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 ni Natural Products Chemistry comprises articles written on bioactive natural products by leading authorities ni their respective fields. Robinson has reviewed the metabolism and pharmacology of alkaloids found ni animals. Chemical ecology can be an attractive tool for identifying antifungal natural products and this area has been reviewed by Graham et .la 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 ni developing countries and there are a large number of deaths each year caused by .ti The review by Kawanishi et .la presents the current status of work done ni this field as well as on natural anti-diabetic compounds. Synthetic approaches involving intramolecular diyl trapping reaction are described by Little et .la for the synthesis of linearly fused tricyclopentanoids. The use of classical and biocombinatorial approaches to bioactive fungal natural products si discussed by Jiang et .la F010p has presented the chemistry of 2- aminocyclopentanecarboxylic acid. Antioxidants continue to attract attention ni medicine and many interesting flavonoids have been found ni nature which possess antioxidant and pro-oxidant properties. These studies have been reviewed by Vanden Berghe and co-workers, while antioxidant activity found ni South American plants si reviewed by Desmarchelier. Other interesting reviews include those on insect juvenile hormones ni plants by Tobe, antiulcer and gastroprotective activity of flavones by Martin et ,la biological activity of simple flavones by Tahara et .la 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 munogyloP species possess interesting biological activities. The bioactive compounds ni such plants are reviewed by Adamczeski. mucirepyH mutarofrep (St. Johns wort) si 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 ni respect of their chemistry and biological activity. I would like to express my thanks to .rD Shakil Ahmad and .rD Durre Shahwar for their assistance ni the preparation of the index. I am also grateful to .rM Waseem Ahmad for typing and to .rM 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 Ph.D. (Cantab), Sc.D. (Cantab) February, 2000 Preface nI his fascinating and beautifully written autobiography (For eht Love of Enzymes, Harvard University Press, Cambridge, MA, 1989), Arthur Kornberg repeatedly emphasizes the central role of chemistry ni understanding life processes. Nowhere si this connection clearer and more direct than ni 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 naturally- occurring 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 ni the popularity or even "trendiness" of natural products research, the field continues to advance around the world. tI si not a coincidence that natural products have played, and will continue to play, a seminal role ni the discovery and development of pharmaceutically and agrochemically important agents. Three billion years of biological evolution have resulted ni the development of metabolic pathways leading to the synthesis of hormones, pheromones, antibacterial, anti-fungal, anti-protozoan, and anti- insectan agents, as well as many other bioactive compounds that are of adaptive value ni 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 si driven by the entirely worthy desire to obtain "useful knowledge," it si clear that scientists entering the field of natural products chemistry are often deeply motivated by their love of nature ni general, of chemistry ni 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 ni 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 ni this series. With respect to the present volume, the reader can expect a veritable chemical feast. Jerrold Meinwald Cornell University Ithaca, NY 14853 USA CONTRIBUTORS Madeline Adamczeski Department of Chemistry, American University, Washington, D.C. 20016-8014, USA Zhiqiang An Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge, MA 02139-1562, ASU 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 ed Buenos Aires, Junin 659 1113 Buenos Aires, Argentina .P Cos Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein ,1 B-2610 Antwerp, Belgium .J Coussio ardet~C 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, 41012- Seville, Spain xii .C Desmarchelier C6tedra de Microbiologta Industrial y Biotecnologta, Facultad de Farmacia y Bioquimica, Universidad ed Buenos Aires, Junin 659 1113 Buenos Aires, Argentina Stephanie .J Eckerman Chemistry Department College of .tS Benedict/St. John's University 73 .S College Avenue, .tS Joseph, NM 56374, USA C.A.J. Erdelmeier .rD Willmar Schwabe GmbH & Co., Research dna Development, Karlsruhe, Germany N.R. Famsworth Program for Collaborative Research ni the Pharmaceutical Sciences, College of Pharmacy, University of Illinois ta Chicago, Illinois, ASU Ferenc pOl~tF Institute of Pharmaceutical Chemistry, Albert Szent- Gy0rgyi Medical University, H-6701, Szeged, BOP ,121 Hungary R.M. Giner Departament de Farmacologia, Facultat ed Farmacia, Universitat de Valencia, Avda. Vicent s~/rdnA Estell~s s/n., 46100 Burjassot (Valencia), Spain Kate .J Graham Chemistry Department College of .tS Benedict/St. John's University 73 .S College Avenue, .tS Joseph, NM 56374, USA .R Hoerr .rD Willmar Schwabe GmbH & Co., Research dna Development, Karlsruhe, Germany J.L. Ingham Department of Food Science dna Technology, University of Reading, Whiteknights, P.O. Box 226, Reading 6GR 2AP, England, U.K. Zhi-Dong Jiang Millennium Pharmaceutical Inc., One Kendall Square Building 300, Cambridge, AM 02139-1562, ASU .K Kawanishi Kobe Pharmaceutical University, Kobe, Japan .E Koch .rD Willmar Schwabe GmbH & Co., Research dna Development, Karlsruhe, Germany .C La Casa Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia Gonzfilez s/n, 41012- Seville, Spain XIU ~176176 .R Daniel Little Department of Chemistry, University of California, Santa Barbara, Santa Barbara, AC 93106, ASU .S zelfi~M Departament ed Farmacologia, Facultat ed Farmacia, Universitat de Valencia, Avda. Vicent Andr6s s~/lletsE s/n., 46100 Burjassot (Val/mcia), Spain M.J. Martin Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia zeli~znoG s/n, 41012- Seville, Spain .V Motilva Department of Pharmacology, Faculty of Pharmacy, University of Seville, Prf. Garcia zeli~znoG s/n, 41012- Seville, Spain Nwaka Ogwuru Department of Chemistry, American University, Washington, D.C. 20016-8014, ASU Michael .M Ott Department of Chemistry, University of California, Santa Barbara, Santa Barbara, AC 93106, ASU .L Pieters Department of Pharmaceutical Sciences, University of Antwerp (U.I.A.), Universiteitsplein ,1 B-2610 Antwerp, muigleB .A Pifleyro-Lopez Departmento de Farmacologia, Y Toxicologia, Apdo. Postal 146, Col. led 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 ed Farmhcia, Universitat ed ,blaV ncia, Avda. Vicent sOrdnA Estell6s s/n., 46100 Burjassot (Valencia), Spain J.L. Rios Departament ed Farmacologia, Facultat ed ,aic.bmraF Universitat de Valencia, Avda. Vicent sOrdnA Estell~s s/n., 46100 Burjassot (Valencia), Spain .T Robinson Lederle Graduate Research Center, Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst xoB 34505, Amherst, AM 01003- 4505, U.S.A. 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, 52 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. ocix~M namhaR-ru-attA ).dE( Studies in Natural Pror Chemistry Vol. 22 (cid:14)9 0002 reiveslE ecneicS .V.B llA sthgir devreser THE METABOLISM AND BIOCHEMICAL ACTIONS OF ALKALOIDS IN ANIMALS T. ROBINSON Lederle Graduate Research Center Department of yrtsimehcoiB and Molecular Biology ytisrevinU of ,sttesuhcassaM Amherst xoB ,50543 ,tsrehmA AM 01003-4505 A.S.U ABSTRACT: 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 11o 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 1,7- dimethylxanthine and 1-methylxanthine 11. After the first demethylation there is an alternate pathway producing 5-acetylamino-6-formylamino-3- methyluracil. This pathway is more active in people with a more active acetylation system ,21 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 .41 In rabbits the major urinary products are, in order, 1-methylxanthine, -1 methyluric 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 .71 Experiments with liver slices and cultured cell lines have corroborated this pathway of purine degradation. In human liver slices 61 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 1,3,7- 0 3HC 0 3H~( Caffeine 3H~( enillyhpoehT 1 1 O 3HCi O H CH3....,J~ 1,7-Dimethylxanthine G 1 enihtnaxlyhteM- O CH3~ ~ H;C)C~"I~ 3 5-Acetylamino-6-formylamino-3-methyluracil H Caffeine Metabolism