA GENUS
595
of the TLC method reported. Some of the results obtained are reproduced in table 16. As was previously suggested, these compounds are unstable under high temperature, in light, and in basic media. While running different tests, we realized how important the control of these parameters was, for the manipulation of these compounds. It became important, too, to avoid basic or extremely acidic conditions. Knowing the values for solubility and acidity was useful for planning separation strategies; half life in different media was used for planning strategies for the derivatization of the compounds. On the other hand, it is important to consider solubility parameters; many of the agents producing toxicity in tumor cells in vitro are not readily soluble in delivery systems that can be applied to animals or humans. In fact, a number of agents had to be dropped from further research because no appropriate formulation could be found. Table 16.
Some Physichochemichal Anthracenones
Parameters
of
Biological
Active
Dimeric
....
Perox A 1
Perox A 2
T544
T496
, . .
7.27+0.09
pKa
7.54+0.18
_u
'
I'
8.47+__0.16
,,
'
,
10.05+-0.29 '|
,
.
Solubility (mg/ml) 20.000
Chloroform
13.640 .
Methanol
10.000
9.520
Dimetilsulfoxide
50.000
200.000 .
10.000
n-octanol .
.
water
.
.
3.300
0.125
100.000
25.000
10.000
20.000
.
.
0.001 .
10.000
11.000 .
n-hexane
100.000 ,
.
.
0.005 .
.
.
0.010
0.024
4.57+0.9
4.85x"0.90
0.090
0.085
0.006
0.005
10.0:1:2.0
> 100
.
Kp butanol/buffer pH 2.5
9
,
pH 6.0
4.00+0.9
9.46+0.29
4.0+0.8
> 100
pH 9.0
1.56+0.3
7.66+ 1.03
2. I +0.4
-
ISOLATION AND PURIFICATION
Secondary drug evaluation efforts require variable quantities of material depending on the potency of the agent under consideration. The challenges of an inadequate supply can adversely affect the preclinical and the subsequent clinical evaluation of an agent (e.g. taxol). For this reason we
596
PII~EYRO-LOPEZ and WAKSMAN
considered it extremely important to evaluate each step of the production of peroxisomicine A 1 and the other dimeric anthracenones. Dimeric hydroxyanthracenones are obtained from semipolar extracts. The general isolation method consists of maceration of the dry plant (fruits or roots) at room temperature. We strongly recommend keeping the temperature as low as possible; if not, degradation products, especially anthraquinones and anthrones, will be produced during extraction; we could verify that even the heat produced in the grinding process can partially decompose these substances. Chloroform, ethyl acetate, dichloromethane and benzene have been reported as extractants for these types of compounds. Peroxisomicines and isoperoxisomicines proved to be very unstable in alcoholic solutions (especially methanol); for this reason, direct methanolic extraction from dry plants and further partition into petroleum, ethyl acetate and butanol led to great losses of material and was discarded. However, during the isolation of peroxisomicine A z and due to the low solubility ofT496 in MeOH (table 17), a methanolic extraction of the semipolar extract is made in order to eliminate almost all of the T496 (which is present in great amounts in K. parvifolia) before the chromatographic step. This extraction, which resulted useful in lowering the costs of the chromatography separation, must be done as quickly as possible and at a temperature not higher than 15~ The first purification of these compounds reported by Dryer [2] used cc in silica gel and mixtures of benzene acetone of ascending polarity for isolation. Although this method allowed him to obtain the desired products, due to the interaction between these compounds and the solid support, there are great losses; irreversible adsorption and decomposition of similar compounds have been widely reported [81]. Extensive tailing of the spots on TLC silica gel also indicates this problem. Some improvement could be made using acetic acid in the eluent mixtures. Most of the reports that have appeared in the literature use gravity chromatography with silica gel columns. Later we recommended the use of flash chromatography and preparative layer chromatography. This method proved to be useful in isolating the compounds on a small scale while avoiding most of the decomposition products. Preparative TLC, however, is not suitable for obtaining large quantities. Besides, some of the isomers isolated in the past years could only be purified by means of reverse-phase chromatography; in some cases the use of LPLC on a Lobar RP-18 was recommended, but other purifications could only be achieved by HPLC. The mixtures of tullidinols, for example, could be obtained more efficiently from semipolar extracts made from roots of K. parvifolia by means of flash chromatography followed by Lobar chromatography. Subsequent separation of tullidinol Bi and BE from this mixture was possible only by means of HPLC using acetonitrile water as eluent. Peroxisomicine A3 is another example of a compound that could only be purified by using HPLC for the last step. Nowadays we are using HPLC in reverse phase
ANTHRACENONES OF THE KARWINSKIA GENUS
597
(C 18) as a last step for purifying peroxisomicine AI; a general purification scheme is seen in Fig. (13). Ground fruit
I
I
1
Residue
Extract .....
MeOH
I
"
I Extract I Flash chromatography
Residue
I
I
I
Fr I T496
Fr 2 pA2 + pAl
Fr3 pal + ipAl
cc
I
l
[ selective extraction
P~
I ' Extract (pAI }
pAl
'
I
Fr 4 pAl + ipAl + lpA2 + pA3 [ selective extraction
I
Residue Extract (isopA I } (pAI + pA3)
IpAl
pA3
l
Residue (pAI + ipA2]
IpA2 and Ipal
Fig. (13). Scheme for purification of peroxisomicines and isoperoxisomicines, pA l" p e r o x i s o m i c i n e A l; pA 2" peroxisomieine A2; pA 3" peroxisomieine A3; ipA 1" isoperoxisomicine A l; ipA2: isoperoxisomicine A 2.
Preservation of these substances has also been problematic. Toxins are kept at -40oC; this low temperature slows down the degradation process but does not completely stop it. Experiments are now in progress to achieve the stability of these compounds in solid state and at low temperatures. The purification of toxins is guided by means of TLC (silica gel) and HPLC. In TLC, the compounds can be observed in the Vis and UV region without chemical treatment and also using a KOH reagent [82]. When sprayed with 5% KOH in ethanol, anthracenones change color to a light pink while their degradation products, especially anthraquinones, turn dark red in the visible spectrum and red-purple fluorescence in UV- 365 nm, which can be easily recognized. DERIVATIZATION
The attempts to prepare derivatives of anthracenonic compounds were unsuccessful for many years. O-acetyl and O-methyl derivatives led to
598
PINEYRO-LOPEZ and WAKSMAN
complex mixtures from which it was not possible to obtain pure substances in reasonable quantities. This fact is also reported in the literature by other authors, and there are very few examples of derivatives obtained from dimeric anthracenones [83]. Knowing the physicochemical behavior of these compounds, some derivatives of peroxisomicine A l could be obtained, purified and identified [84]. In all cases, we took great care to work at low temperatures, protected from light, under a N2 or Ar jet stream, and in neutral or lightly acidic conditions. We maintained these environmental conditions in order to obtain derivatives of peroxisomicine A1 while avoiding oxidation that would have produced anthraquinones or anthrones. The best method for checking the presence of anthraquinones was TLC followed by spraying with 5% KOH, as has already been mentioned. This procedure allowed us to select the most advantageous conditions for arriving at a minimum of degradation products in each reaction. Methyl ethers could be obtained by using diazomethane generated by reaction with diazald [85]. The crude mixture obtained was dissolved in ethyl acetate and precipitated with hexane. By means of cc using ethyl acetate as eluent, followed by LPLC in Lobar Lichroprep Si 60 (40-63 mm), four principal compounds were purified. Mass spectra analysis as well as 1HNMR including long-range correlations and NOE enhancements led to the following structures (31a, b, e, d). H
~
CH3
O
CI
~._
CH3 OH
ORa
OH OH
ON
0
0
31a: 31b: 31c: 31d:
Rl=R4=Me; R2=R3=H RI=Ra-Me; R2=R4=H RI=Ra=R4=Me;R2=H RI=Rz=R4=Me;Ra=H
32
The reaction of peroxisomicine Al with n-chlorosuccinimide in DMF produced a mixture, which was lyophilized to eliminate the solvent and then purified by means of LPLC in Lobar reverse phase (using a mixture of
ANTHRACENONES OF THE KARWINSKIA GENUS
599
acetonitrile, water, acetic acid as eluent), followed by preparative HPLC in a similar method. We obtained one major product which, according to spectroscopic analysis, has structure 32. The position of substitution coincided with the one predicted by means of composite isosurfaces on a geometry-optimized molecule obtained through the AM 1 molecular orbital method. In this case, the electrostatic potential and the HOMO orbitals were encoded onto the electron density isosurface, which allowed us to delineate the areas which are electron-rich and hence subject to electrophilic attack. All reactions tried out on the carbonyl group have been unsuccessful. Gill et al. [86] also reported the difficulty of reducing carbonyl in some hydroxyanthracenones isolated from Macromycetes; which is attributed to strong hydrogen bonding. We pursued the strategy of methylating the 9 and 9' positions of the peroxisomicine Al molecule with diazald. The dimethylated compound was then subjected to the reduction step with LiBH4 in THF. The product resulting from this reaction was purified by several extractions and further by means of LPLC in silica gel using a mixture of ethyl acetate and acetone in ascending polarity as eluent. The main product had the structure 33. Subsequently, this product was demethylated to achieve the desired result.
oH
]~n ~/oH
OH OH OH
OH
83 I
III
II
All derivatives obtained were cytotoxic on hepatoma cell lines, but only compound 33 conserved the selectivity found for peroxisomicine Al.
600
PllC/EYRO-LOPEZ and WAKSMAN
IN VITRO CULTIVATION When we had demonstrated that the production of secondary metabolites in plants of the genus Karwinskia was significantly influenced by exogenous conditions, cultivation of these plants in vivo as well as in vitro was initiated under controlled conditions. The results obtained concerning variations in toxin content of plants of this genus, seasonal variations in the compounds found, and the possibility of having better sources for peroxisomicine A1 were the factors that motivated these studies. K. humboldtiana showed high explant capacity in vitro for multiplication of shoots and their roots. The presence of T514 in plants cultivated in vitro was ascertained [87]. Since K. parvifolia has the highest T514 content, it seemed relevant to produce an in vitro culture of this species, too, and compare its regeneration capacity to that of K. humboldtiana. The vitality of embryos varied between the two species, and distinctions were also made in the differentiation ability. The multiplication of shoots of K. parvifolia depended on the origin of the seeds. The regeneration potential, multiplication of shoots and subsequent rooting, was 95% in humboldtiana and 58% or less in parvifolia. This could be attributed to differences in the vitality of the embryos isolated. The results obtained up to now indicate considerable differences in the in vitro regeneration ability between the two Karwinskia species as well as in the vitality of K. parvifolia embryos isolated from seeds of different origin, which were probably influenced by local environmental conditions of these plants [88]. Surveys are now underway for finding the best condition for in vitro cultivation of each plant to lead to the specific production of peroxisomicine A i. A study was made for comparing leaf anatomy, toxin content, and some physiological characteristics of K. humboldtiana plants coming from natural, greenhouse and in vitro conditions. It was found that the leaf structure had changed; leaves of in vitro cultured plants contained a less developed protective wax layer in comparison to those from natural conditions. For K. parvifolia these differences were not so obvious. The toxin content in leaves from in vitro cultured plants was comparable to that in leaves grown under natural conditions [89]. MECHANISM OF ACTION Although the complete mechanism remains unknown, the following elements play some kind of role that has not been totally explained: Action on P e r o x i s o m e s
Depending on the dose applied, peroxisomicine Al (T514) produces a patent diminution of peroxisomes, both in vivo and in vitro. In in vitro
ANTHRACENONES OF THE KARWINSKIA GENUS
601
cultures of Hansenula polymorpha and Candida boidinii at sublethal doses, a specific effect on the peroxisomal integrity of both strains tested was observed; peroxisomal lesion begins at the membrane, which breaks; then the peroxisomal content (enzymes) flows out, and the rest is included in the digestive vacuoles [90]. Damage to other cellular membranes was not detected under these conditions. In the in-vivo experiment, intraperitoneal administration of peroxisomicine Al (T514) to rats produces an evident reduction of hepatic peroxisomes that starts 30 minutes after administration. Peroxisomicine A2 and tullidinol, also reduce the viability of Candida boidinii, with damage to the peroxisomal membrane similar to that observed with peroxisomicine Am. C. boidinii cells exposed to tullidinol showed the strongest damage to peroxisomal membrane without causing toxic effects on the yeast itself. These data were confirmed by immunocytochemical experiments [91 ] There are reports in literature stating that some tumoral lines have a significantly lower number of peroxisomes and a reduced level of catalase [92]. Possibly, the effect of peroxisomicine Al (T514) on peroxisomes and on catalase could explain this selective toxicity. A c t i o n on C a t a l a s e
In vitro, peroxisomicine A! (T514) in very low concentration inhibits the activity of murine, bovine and canine catalase in a non-competitive way [93]. Catalase is a marker peroxisomal enzyme. The degree of inhibition of all three catalases assayed was very similar among them. This effect can be used for quality control; as the compound loses its selective cytotoxicity, the inhibiting capacity also diminishes Other dimeric anthracenones were also tested with bovine catalase; all of them showed an inhibitory effect of the enzyme's activity. The degree of inhibition seems to be related to the compound's anthracenone moiety (table 17). The most active compounds are those with two anthracenonic groups (9,10,11,12a); there is no significant difference between peroxisomicine A1 and A2 , which produce the highest degree of inhibition. On the other hand, those dimers with anthraquinone or anthrone moiety, such as compounds 2 and 13 do not produce a significant catalase inhibition. The commercially available anthraquinones chrysophanol and emodine do not show any significant inhibition, either. However, it has not been possible to establish this effect in vivo (CD-1 mice), whereas a decrease in the number of hepatic peroxisomes could be demonstrated [94]. In methylotrophic yeast with peroxisomal damage induced by peroxisomicine A l, A2 and tullidinol no evidence of catalase inhibition was seen, either. Considering these results, it has been suggested that catalase is not directly involved in the selective effect of these compounds on peroxisomes.
PII~IEYRO-LOPEZ and WAKSMAN
602
Table 17.
Inhibitory Effect (IC50) of Anthracenonic C o m p o u n d s on Catalase Activity I
I
I
I
I
I,
I
I
Compounds |l
,,
.I
i
,,,
i
, i
ICso ,
,,
,
Peroxisomicine A 1 ( 9 )
3.34+0.99
Peroxisomicine A 2 (10)
3.64+1.10
Phlegmaeine A 1*
6.33+_2.79
Phlegmaeine A2.
8.54+3.23
Peroxisomieine A 3 (11)
9.07:t:0.54
lsoperoxisomicine AI (12a)
17.59:1:1.00
Tuilidinoi ( 1 )
40.08+1.57
T516( 3 )
69.77-1-1.99
T496( 2 )
99.50+19.03
T 510 ( 13 )
120.00+10.44
Aminotriazol
4x 105
*These two compounds are the enantiomers of 22a and 22b, obtained from a mixture isolated from Cortinarius by Prof Steglich
The catalytic activity of other enzymes such as fumarase, cytochrome oxidase and peroxidase, was also tested in vitro in the presence of peroxisomicine Al. This toxin was not able to inhibit the activity of the three mentioned enzymes. Free Radicals Because of its chemical structure, peroxisomicine Al (T514) could be thought of as acting through free radicals. The fact that its diastereoisomer, peroxisomicine A:, lacks selective toxicity, however, suggests that the mechanism of action is a different one, and that it possibly acts through receptors. CONCLUSIONS It can therefore be concluded that pharmaceutical research should not be limited to only those plants that pertain to popular medical culture but should also include the so-called "poisonous" plants, which may offer therapeutic options, at least in the case of chemotherapy.
ANTHRACENONES OF THE KARWINSKIA GENUS
603
ACKNOWLEDGMENT The authors are grateful to Mr. Rogerio Gonz~lez-Alanis for his assistance in the preparation of this manuscript, to Dr. Med. Victoria BermfidezBarba for her assistance in providing figures # 1, 11 and 12 and to CONACYT (M6xico) for financial support through grants and scholarships. REFERENCES
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
[11] [12] [13] [14] [15] [16] [17]
[18] [19]
[20] [21] [22]
Calderon-Gonzfilez, R; Rizzi-Hemfindez, H. N. Engl. d. Med., 1967, 277, 69-71 Dreyer, D.I.; Arai, I; Bachman, C.D.; Anderson, W.R.; Smith, R.G.; Daves, G.D.J.Am. Chem. Soc., 1975, 97, 4985-4990 Escobar-Izquierdo, A.; Nieto, D. Gac. Med. Mex., 1965, 95 163-177 Femfindez-Nava, R. An. Inst. BioL UNAM. Ser Bot., 1992, 63, 1-23. Femfindez-Nava, R. Acta Bot. Mex., 1988, 2, 11-20. Femfindez-Nava, R; Waksman, N. Phytol., 1992, 73, 435-438. Arellano-Cervantes, E; Mendoza-Cruz, J.F. Dominguez-Chfivez, F.J. Bol. Med. Hosp. Infant. Mex., 1994, 51, 105-112 Clavijero, F.J. Historia de la Antigua o Baja California, Ed. Porrfia: M6xico, 1982 Bustamante-Sarabia, J; Olvera- Rabiela, J.E.; Nieto-Cafiedo, L.C. Gac. Med. Mex., 1978, 114, 241-244 Weller, R.; Mitchell, J.; Daves, G.D. In Buckthorn (Karwinskia humboldtiana) toxins; Spencer P; Schaumburg, H; Williams-and Wilkins: Baltimore, 1980; 336-340 Dominguez, X.A.; Temblador, S.; Cedillo-L, M.E.. Rev. Latinoam. Quire., 1976, 7, 46-48 Bermfidez, M.V.; Lozano-Mel4ndez, F.E.; Salazar-Leal, M.E.; Waksman, N.; Pifleyro-L6pez, A. Gac. M~d. Mex., 1995, 131, 100-106. BermOdez-De Rocha, M.V.; Lozano-Melendez, F.E.; Tfimez-Rodriguez, V.A.; Diaz-Cuello, G.; Pifleyro-L6pez, A. Salud P~blica de Mdxico, 1995, 37, 57-61. Dewan, M.L.; Henson, J.B.; Dollahite, J.W.; Bridges, C.H. Amer. J. Path., 1965, 46, 215- 226 Castillo-Nfijera F. Mem. V Congreso M4dico Mexicano, 1920, 1,240-244. Padron, F.; Velfizquez, T. Rev. M~x. Pediatr., 1956, 25, 225-237. Padron-Puyou, F.. Gac. Med. Mex., 1951, 81,299-311 Del Pozo, E. Gac. Med. Mex., 1965, 95, 179-182 Pifieyro-L6pez, A.; Gonz~ilez Alanis, R.; Martinez de Villarreal, LE. Investigaci6n Cientifica de la herbolaria Medicinal Mexicana; Secretaria de Salud; M4xico, 1993, pp. 209- 231. Standley, P.C. In Rhamnaceae in trees and shrubs of Mdxico; contr. U.S. Natural. Herb, 1923, 23, 715. Aguilar, A.; Zolla, C. Plantas t6xicas de M~xico, I.M.S.S.: M6xico, 1982 Waksman, N.; Santoyo, A.; Ramirez, R.; Femandez-Nava, R.; Pifleyro-L6pez, A. Polibot6nica, 1997, 5, 13-19.
604
[23] [24] [25] [261 [27]
[28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41]
[42] [43]
[44] [45] [46] [47]
[48] [49]
[5o1 [51] [521 [53]
PII~IEYRO-LOPEZ and WAKSMAN
Standley, P.C.; Steyermark, J.A. Flora of Guatemala, Chicago Natural History Museum" Chicago, 1949 Arai, I.; Dreyer, D.L.; Anderson, W.R.; Daves, G.D.J. Am. Chem. Soc., 1978, 97, 163- 164 Waksman, N.; Martinez. L. Rev. Latinoamer. Quire., 1989, 20, 27-29. Eliel, E.; Wilen, S., Stereochemistry of Organic Compounds, John Wiley and Sons, 1994 Waksman, N.; Ramirez-Dur6n, R. Rev. Latinoamer. Quire., 1992, 23-22, 25-27. Rodriguez de Barbarin, C., Bailey, N., Ramirez-Dur6n, R., Martinez-Villarreal, L, Pifleyro-L6pez, A and Waksman, N., Ciencia U.A.N.L., 1998, 1, 37-42. Rivas, V., Pifteyro, A. and Waksman, N., manuscript in preparation. Gill, M.; Steglich, W. Prog. Chem Org. Nat. Prod., 1987, 51, 1-317. Rivas, V.; Torres, R.; Waksman, R. Planta Medica, 1990, 56, 562-563. Mitscher, L.A.; Gollapudi, S.R.; Obum, D.S.; Drake, S. Phytochemistry, 1985, 24, 1681- 1683. Yussim, L.F.; Lara, O.R.; Benavides, A.; Hem~indez, B.; Fernandez, R.; Tamariz, J.; Zepeda, G. Phytochemistry, 1995, 40, 1429-1431 Waksman, N.; Benavides, G. and Rivas, V., Phytochemistry, (in press). Atherton, J.; Bycorfi, B.; Roberts, J.; Roffey, P.; Wilcox, M. J. Chem. Soc. (C), 1968, 2560-2564. Billen, G.; Karl V.; Scholl, T.; Stroech, K.D.; Steglich, W. In Natural Products Chemistry III; Atta-ur-Rahman and PW, Le Quesne, Ed.; Springer: Berlin, 1988; 305-315. Gill, M. Nat. Prod.Reports, 1994, 67-91 Yenesew, A.; Ogur, J.; Duddeck, H. Phytochemistry., 1993, 34, 1442-1444 Dagne, E.; Casser, I.; Steglich, W. Phytochemistry., 1992, 31, 1791-1793 Takahashi, S.; Takido, M.; Sankawa, W.; Shibata, S. Phytochemistry, 1976, 1295-1296 a)-Delle-Monache, F.; Ferrari, F.; Marini, G.; Maxfield, P.; Cerrim, S.; Fedeli, W.; Gavuzzo, E.; Vaciago, A. Gaz. Chim. Ital., 1979, 109, 301-310. b)Casinr G.; Geroni, C.; Botta, B.; Delle-Manache, G.; Delle-Monache, F., J. Nat. Prod., 1986, 49, 129-131. Marston, A.; Chapuis, J.; Sordat, B.; Msonthi, J.; Hostetman, K. Planta Medica, 1986, 207- 210. Dagnr E.; Van-Wyck, B.; Mueller, M.; Hagg, S.W. Phytochemistry,, 1996, 41, 795-799. Anton, R.; Berrurier, M. Pharmacology, 1980, 20: ( Suppl. 1), 104-112 Lewis, D.; Shibamoto, T. Toxicon., 1989, 5, 519-529 Takahashi, S.; Kitanaka, S.; Takido, M; Sankawa, U.; Shibata, S., Phytochemistry., 1977, 16, 999-1002 Steglich, W.; TSpfer-Petersen, E. Z. Nat. Forsch., 1972, 2 7b, 1286-1287 Kitanaka, S.; Takido, M. Phytochemistry, 1982, 2103-2106 Endo, M; Naoki, H., Tetrahedron, 1980, 36, 2449-2452 Kitanaka, S; Takido, M., Chem. Pharm. Bull., 1989, 37, 511-512 Kitanaka, S; Takido, M., Chem. Pharm. Bull., 1990, 38, 1292-1294 BermOdez, M.V.; Gonzfilez-Spencer, D.; Guerrero, M.; Waksman, N.; Pifleyro, A. Toxicon, 1986, 24, 1091- 1097. Montoya-Cabrera, M.A.; L6pez-Martin, G.; Hem,'tndez-Zamora, A. Rev. Med. I.M.S.S., 1982, 20, 707-710.
ANTHRACENONES OF THE KARWINSKIA GENUS
[54]
[55] [56] [57]
[58] [59]
[60] [61] [62] [63] [64] [65] [66] [67]
[68] [69] [70] [71] [72] [73] [74]
[75] [76] [77] [78] [79]
[80] [81] [82] [83]
[84] [85]
605
Carrada-Bravo, T.; L6pez-Leal, H.; V/tzquez-Arias, G.; Ley-L6pez, A. Bol. Med. Hosp. Infant. Mex., 1983, 40, 139-147. Puertolas, M.; Nava, O.; Medina, H.; L6pez, F.; Oyervides, J. Rev. Med. LM.S.S. 1984, 22, 22-27. Guerrero, M., Pifleyro, A.; Waksman, N. Toxicon., 1987, 25, 565-568 Charlton, K.M.; Pierce, K.R.Texas Rep. Biol.Med., 1969, 27, 389-399 Charlton, K.M.; Claborn, L.D.; Pierce, K.R. Am. 3'. Vet. Res., 1971, 32, 13811387 Charlton, K.M.; Pierce, K.R. Path. Vet., 1970, 7, 385-407. Charlton, K.M.; Pierce, K.R. Path. Vet., 19711, 7, 408-419. Charlton, K.M.; Pierce, K.R. Path. Vet., 1970, 7, 420-434. Charlton, K.M.; Pierce, K.R.; Storts, R.W.; Bridges, C.H. Path. Vet., 1970, 7, 435-447. Dollahite, J.W.; Henson, J.B. Am. J. Vet. Res., 1965, 26, 749-752 BermOdez, M.V.; Martinez, F.J.; Salazar, M.E.; Waksman, N.; Pifleyro, A. Toxicon., 1992, 30, 1493-1496. Martinez, F.J.; Ramirez-Dur6n, R.; Waksman, N.; Pifleyro-L6pez, A. Toxicol. Lett., 1997, 90, 155-162. Mufloz-Martinez, E.J.; Chavez, B. Exp. Neurol., 1979, 65, 255-270. Aoki, K., Mufioz-Martinez, E.J.J. Neurobiol., 1983, 14, 463-466. Aoki, K.; Mufioz-Martinez, E.J.J. Neurochem., 1981, 36, 1-8. Mufioz-Martinez, E.J.; Cuellar-Pedrosa L. H.; Rubio-Franchini C.; J~tureguiRincon J.; Joseph-Nathan P., Neurochem Res., 1994, 19, 1341-1348. Pifleyro-L6pez, A.; Martinez, L.; Gonz~ilez-Alanis, Ro Toxicology, 1994, 92, 217227. Pifieyro-L6pez, A., Europ/fisches Patenblatt, 48/1995, 29, 1.1995 Garza-Ocaflas, L.; Jiang, T.; Acosta, D.; Torres-Alanis, O.; Waksman, N.; Pifieyro-L6pez, A. Toxicon, 1994, 32, 1287-1291. Garza-Ocafias, L.; Hsieh, G.C.; Acosta, D.; Torres-Alanis, O.; Pifleyro-L6pez, A. Toxicology, 1992, 73, 259-267. Takido, M; Kitanaka, S. (Taisho Pharmaceutical), Chem Abst., 1989, 110, 82474 Velazco, M.R.; Montero, R.; Rojas, E.; Gonsebatt, M.E.; Sordo, M.; Pifleyro, A.; Ostrosky- Wegman, P., Anti-cancer Drugs, 1996, 7, 1-5. Martinez, L., Velazco-Campos, R.; Pifleyro-L6pez, A.; Gonz~ilez-Alanis, R. Toxicon., 1990. 28, 449-452. Valadez-Alvizo, R.; Rodriguez-Alvarado, A.; Martinez, L.; Gonz,~lez-Alanis, R.; Pifieyro- L6pez, A. Toxicon, 1993, 31, 1329-1332. Flores-Otero, G.; Cueva, J.; Mufloz-Martinez, E..J.; Rubio-Franchini, C. Toxicon., 1987, 25, 419-426. Salazar, M.L.; Pifleyro, A.; Waksman, N. J. Lig. Chrom. & Rel. Technol., 1996, 19, 1391- 1403. Waksman, N.; Torres M.; Salazar M.; Pifleyro, A., Revista Sociedad Quimica de Mexico, 1994, 38, 254 Marston, A.; D~costerd, L.; Hostettmann, K. In Bioactive Natural Products, Colegate, S. and Molyneux, R., Ed; CRC Press, 1993, 233-237 Wagner,P.; Bladt, S.; Zgainski, E.M., Plant Drug Analysis, Springer Verlag, 1984 Gill, M.; Gimenez, A., Phytochemistry, 1991, 30, 951-955 Ramirez, R., Dissertation,1998,U.A.N.L., M6xico Black, T.H., Aldrich Chimica ,4cta, 1983, 16, 3.
606
[86] [87]
[881 [89] [90] [91] [92] [93] [94]
PII~IEYRO-LOPEZ and WAKSMAN
Gill, M.; Gimenez, A.; Jhingram, A., Palfreyman, A., Tetrahedron, Assimetry, 1990, 1, 621- 634 Lux, A.; Liskova, D.; Pifieyro-L6pez, A.; Ruiz-Ord6fiez, J.; Kakoniova, D., Biologia Plantarum, 1997/1998, 40, 143-147. Liskova, D.; Ruiz-Ord6fiez, J.; Lux, A.; Pifieyro-L6pez, A. Plant. Cell Tissue Organ. Cult., 1994, 36, 339-343. Hanackova, Z.; Waksman, N.; Mikus, M.; Lux, A.; Liskova, D., Physiologia Plantarum, 1997, 29, 95-100 Sepfilveda-Saavedra, J.; Vander Klei, I.J.; Keizer, I.; Pifieyro-L6pez, A.; Harder, W.; Veenhuis, M. FEMS Microbiol. Lett., 1992, 91,207-212 Salazar-Aranda, R.; Sepulveda-Saavedra, J.; Waksman de Torres, N.; PifieyroLop6z, A.; Moreno-Sepulveda, M.,FEMS Microbiol. Left., 1998, 158, 255-260. Lazarow B Paul. In The Liver Biology and Pathology; Arias Irwin M., Jakoby William B., Popper Hans, Schlacter David, Schafritz David A, Eds.; Raven Press: New York, 1988; 241-254. Moreno-SepOlveda, M.; Vargas-Zapata, R.; Esquivel-Escobedo, D.; Waksman, N.; Pifieyro- L6pez, A. Planta Med., 1995, 61, 337-340. Moreno-Sep61veda, M.; Vargas-Zapata, R.; Ballesteros-Elizondo, R.; PifieyroL6pez, A.; Sepfilveda-Saavedra, J. Toxicon, 1997, 35, 777-783.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 22 9 2000 Elsevier Science B.V. All rights reserved
607
BIOACTIVE NATURAL PRODUCTS DERIVED FROM POLYGONUM SPECIES OF PLANTS: THEIR STRUCTURES AND MECHANISMS OF ACTION
andMADELINE A D A M C Z E S K I *
N W A K A OGWURU$
Department of Chemistry, American University, Washington, D.C. 20016-8014, USA ABSTRACT" This review encompasses the natural products literature (with the exception of the patent literature or conference abstracts) of some of the different species of plants in the Polygonum species. Some of the plants in this genus originated in Japan and were later introduced to other parts of the world. These plants are commonly used in Chinese and Japanese folk medicine for the treatment of bronchial and pulmonary disorders, suppurative dermatitis, gonorrhea, and hyperlipemia. The main focus will be on plant species from the genus Polygonum which is also synonymous with the genuses, Fallopia and Reytonouria. A comprehensive analysis detailing the research on the chemistry of the different classes of natural products from extracts of Polygonumplant species and their modes of action will be reported and the structure-activity relationships will be addressed wherever possible. In addition, the evaluations of bioactive compounds of Polygonumspecies will be discussed in terms of potential biological uses.
INTRODUCTION
Taxa, Sex, and Ploidy of Various Species of Polygonum
Polygonum genus of plants have a distinct morphology characterized by large ovate to elliptical leaves [ 1]. The polygonaceous plants are widely distributed throughout eastern Asia and eastem North America [2], with the Polygonum genus originating from Japan. Polygonum species of plants are perennial herbaceous knotweeds with relatively thick rhizomes and are also synonymous with Fallopia and Reynoutria. It is important to note that there are several subspecies with varying n u m b e r of c h r o m o s o m e s . For example, Polygonum cuspidatum, also
i
.
,|,l
..,
.
,.,
,
,,
~Currently at the University of the District Of Columbia in the Department of Chemistry and Physics, Washington DC, 20008 and *Department of Chemistry, American University, 4400 Massachusetts Ave., N.W., Washington, D.C. 20016-8014, USA. *New address: San Jos6 City College, 2100 Moorpark Ave. Dept. of Science and Mathematics, San Jos6, CA 95128-2799, USA
608
OGWURU and ADAMCZESK!
known as both Fallopia japonica and Reynoutria japonica has the chromosome number 2n = 44 and 2n = 88. Other species of Polygonum have been reported with chromosome number 2n = 44 and 2n = 66. Polygonum cuspidatum (Fallopiajaponica) originated from Japan and the Sackhalin Island. The plant is erect and closely related to the black bindweed and the Russian vine plant that also belong to the Fallopia genus. Bailey [3-5] investigated three naturalized knotweeds, namely, Polygonum sachalinense (Fallopia sachalinensis), F. japonica var. compacta and F. japonica var. japonica in the British Isles and their progenies and determined their taxa, sex and chromosome numbers (see Table 1). Table 1
Distribution of Polygonum British Isles
(Fallopia) Species and their Hybrids in the
Taxon
Sex |
|l
i
Chromosome number i
i
i
F. japonica vat compacm
male sterile
44
P. sachalinense (F.sachalinensis)
hermaphrodite male sterile
44
F. sachalinensis x
hermaphrodite male-fertile hermaphrodite male-sterile
44 44 44 44
P. cuspidatum x P. sachalinense
hermaphrodite male-sterile
66 66
F. japonica (P. cuspidatum) x F. japonica vat compacta
male-fertile
66
P. cuspidatum (F. japonica) x P. sachalinense (F. sachalinensis)
hermaphrodite
88
P. cuspidatum
male-sterile
88
F. japonica vat compacta
i
i
The Fallopiajaponica var compacta examined were either male-sterile or female-sterile. The introduction of new species was limited to the genetic base of the fertile hybrid progeny. Some hybrids were sterile, while some had low fertility (e.g. the hybrid between Polygonum cuspidatum, also known as Fallopiajaponica and Polygonum sachalinense, also known as Fallopia sachalinensis yields a hexaploid). Pharmaceutical Uses of Polygonum In Chinese and Japanese traditional medicine [6-8], Polygonum cuspidatum is used to treat hyperlipemia, suppurative dermatitis, gonorrhea, favus,
BIOACTIVE NATURAL PRODUCTS FROM POLYGONUM SPECIES
609
and athletes foot. Compounds that have been isolated from the root of Polygonum cuspidatum encompass anthraquinone-derivatives, [7-9] such
as: emodin (5), [ 10,11], physcion (6) [ 12], glucosides of physcion (8) and emodin (7) [9,13], stilbene-derivatives (15-21) including: resveratrols,[9], [ 10], chrysophanic acid (1,8- dihydroxy-3-methyl-9,10-anthracenedione); tannin-derivatives; sesquiterpenes including, the drimane class (22-27); and glycosidic aromatic esters including: vanacoside- (28-30) [14], phenolic [ 15], and the flavonoids (31-43) [ 16-20]. Two independent research groups [21,22] reported that emodin has anticancer properties against lymphocytic leukemia in mice. Two other laboratories [23,24] demonstrated that piceid possessed antihyperlipemia activity on mice and provided some protection from liver injury in rats fed peroxidized oil [ 15]. Several research groups [7,8,14,23,25,26] showed that biologically active resveratrol possessed both antibacterial and antifungal activities, while Goda et al. [27] and Kimura et al. [28] provided evidence of inducing platelet hyperaggregation and lipid lowering activity in rats with hyperlipemia. S T R U C T U R E S OF SEVERAL DISTINCT CLASSES COMPOUNDS ISOLATED FROM POL YGONUM
OF
Several representative classes of compounds have been isolated from Polygonum species of plants, including: quinone, phenol, stilbene, tannin,
terpenoid, flavonoid and catechol compounds. The structures of some of the common compounds occuring in Polygonum are given below. Quinones and Phenols
Kimura et al. [ 15] isolated several phenolic constituents from the roots of Polygonum cuspidatum. The structures were elucidated and the compounds were identified as fallacinol (1), citreorosein (2), questin (3), questinol (4), protocatechuic acid, (+)-catechin, 2,5-dimethyl-7-hydroxy chromone, 7-hydroxy-4-methoxy-5-methylcoumarin, torachroysone-8-OD-glucoside, 2-methoxy-6-acetyl-7-methyljuglone (9), 2-methoxy-6acetyl-l-monoacetate (10) and 2-methoxy-7-methyljuglone (11). Yeh et al
