The Alkaloids: Chemistry and Pharmacology, Volume 43

THE ALKALOIDS Chemistry and Pharmacology VOLUME 43

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THE ALKALOIDS Chemistry and Pharmacology Edited by Geoffrey A. Cordell College of Pharmacy University of Illinois at Chicago Chicago, Illinois

VOLUME 43

Academic Press, Inc. Harcourt Brace Jovanovich, Publishers

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This book is printed on acid-free paper. @ Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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CONTENTS

CONTRIBUTORS ......................................................... PREFACE...............................................................

vii ix

Chapter 1 . Allelochemical Properties or the Raison d'Etre of Alkaloids MICHAELWINK I . Introduction ...................................................... I1 . Allelochemical Properties of Alkaloids ............................... 111. Raison d'Etre of Alkaloids ......................................... IV . Conclusions ...................................................... References .......................................................

I 5 86

I03 104

Chapter 2. Mammalian Alkaloids I1 ARNOLDBROSSI 1. Introduction ...................................................... I1 . Mammalian Indole Alkaloids ....................................... 111. Mammalian Isoquinoline Alkaloids .................................. IV . Mammalian Morphine ............................................. V . Alkaloid Formation in Mammals as a Therapeutic Concept ............. VI . Addendum ....................................................... VII . Conclusions ...................................................... References .......................................................

Chapter 3. Amphibian Alkaloids JOHNW . DALY.H . MARTINGARRAFFO.A N D THOMASF. SPANDE I . Introduction ...................................................... I1 . Steroidal Alkaloids ................................................ 111. Bicyclic Alkaloids ................................................. IV . Tricyclic Alkaloids ................................................ V . Monocyclic Alkaloids .............................................. .................................... VI . Pyridine Alkaloids ........ VII . Indole Alkaloids .............................................. VIII . Imidazole Alkaloids ............................................... IX . Morphine ........................................................ X . Guanidinium Alkaloids ............................................. V

186

187 199

242 251 255 257 263 263 264

CONTENTS

vi

XI. Other Alkaloids ................................................... XII. Summary ......................................................... Appendix ........................................................ References .......................................................

CUMULATIVE INDEX OF

INDEX

TITLES ...........................................

.................................................................

269 215 211 28 1

289 291

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ARNOLD BROSSI(1 19), Department of Chemistry, Georgetown University, Washington, D. C. 20057 JOHNW. DALY(189, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 H. MARTINGARRAFFO (185), Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 THOMASF. SPANDE (189, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 MICHAEL WINK (I), Universitat Heidelberg, Institut fur Pharmazeutische Biologie, 6900 Heidelberg, Germany

vii

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PREFACE

Over the 43 years since the first volume in this series was published, most of the chapters have provided critical reviews of the many aspects of the chemistry and biology of alkaloids from the plant kingdom. In Volume 43 of “The Alkaloids, Chemistry and Pharmacology” all three chapters adopt a quite different perspective. In Chapter 1 “Allelochemical Properties or the Raison d’Etre of Alkaloids,” Michael Wink examines in considerable detail, and with some healthy speculation, why it is that organisms, such as plants, actually produce alkaloids. Is it, for example, as was previously thought, that alkaloids are the waste products of the organism? Wink says that based on the biological activities observed for many alkaloids and the ecological niche that certain plants occupy, emphatically not. Rather, alkaloids are indeed important compounds to the organism, possibly as antimicrobial or antipredation agents, or as competitive inhibitors for other plants or organisms. The remaining two chapters provide fascinating insights into the alkaloids of mammals and of amphibians. As a follow-up to a chapter published in Volume 21 of this series, Arnold Brossi offers a critical review of the current status of the knowledge of mammalian alkaloids, such as those derived from tryptophan and from phenylalanine, and in particular he reviews the literature regarding the fascinating subject of whether morphine-like alkaloids are indeed mammalian metabolites. Also following up on an earlier review in Volume 21 by Witkop and Gossinger, John Daly, Martin Garraffo, and Thomas Spande present a summary of the diverse groups of biologically active alkaloids that have been isolated and detected from various amphibians. Much of this work, conducted on minute amounts of material, is from the authors’ laboratory and has not been available previously. In summary, this volume maintains the tradition of the series of providing outstanding new reviews of general and specific interest and of updating established areas where there have been significant recent results. Geoffrey A. Cordell University of Illinois at Chicago ix

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-CHAPTER1-

ALLELOCHEMICAL PROPERTIES OR THE RAISON D’ETRE OF ALKALOIDS MICHAEL WINK Universitat Heidelberg Institut fur Pharmazeutische Biologie 6900 Heidelberg, Germany

I. Introduction .......................................................................................... 11. Allelochemical Properties of Alkaloids ...................................................... A. Plant-Herbivore Interactions ............................................................... B. Plant-Microbe Interactions ............................................................... C. Antiviral Properties .......................................................................... D. Allelopathic Properties ..................................................................... 111. Raison d’8tre of Alkaloids ..................................................................... A, Concentrations in Plants and Allelochemical Activities.. ......................... B. Presence of Alkaloids at the Right Site and Right Time .......................... C. Importance of Alkaloids for Fitness of Plants ....................................... D. Exceptions to the Rule: Role of Adapted Specialists .............................. IV. Conclusions ........................................................................................ References .........................................................................................

1 .5

8 61

79 82 86 87 89 92 % 103 104

I. Introduction

Plants constitute the major group of photoautotrophic organisms on our planet that are able to use solar energy to fix carbon dioxide into hydrocarbons, such as glucose, and to produce ATP and NADPH, as “fuel” and reduction equivalents, which serve to build up all the other essential components of a cell. Animals and most microorganisms (except the chemo- or photoautotrophic bacteria) are heterotrophic organisms, which rely on complex, plant-made organic molecules for their energy requirement or other metabolic functions. Thus plants serve as a major and ultimate source of food for animals and microorgansims, whether they like it or not. We can safely assume that plants struggle for life and that they have evolved strategies against herbivorous animals or phytopathogenic micro1

THE ALKALOIDS, VOL. 43 Copyright 8 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MICHAEL WINK

organisms. We must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients. How do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? Because plants do rather well in Nature, this question has often been overlooked. We are well aware of the defensive strategies of higher animals against microbes and predators (1,2,4,15,17,28,494).The complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. Against predating animals, Nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called “poisons”) (1). It is evident that most of these possibilities are not available for plants with their sessile and “passive” life-style. What then is their evolutionary solution? We can distinguish the following defense mechanisms in plants (3,4,7,15,17);the mechanisms are not independent and may act cooperatively and synergistically. We should be aware that many species have additionally evolved specialized traits in this context. 1. Mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). 2. Formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. 3. If plants are wounded or if parts of them are eaten, this is usually not as fatal as the similar situation in animals, since plants can easily replace a lost leaf or branch (so-called open growth). 4. A most important strategy, however, is the production and storage of defense chemicals, which are abundant and a typical trait of all plants. a. Plant surfaces are usually covered by a hydrophobic layer consisting of antibiotic and deterranthepellent cuticular waxes which may contain other biologically active allelochemicals such as flavonoids (3-5,7). b. Cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. Callose and lignin are often accumulated at the site of infection or wounding (6,7)and form a penetration barrier. c. Synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. These proteins are either stored in the vacuole

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

3

or are secreted as exoenzymes into the cell wall or the extracellular space (8,9).These compounds are thus positioned at an “advanced and strategically important defense position.” In addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. As a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced (2-4, 10-21). These allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides,glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~ O U synthesis O may be induced by elicitors (so-called phytoalexins), infection, or herbivory (4,7,22-24). These products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection (25,26)]or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots (2,3,15)]. In animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals (1,4,12,16,17,28-30,494-496,503). In many instances, the animals have obtained the toxins from their host plants (4, 12,15,17,27-33).Hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ‘‘toxins” in this context) in animals is that of defense against predators or microorganisms (1,17,28,494-496). These defense compounds are better known as natural products or secondary metabolites. The latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites (34,35,38). Unfortunately the term “secondary” has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. As discussed in this chapter, just the opposite is true. More than 30,000 natural products have been reported from plants so far (2,4,17).Owing to the sophistication in phytochemical methods, such as chromatography (HPLC, GLC) and spectroscopy (NMR, MS), new products are reported at rapid intervals. Because only 5-10% of all higher plants, which consist of over 300,000 species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large. It is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. Within a taxon secondary metabolites

4

MICHAEL WINK

often share a common distribution pattern and are therefore of some importance for phytochemical systematics. Classic taxonomy, however, has taken little account of alkaloid distribution: If the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. Because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated (233). For many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of Nature (34,35). In 1887 and 1888, Errera and Stahl (92,308,504) published the idea that natural products are used by plants for chemical defense against herbivores. Since the leading plant physiologists of that time were mostly anti-Darwinian, they were not willing to accept the defense argument, which was too much in line with the Darwinian concept. Therefore, this early defense concept was negated and remained forgotten for nearly 60 years. In 1959, Fraenkel(10) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. During the next three decades this concept was improved experimentally, and we can summarize the present situation as follows (2-4,11-223,210). Although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century (34,35). In many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy). In some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the Centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes (15,17). Physiological roles, such as UV protection [by flavonoids or coumarins (4,17)],nitrogen transport or storage (14,36,37),or photosynthesis (carotenoids), may be an additional function. Allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

5

antimicrobial). Thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). To understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach. It might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. However, we have to understand and accept that chemical defense is not an absolute process. Rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. Plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. During evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them (14,15,32).This is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. The number of these “specialists” is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. We can compare this situation with our immune system: It works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. Nobody would call the immune system and antibodies useless because of these few adapted specialists! We should adopt the same argument when we consider plants’ defenses by secondary metabolites (2). Since secondary metabolites have evolved in Nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. In addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds.

11. Allelochemical Properties of Alkaloids

About 20-30% of higher plants accumulate alkaloids (505,506). The incidence of alkaloid production varies between taxa to some degree; for example, about 60-70% of species of the Solanaceae and Apocynaceae are

6

MICHAEL WINK

alkaloidal, whereas other families contain few alkaloid-producing species. Some alkaloids have a wide distribution in Nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. Alkaloids are not restricted to higher plants (althoughthey are here most numerous); they are also present in club mosses (Lycopodium), horsetails (Equisetum), fungi, and animals such as marine worms (e.g., Nereidae), bryozoans, insects (e.g., Coccinellidae, Solenopsidae), amphibians (toads, frogs, salamanders), and fishes. Alkaloids thus represent one of the largest groups of natural products, with over 10,000 known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan (38-40). In addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates (38-40). Whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. This is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals. Alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as W. 0. James and Kurt Mothes ( 3 4 3 , 505,526)l.Because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. The waste product argument probably came from animal physiology: Carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. A similar situation or need, however, is not applicable for plants. In fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves (2,37,506). If alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. On the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals (16,28,494496). Thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. This idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and Homo sapiens. As a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. (1) The alkaloid should have significant effects against microbes and/or animals in bioassays.

1.

ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

7

(2) The compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. (3) The compound should be present in the plant at the right time and the right place. (4) Evidence should be provided that a particular compound is indeed important for the fitness of a plant. Although more than 10,000 alkaloids are known, only few (-2-5%) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. In most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities. Furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. These objections have to be kept in mind, because an alkaloid is sometimes termed “inactive” in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). In many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. If the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. Another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. However, if a compound is active against Escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. Nevertheless, most of the data obtained in these studies (Tables I-VIII) provide important information which at present permits extrapolation to the function of alkaloids in plants. In this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. In the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail. It is nearly impossible to cover the literature exhaustively. Therefore, an overview of the allelochemical properties of alkaloids is presented. Because of the large amount of data (literature up to 1990 is included), the selection of examples must remain subjective to some degree. Nevertheless, the author would be grateful to receive information or publications about relevant omissions.

8

MICHAEL W I N K

A. PLANT-HERBIVORE INTERACTIONS Because Homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (Tables I through V) and alkaloid-containing plants. Previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products (38,39). In the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates. 1 . Invertebrates

Among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. Entomologists estimate that the number of insects is at least 1 million, but tropical rain forests may harbor up to 20-30 million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists. Most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( I ,4,10,14,15,28-33, 494496,503). Whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. In the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). At the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds. The close association between plants, especially the angiosperms, and insects evolved during the last 200 million years. Some scientists have called this phenomenon a “coevolutionary” process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved (18,505,506).Applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. Times can also be much longer. If plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. For example, Lupinus polyphyllus from North America has a number of specialized herbivores, but is rarely attacked by herbivores in Europe. This lupine left its enemies behind when it was transferred to Europe three centuries ago. About 10 years ago, however, the North American lupine aphid (Macrosiphum albifrons) was introduced to Europe accidentally. This aphid is specialized to alkaloid-rich lupines with lupanine as a major

1.

ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

9

alkaloid. At present, this aphid has spread over most of Europe and is now colonizing its former host, L. polyphyllus (2,503). Insect herbivores can be divided into two large groups whose strategies with respect to the plant’s defense chemistry differ substantially (15). The polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagousinsects are often specialized on one or a small number of (often systematically related) hosts. Polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors (501) that allow the distinction between plants with high or low amounts of “toxins.” The receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates (507). These “generalists,” as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see Table XI). Alternatively, they change host plants rapidly and thus avoid intoxication. In addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products (4,15,17,508). In contrast, mono- and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. For these “specialists” the originally noxious defense compounds are often attractive feeding and oviposition stimulants. These insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes (1,4,10-12,1447,28,31,33,494-496).These observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as Mothes (35)and Robinson (505). However, these specialized insects are exceptions to the general rule. For these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants (32).As compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species. Quite a number of alkaloids have been tested toward herbivorous insects (Table I). In general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>I%, w/w). Given the choice, insects tend to select a diet with no or only a small dose of alkaloids. Also,

TABLE I ACTIVITYOF ALKALOIDS AGAINST HERBIVORES (MOSTLYINSECTSAND OTHERINVERTEBRATES) ED, Alkaloid Alkaloids derived from tryptophan Acetylokaramine Ajmalicine Ajmaline Brucine

-

Cinchonidine

0

Cinchonine Dictamnine Ergocryptine Ergometrine Ergonovine Ergotamine Gramine Harmaline Harman

Effect Insecticidal in Eombyx Feeding deterrent to polyphagous Synfomis (Lepidoptera) larvae Feeding deterrent to polyphagous Synfomis larvae Feeding deterrent to polyphagous Synfomis larvae Feeding deterrent in bees (Apis mellifera) Insecticidal for bees Phagorepellent in Pieris, Eombyx (Lepidoptera) Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Feeding deterrent in Agelaius (Aves) Feeding deterrent in bees Feeding deterrent in Leptinofarsa (Coleoptera) Insecticidal Toxic to Oncopelfus Inhibition of insect spermatophore formation Feeding deterrent to polyphagous Syntornis larvae Toxic to Oncopelrus Feeding deterrent to polyphagous Synfomis larvae Feeding deterrent in aphids Insecticidal for Schiznphis (Aphidoidea) Phototoxicity in larvae of Trichoplusia (Lepidoptera) Feeding deterrent to polyphagous Syntomis larvae Phototoxicity in larvae of Trichoplusia Deterrent to polyphagous larvae

(dml, d g , or %I 10 1% 0.1% 1% 0.05%

0.2%

-

0.1% 0.04% 40 mg/kg 0.007%

-a 1%

0.1%
-

Ref. 166 32 32 32 152 152 161 32 152 175 152 162 176 167 164 32 167 32 155,156 157 66 32 66 151

Harmine

--

H ypaphorine Kokusagine Maculine Melicopicine 5-Methoxy-N,Ndimethyltryptamine 6-Methoxybenzoxazolinone 6-Methoxydictamine 2-Methyl-6methoxytetrahydro-pcarboline Norharman Okararnines A, B Phy sostigmine Quinidine Quinine

Reserpine

Photoxicity in larvae of Trichoplusiu Phototoxic to Aedes (Diptera) larvae Deterrent to polyphagous larvae Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Feeding deterrent for seed predators Insecticidal Insecticidal Antifeedant in Spodoprera (Lepidoptera) Antifeedant in larvae of Anthonornus

66 57

Insecticidal Insecticidal

165 I 76

Antifeedant in larvae of Anthonomus (Coleoptera) Phototoxicity in larvae of Trichoplusiu Toxic to Oncopelrus Insecticidal in Bombyx Feeding deterrent to polyphagous Synromis larvae Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Insecticidal for bees Feeding deterrent in Phormia (Diptera) Inhibition of insect spermatophore formation Feeding deterrent in Locusra (Orthoptera) Phagorepellent in Pieris, Bombyx, Lymanrria (Lepidoptera) Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Toxic for bruchids (Coleoptera) Feeding deterrent to polyphagous Synromis larvae

151 32 152 i63

I 76 I 76 97 153

380

-

0.1-3 0.01%

0.01%

0.02% 0.02% 0.6 mM 0.01% dry wt

153 66 167 166 32 32 152 152

154,160 164

0.01%

171 161,I74 32

0.04%

152

0.1% 1%

159,158 32

-

(continued)

TABLE I (Continued)

Alkaloid

Effect

Toxic for bruchids Feeding deterrent in Phormia Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in bees Insecticidal for bees Phagorepellent in Pieris, Bombyx, Lymanrria Feeding deterrent in Leptinotarsa Antifeedant in Spodoprera Tecleanthine Toxic for bruchids Feeding deterrent in Schisrocerca (Orthoptera) Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in bees Feeding deterrent to polyphagous Synromis larvae Vincamine Feeding deterrent in bees Insecticidal for bees Feeding deterrent in Phormia Yohimbine Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Alkaloids derived from phenylalanineltyrosine Feeding deterrency, growth inhibition in larvae of Hyphanrria, Aristolochic acid Spodoprera, Lymanrria Feeding deterrent in Locusta Toxic for Eurytides, Papilio (Lepidoptera) Photoxicity in Aedes larvae Berberine Feeding deterrency, growth inhibition in larvae of Hyphantria, Spodoptera Lymantria Strychnine

~

ED, (pglml. pglg, or %)

Ref.

0.008%

158 160 32 152 152 161 162 97 158 159 32 152 32 152 152 154 32 152

0.25-0.5%

168

0.000001%dry wt 0.5% dry wt 8.8 light1250 dark 0.25-0.5%

171 168 172 168

0.1% 10 mM

I% 0.02% 0.2% 0.1%

-

1%

0.2% 0.01% 0.08% 0.04% 2.5 mM 1%

Boldine Canadine Chelidonine Cocculolidine Codeine Colchicine

Emetine L-Ephedrine

Glaucine Isoboldine Laudanosine Lycoricidine Lycoricidinol

Toxic to larvae of Euxoa (Lepidoptera) Feeding deterrent in Phormia Toxic for Euryrides, Parides (Lepidoptera) Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Insecticidal for bees Phagorepellent in Pieris, Bombyx Feeding deterrent in Leprinorarsa Feeding deterrent to polyphagous Synromis larvae Feeding deterrent to polyphagous Synromis larvae Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in Spodoptera, Oraesia (Lepidoptera) Feeding deterrent in Phormia Feeding deterrent in Locusra Toxic for bruchids Feeding deterrent to polyphagus Synromis larvae Feeding deterrent in bees Insecticidal for bees Feeding deterrent in Agelaius Insecticidal to Leptinotarsa Feeding deterrent to polyphagus Synromis larvae Toxic for bruchids Feeding deterrent to polyphagus Syntomis larvae Feeding deterrent in bees Feeding deterrency, growth inhibition in larvae of Hyphantria, Spodoptera, Lymantria Feeding deterrent in Prodenia, Oraesia Feeding deterrency, growth inhibition in larvae of Hyphantria, Spodoptera, Lymantria Antifeedant in Eurema (Lepidoptera) Antifeedant in Eurema

0.3% 0.6 m M 0.5% dry wt

I 73 154 168

32

I% 0.01%

152 152 161 162

0.003% 0.01% 0.1% 0.1%

32 32 32 I 70

10 mM 0.001% dw 0.1% 0.01%

154 171

158 32 152

0.2% 0.03% 22 mg/kg 0.1% 0.1% 0.1%

152

175 162

32 158

32

0.09%

152 168

0.25-0.5%

-

I 70

0.25-0.5%

168 169 169 ~

(continued)

TABLE I (Continued)

Alkaloid Morphine Noscapine Papaverine

L

P

Salsoline Sanguinarine

Effect Phagorepellent in Pieris Feeding deterrent in Leptinotarsa Feeding deterrent to polyphagous Syntomis larvae Feeding deterrency, growth inhibition in larvae of Hyphantria, Spodoptera, Lymantria Feeding deterrent in Phormia Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in Leptinotarsa Feeding deterrent to polyphagous Syntomis larvae Feeding deterrency, growth inhibition in larvae of Hyphantria, Spodoptera Lymantria Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in Leptinotarsa

0.01% 0.25-0.5% 10 mM

0.1%

-

0.1% 0.25-0.5%

161 162 32 168 160 32 162 32 168

~

Quinolizidine alkaloids Anagyrine

Nematicidal in Bursaphelenchus

32 162

6

216

0.1 m M 0.1%

181 32 179 185 216 219

13-trans-

Cinnamoyloxylupanine C ytisine

2,3-Dehydro-0-(2pyrrolylcarbony1)virgiline

Feeding deterrent in Choristoneura fumifPrana Feeding deterrent to polyphagous Syntornis larvae Feeding deterrent in Acyrthosiphon pisum Feeding deterrent in Formica rufa (Hymenoptera) Nematicidal in Bursaphelenchus Feeding deterrent in molluscs (Helix) Molluscicidal in Biomphalaria

0.02% ED,, 0.1% 1 2.5 mM

220

Lupanine

Lupinine Matrine N-Methylcytisine 17-Oxosparteine Sparteine

Feeding deterrent to polyphagus Syntomis larvae Reduction of growth and survivorship in Spodoptera Lethal to Plutella maculipennis Lethal in Dysdercus (Homoptera) Lethal in Phaedon (Coleoptera) Lethal in Ceratitis (Diptera) Feeding deterrent in Formica rufa Feeding deterrent in molluscs (Helix) Insecticidal in Melanoplus (Orthoptera) Feeding deterrence in Acyrthosiphon pisum Active against Dipylidium, Fasciola, Angiostrongylus Nematicidal in Bursaphelenchus Active against Dipylidium, Fasciola, Angiostrongylus Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in Acyrthosiphon pisum Feeding deterrent for Entomoscelis (Coleoptera) Toxic for bruchids Feeding deterrent in Phormia Feeding deterrent to polyphagus Syntomis larvae Feeding deterrent in bees Insecticidal for bees Phagorepellent in Pieris Reduction of growth and survivorship in Spodoptera Feeding deterrent in Manduca sexta (Lepidoptera) Lethal to PIutella maculipennis Lethal in Dysdercus Lethal in Ceraritis Feeding deterrent in Formica rufa Feeding deterrent in molluscs (Helix)

0.1%

-

LD,, 6 mM LD,, 12 mM LDlm 12 mM LDl,3 mM ED,, 1% 1-7 mM 0.08% 1-2 0.1% 0.01% 1-10 m M 0.1% 10 mM 0.1%

0.03% 0.05% -

0.05% LD,, 50 mM LD,, 50 mM LDl,9 mM ED,, 1% 0.7-0.8% mM

32 180 183,184 183,184 183,184 183,184 185 219 178 179 217,218 216 217,218 32 179 177 158 160 32 152 152 161 180 182 183,184 183,184 183,184 185 219

(continued)

TABLE I (Continued) ED50 Alkaloid 13-Tigloylox ylupanine

e

m

Steroidal alkaloids Cevadine Chaconine Conessine Demissidine Protoveratrine B Solacaudine Soladulcine Solamargine Solanidine Solanine

Solanocapsine Solasonine Tomatidine

Effect Feeding deterrent in Choristoneura fumiferana Lethal to Plutella maculipennis Lethal in Dysdercus Lethal in Phaedon Lethal in Ceraritis Insecticidal Feeding deterrent in Choristoneura (Lepidoptera) Molt inhibition in Periplaneta Phagorepellent in Pieris, Bombyx, Lymantria, Dysdercus Feeding deterrent in Leptinotarsa Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in Leprinorarsa Feeding deterrent in Leptinorarsa Insecticidal in Earias Feeding deterrent in Choristoneura Feeding deterrent in Chlorisroneura Feeding deterrent in Pieris Feeding deterrent in Leptinotarsa Feeding deterrent for Manduca Insecticidal in Earias Feeding deterrent in Choristoneura Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in Leptinotarsa

(pg/ml, pgk, or %)

Ref.

8% at 1.4 mM LD,, 12 mM LD,, 6 mM LD,, 6 mM LD,, 6 mM

181 183,184 183,184 183,184 183,184

-

194 I 90 I 95 161,196 189 32 189,191 189 I 92 190 190 I 74 189 I 93 I 92 190 32 189

0.1 mM

-

0.01%

0.1 mM 1 mM 0.4 p M

-

5 mM 1 mM 1%

Tomatine

Veratridine Veratrine Tropane alkaloids Atropine

4

Cocaine H yoscyamine

Scopine Scopolamine

Tropine Polyhydroxy alkaloids Castanospermine Deox ynojirim ycine 6-Epicastanospermine

Feeding deterrent for Locusta Growth inhibition in Heliothis (Lepidoptera) Feeding deterrent in Chorisroneura Feeding deterrent in Melanoplus Deterrent in Locusta Feeding deterrent in Phormia Growth inhibition in Hyposoter (Hymenoptera) Phagorepellent in Pieris Phagorepellent in Leptinotarsa Insecticidal Feeding deterrent in Schistocerca Insecticidal to Leptinotarsa Feeding deterrent in Phormia Toxic for bruchids Phagorepellent in Pieris Feeding deterrent in Leptinotarsa Feeding deterrent to polyphagous Feeding deterrent in bees Insecticidal for bees Feeding deterrent to polyphagous Feeding deterrent to polyphagous Feeding deterrent in bees Phagorepellent in Pieris, Bombyx Feeding deterrent to polyphagous Feeding deterrent in bees

0.1% 0.9 mM 0.1 mM

-

0.15% dry wt 10 mM 20 pmol/g

-

0.6 mM 0. I%

Syntomis larvae Syntomis larvae Syntomis larvae

0.1% 0.005% 0.1% 0.1% 0.01% 0.03%

-

Syntomis larvae

Feeding deterrent in aphids and greenbugs Feeding deterrent in aphids and greenbugs Feeding deterrent in aphids and greenbugs

0.1%

0.2% 0.1 mM 2.5 mM 5 mM

186 187 190

I 78 I71 160 188 161 189 I94 159 162 154,160 158 161 162 32 152 152 32 32 152 161 32 152 197 197 I97 (continued)

TABLE I (Continued)

Pyrrolizidine alkaloids Crispatine N-Form ylloline Heliotrine

m

I

Jacobine Jaconine Lasiocarpine Perloline Senecionine Senkirkine Miscellaneous alkaloids Aconitine 2.5-Alkylpyrroline (ant) Anabasine Anacycline Anonaine Arecoline

Feeding deterrent in Choristoneura Toxic to Oncopelrus Feeding deterrent in Choristoneura Feeding deterrent in bees Insecticidal for bees Feeding deterrent in Locusta Feeding deterrent in Locusta Feeding deterrent in Choristoneura Feeding deterrent in Locusta Toxic to Oncopeltus Feeding deterrent in Choristoneura Deterrent in Locusra Feeding deterrent in Choristoneura Feeding deterrent to polyphagous Syntomis larvae Insecticidal to Leptinotarsa Toxic to Locusta, Pieris, Musca Insecticidal Feeding deterrent to polyphagous Syntomis larvae Insecticidal Insecticidal Feeding deterrent in Phormia Feeding deterrent to polyphagous Syntomis larvae

1.6 mM

1.6 mM 0.09% 0.1% 0.001% dry wt 0.05% dry wt 1.2 mM

0.1% dry wt

-

1.6 rnM 0.001% dry wt 1 mM 1%

-

0.1%

-

10 mM 0.1%

198 167 198 152 152 171 171 198 171 167 I98

I71 I98

32 162 213 211 32 211 194 160 32

Caffeine

Capsaicin Celastrus alkaloids

Cocculolidine Coniine

W

C ycloheximide Cyclopyazonic acid Delphinine Demethylhomol ycorine Deox yvasicine Dihydrowisanine 2,5-Dihydroxymethyl3,4-dihydroxypyrrolidine DIMBOAIMBOAb Echinacein Halostachine Isoboldine Lobeline

Feeding deterrent in Phormia Feeding deterrent in Lepidoptera, Coleoptera, Diptera Toxic for bruchids Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in bees Insecticidal for bees Feeding deterrent in Agelaius Phagorepellent in Bombyx, Lymantria Phagorepellent in Leptinotarsa Antifeedants in Pieris (Lepidoptera), Ostrina, Tribolium (Coleoptera) Insecticidal Feeding deterrent in Phormia Feeding deterrent in Agelaius Feeding deterrent to polyphagous Syntomis larvae Insecticidal in Bombyx Insecticidal to Leptinotarsa Antifeedant in Eurema Antifeedant in Aulacophora, Dysdercus. Epilachna (Coleoptera) Insecticidal in Sirophilus (Coleoptera), feeding deterrent Toxic to Callosobruchus (Coleoptera) Feeding deterrent to locusts Resistance toward Ostrinia, Sesamia (Coleoptera), Schizaphis, Metopolophiurn. Rhopalosiphon, Sirobion (Aphidoidea) Insecticidal Toxic to Oncopelfus Insecticidal to Leptinotarsa Insecticidal, deterrent in Spodoptera Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in bees

2.5 mM 0.007-3% 1% 0.1%

0.03% 0.2% 14 mglkg -

154,160 202 158 32 152 I52 I 75 161 199 203

-

170

5 mM 71 mglkg

154 I 75

0.1%

32 207 162 169

209 204

0.03% -

-

1%

0.008%

212 212 106

211 167 162 I 70 32 152 (continued)

TABLE I (Continued)

Alkaloid Methoxy-3-alkylpyrazines Methyllycaconitine Muscirnol Nicotine

Nornicotine Pellitorine Pergularinine

Effect Evocative, alerting odor to herbivores and predators Insecticidal in Spodoptera, Heliothis, Musca Induction of food aversion in Opossum Antifeedant in larvae of Anthonomus Feeding deterrent in Locusta Insecticidal in Culex (Diptera), Spodoptera Toxic for bruchids Feeding deterrent to polyphagous Syntomis larvae Feeding deterrent in bees Insecticidal for bees Feeding deterrent in Agelaius Nernaticidal in Bursaphelenchus Feeding deterrent in Melanoplus Insecticidal Antifeedant against Spodoptera

214 200

0.02%-0.002% 0.1% 0.1%

0.03% 0.2% 50 rnglkg 1

210

153 186,I71 201 158 32 152 152 175 216 178 211 208

Pilocarpine

Pipercide Piperine Roemerine Ryanodine Spilanthol Stemofoline Stemonine Stemospironine Theobromine Tylophorinine Tripiperideine Tylophorine Vasicine Vasicinol Vasicinone Wisanine Xestoaminol A

.

Feeding deterrent in Phormia Feeding deterrent to polyphagous Synromis larvae Phagorepellent in Pieris, Bombyx Insecticidal Insecticidal in Sitophilus, feeding deterrent Feeding deterrent to polyphagous Synromis larvae Insecticidal Contact poison Insecticidal Insecticidal to Bombyx, Mamestra (Lepidoptera) Insecticidal to Bombyx, Mnrnesrra Insecticidal to Bombyx, Mamestra Toxic for bruchids Antifeedant against Spodoptera Feeding deterrent to polyphagous Synromis larvae Antifeedant against Spodoptera Antifeedant in Aulacophora, Dysdercus, Epilachna Antifeedant in Aulacophora, Dysdercus, Epilachna Antifertility effects in Dysdercus and Tribolium Antifeedant in Aulacophora, Dysdercus. Epilachna Insecticidal in Sitophilus, feeding deterrent Nematicidal in Nippostrongylus

No ED, value recorded. Hydroxamic acids (4-hydroxy-7-methoxy-I ,4-benzoxazin-3-one).

0 -

2.5 mM

I54

0.1%

32 161

0. I%

1% 8.6 pprn 0.1% 2.9 ppm

205 204 32 194 194 211 206

206 206 158

208 32

208 209 209 209 209

204 112

22

MICHAEL WINK

specialists avoid most “toxins” except those of their host plants. These data indicate that under natural conditions plants with a high content of alkaloids should be safe from most herbivorous insects, with the exception of particular monophagous species or a few very potent polyphagous ones. If insects have no choice or if they are very hungry, the deterrency threshold value is much reduced, and they often feed on a diet with alkaloids that they would normally avoid (15,32). In this case we have the chance to test the toxicity of an ingested alkaloid. If insects do not take up alkaloid-containing food, alkaloid toxicity can be assessed to some degree by topical application or by injection (Table I). As can be seen from Table I a substantial number of alkaloids display significant insect toxicity, including nicotine, piperine, lupine alkaloids, caffeine, gramine, strychnine, berberine, ephedrine, and steroidal alkaloids. Only the specialists can tolerate the respective alkaloids. The tobacco hornworm (Manduca sexta), for example, can grow on a diet with more than 1% nicotine without any adverse effects. Most of the nicotine is either degraded or directly eliminated via the Malpighian tubules and in feces (182). Because nicotine binds to the acetylcholine (ACH) receptor, it is likely that in Manduca this receptor has been modified in such a way that ACH can still bind, but not nicotine (so-called target site modification). The toxic effects of alkaloids in insects (Table I) can be caused by their interference with diverse cellular and intracellular targets. Since most mechanisms have not yet been elucidated for insects, this issue is discussed below in the section on vertebrate toxicity (see Table IV). With some caution we can extrapolate to insect toxicity. 2. Vertebrates

Because Homo sapiens and domestic animals are largely herbivores, a voluminous body of information on the adverse effects of secondary metabolites has accumulated over the centuries. Many allelochemicals and alkaloids are feeding deterrents for vertebrates, owing to their bitter or pungent taste or bad smell, and instinctively a foul-smelling, bitter, or pungent diet is normally avoided. Examples of bitter alkaloids (at least for man) are quinine, strychnine, brucine, and sparteine, and for pungent alkaloids are capsaicin, and piperine. It should be recalled that these taste properties are not identical for all animals. For example, geese, which are obligate herbivores, hardly avoid food with alkaloids or smelly compounds (amines, mercaptoethanol) that man would hardly touch (185). Conversely, fragrances that are attractive to us are highly repellent to geese (185). Even within a given population taste can differ significantly. It has been observed that a substantial proportion of Homo sapiens cannot detect

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

23

the smell of HCN, whereas others are highly sensitive. Furthermore, olfactory sensitivity can differ with age, sex, and hormonal cycles. Bitterness varies with the chemical structure of an alkaloid. With the quinolizidine alkaloids (QAs) the following scale was assessed for man: Mean detection levels are 0.00085% for sparteine, 0.0021% for lupanine, and 0.017% for hydroxylupanine (503).Whereas we know a few parameters of olfactory qualities in Homo sapiens, often much less or hardly anything is known for most other vertebrates. Alkaloids are famous for their toxic properties in vertebrates, and plants that produce alkaloids are often classified by man as poisonous or toxic plants. For a number of alkaloids the respective LD,, values have been determined with laboratory animals, especially mice, but also rats, guinea pigs, cats, rabbits, dogs, or pigeons. Table I1 presents an overview for 132 alkaloids, including the very poisonous alkaloids aconitine, coniine, atropine, brucine, curarine, ergocornine, physostigmine, strychnine, colchicine, germerine, veratridine, cytisine, delphinidine, and nicotine. Toxicity is usually highest if the alkaloids are applied parenterally [intravenously (i.v.), intraperitoneally (i.p.), and subcutaneously (s.c.)] as compared to oral application [per 0s (P.o.)]. Also, some of the alkaloids which are made or stored by animals are strong vertebrate poisons, including batrachotoxin, batrachotoxinin A, anabasine, glomerine, maitotoxin, nereistoxin, palytoxin, saxitoxin, and tetrodotoxin (1,28,29,259).Although the general toxicity of alkaloids differs from species to species, the data in Table I1 generally show that many alkaloids are more or less toxic to vertebrates. 3. Mode of Action of Alkaloids in Animals

The toxic effects observed with intact animals has its counterpart in the cytotoxic effect, which has been recorded for nearly 180 alkaloids (Table 111). These data have been obtained by screening many natural products for anticancer activity. However, an alkaloid that can kill a cancer cell is usually also toxic for “normal” cells. Therefore, the data shown in Table I11 are another indication of the general toxicity of alkaloids toward animals. Because this toxicity applies also for herbivores, the production of alkaloids by plants can certainly be interpreted as a potent antiherbivore mechanism. For a number of alkaloids the mechanisms underlying the toxic effects have already been elucidated in some detail. We can distinguish molecular targets and processes that are important for all cells, such as synthesis of DNA, RNA, and proteins, replication, transcription, translation, membrane assembly and stability, electron chains, or metabolically important enzymes or proteins including receptors, hormones, and signal compounds (Table IV). In the following we discuss some of these toxic effects.

TABLE I1 TOXICITY OF ALKALOIDS I N VERTEBRATES Alkaloid Alkaloids derived from tryptophan Annomontine Aspidospermine Brucine Cinchonidine

Test System Mouse Mouse Rat Rat Agelaius

N P

Cinchonine Curarine Ellipticine Ergocornine Ergocryptine Ergometrine Ergotamine Harman Harmine Methoxyannomontine Phy sostigmine Psilocybin Quinidine Quinine Reserpine Roquefortine A Roquefortine C

Rat

-

Mouse Rabbit Rabbit Mouse Mouse Rat Rabbit Mouse Mouse Mouse Mouse Mouse Rat Rabbit Rat Agelaius Agelaius

Mouse Mouse

LD LDSop.0. >lo00 rng/kg LD, i.p. 40 mg/kg LD, p.0. 1 mg/kg LD, i.p. 206 mg/kg LD, p.0. 100 mg/kg LD, i.p. 152 mg/kg LDlm i.p. 0.34 mg/kg LD, i.v. 19-22 rng/kg, p.0. 178-204 rng/kg LD, i.v. 1.2 mg/kg LDso i.v. 1 . 1 mg/kg LD, i.v. 0.15 mglkg LD, i.v. 62 rng/kg LD, i.v. 80 mg/kg LD, i.v. 3.5 mg/kg LD, i.p. 50 mg/kg LD, i.v. 38 mg/kg LD, i.p. 30-100 mg/kg, p.0. >lo00 mglkg LD, p.0. 4.5 mg/kg LD, i.v. 285 mg/kg LD, i.v. 280 mg/kg LD, i.v. 12.5 mg/kg LD, i.v. 30 mglkg, p.0. 263 mg/kg LD, p.0. 100 mg/kg LD, p.0. 100 mg/kg LD, i.p. 340 mg/kg LD, i.p. 169-184 mg/kg

Ref. 25 7 149 149 149 175 149 258 149 149 149 259 149 149 259 149 149 257 149 149 149 149 149 I 75 I 75 259 259

Strychnine

Toxiferine Vinblastine Vincamine Vincristine Alkaloids derived from phenylalanine and tyrosine Aristolochic acid

VI N

Agelaius

Starling Rat Dog Mouse Mouse Mouse

Mouse

Berberine Bulbocapnine Canadine

Mouse Mouse Mouse

Chelerythrine C helidonine Codeine Colchiceine Colchicine

Galanthamine

Mouse Mouse Mouse Mouse Mouse Rat Man Agelaius Starling Mouse Rat Mouse Mouse

Glaucine

Mouse

Corydaline Emetine

LD, LD, LD, LD,, LD,, LD, LD, LD,

p.0. 6 mg/kg

i75

p.0. 6 mg/kg 175 i.v. 0.9 mg/kg 149 p.0. 0.3-1.2 mg/kg, S.C. 0.003-0.02 mg/kg 259 i.p. 0.03 mg/kg 258 i.v. 9.5 mglkg 149 i.v. 75 mglkg, p.0. lo00 mg/kg 149 i.p. 5.2 mg/kg 149

LD, i.v. 38(m)-70(f) mg/kg, p.0. 56(m)-106(f) mg/kg LD5o i.p. 23 mg/kg LD, p.0. 413 mg/kg LDm p.0. 940 mg/kg, S.C. 790 mglkg, i.v. 100 mg/kg LDlmS.C. 95 mg/kg LD, i.v. 35 mg/kg LD, S.C. 300 mglkg LD5o i.p. 84 mg/kg LD, i.v. 4.1 mg/kg LD, i.v. 1.6 mglkg LDlWp.0. 0.1-0.3 mg/kg LD, p.0. 32 mglkg LD, p.0. 21 mglkg LD, i.v. 135 mg/kg LD, i.v. 12.1 mg/kg LD, S.C. 32 mg/kg LD50 i.v. 8 mg/kg, p.0. 18.7 mg/kg, S.C. 1 1 . 1 mg/kg LDS0i.v. 98 mg/kg, p.0. 401 mg/kg

149 149 259 149 259 149 149 149 149 149 259 175 17.5 149 149 149 149 149

(continued)

TABLE I1 (Continued) Alkaloid

I sot hebaine Mescaline Morphine Nuciferine Papaverine Protopine Sanguinarine oI N

Tazettine Tetrahydropalmatine Thebaine

Tubocurarine Steroidal alkaloids Batrachotoxin (frog) Batrachotoxinin A Chaconine Germerine Jervine Protoveratrine Rubijervine

Test System

LD

Mouse Ratlmouse Mouse Rat Mouse Mouse Rat Mouse Mouse Mouse Mouse Frog Rabbit Rabbit Mouse Rat

LD, LD, LD, LD, LD, LD, LD,, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD,

Mouse Man Mouse Rat Rat Mouse Rabbit Rat

LD, S.C. 2 /&kg Lethal dose 200 pg LD, S.C. 1 mg/kg LD, i.p. 84 mg/kg LD, S.C. 3.7 mglkg LD, i.v. 9.3 rng/kg Lethal dose 0.1 mglkg LD, i.v. 70 mg/kg

Mouse Agelaius

i.p. 26 mg/kg p.0. 100 mg/kg i.v. 226-318 mg/kg p.0. 240-280 mg/kg i.v. 27.5 mg/kg, S.C. 150 mg/kg i.v. 20 mg/kg, S.C. 370 mg/kg 100 mg/kg i.p. 36-102 mg/kg i.v. 29 mg/kg, p.0. 1658 mg/kg S.C. 102 mg/kg, i.v. 16 mg/kg i.v. 100 mg/kg, i.p. 420 mg/kg i.p. 111 mg/kg i.p. 20 mg/kg i.p. 50 mg/kg i.p. 3-4 mglkg S.C. 14 mglkg p.0. 33.2 mg/kg p.0. 21.8 mg/kg

Ref. 260 I 75 149 260 149 149 259 260 149 149 259 260 259 260 260 149 149 149 149 259 149 259 259 149 259 149

Samandarine Solanine

Tomatidine Tomatine Veratridine Tropane alkaloids Apoatropine Atropine Cocaine -4 N

Pyrrolizidine alkaloids 7-Angeloylheliotridine Echimidine Echinatine Europine Heliotrine Heliotrine N-oxide Jacobine Lasiocarpine Monocrotaline Retronecine Retrosine Retrorsine N-oxide

Frog Mouse Rabbit Hens’ eggs Monkey Rat Mouse Rabbit Agelaius Rat Mouse

LD,, 19 mglkg LDIM3.4 mglkg LD,, 1 mg/kg LD,, 0.3-1.5 mg/egg L D I Mi.p. 40 LD, i.p. 67 mg/kg, p.0. 590 mg/kg LDN i.p. 42 mg/kg Lethal dose 20-30 mg/kg i.p. LDN p.0. 100 mg/kg LD, p.0. 900-1OOO mg/kg LD, i.p. 1.4 mg/kg

263 263 263 26 I 262 262 259 259 I 75 149 149

Mouse Rat Man Rat Man

LD, p.0. 160 mg/kg, i.p. 14.1 mg/kg LD, p.0. 750 mg/kg Paralytic dose >I0 mg LD, i.v. 17.5 mglkg Lethal dose >30 mg i.v.

149 149 259 149 259

Rat Rat Rat Rat Rat Rat Rat Rat Rat Mouse Rat Rat

LD, i.p. 260 mg/kg LD, i.p. 200 mg/kg LD, i.p. 350 mglkg LD, p.0. 1OOO mglkg LD, i.p. 300 mg/kg LD, i.p. 2500(f)-5OOO(m) mg/kg LDSoi.p. 138 mg/kg LD, i.p. 260 mglkg LDN i.p. 175 mg/kg, p.0. 71 mglkg LD, i.v. 634 mg/kg LD, i.p. 30-150 mg/kg LD, p.0. 250 mg/kg, i.p. 48 mg/kg

259 259 259 264 259 259 259 259 149,259,265 149 265 265 (continued)

TABLE I1 (Continued) Alkaloid Senecionine Seneciph ylline Supinine Quinolizidine alkaloids Cytisine

Test System Rat Mouse Rat Rat

LD, LD, LD, LD,

Cat Dog Goat Mouse

LDlw S.C. 3 mg/kg LD,, S.C. 4 mg/kg LD,, S.C. 109 mg/kg LD, i.v. 1.7 mg/kg, i.p. 9.3 mg/kg, p.0. 101 mg/kg LD, i.p. 200-400 mg/kg LD,, i.p. 228 mg/kg, S.C. 456 mg/kg LD, i.p. 199 mg/kg LD, i.p. 172 mg/kg LD,, i.p. 28-30 mglkg LD,, i.p. 22-25 mg/kg LD, i.p. 80 mg/kg LD, i.p. 180-192 mg/kg LD, i.p. 210 mg/kg LD,, i.p. 175 mg/kg, p.0. 410 mg/kg LD5, i.p. 150 mg/kg LD, i.p. 750 mg/kg, i.v. I50 mg/kg LD, i.v. 21 mg/kg, i.p. 51 mg/kg LD, i.v. 29 mg/kg LD, i.p. 177 mg/kg, p.0. 1464 mg/kg LD, i.p. 690 mg/kg

m N

Ep iIup in ine 13-H ydroxylupanine

Lupinine Lupanine

Matrine Matrine N-oxide N-Methylycytisine Nupharidine 17-Oxolupanine

LD

Rat Guinea Rat Mouse Guinea Guinea Mouse Rat Guinea Mouse Mouse Mouse Mouse Mouse Rat Mouse

pig pig pig

pig

50 mg/kg, i.p. 85 mg/kg i.v. 64 mg/kg i.p. 77 mg/kg i.p. 450 mg/kg

Ref. 259 149 259 259 278 278 278 149 275 268 2 75 2 76 268 268 273 273 273 274 31 I 31 I 149 259 275 277

Sparteine

Guinea pig Rat Mouse Rabbit Rabbit Dog Pigeon

Miscellaneous alkaloids Aconitine

Actinobolin t 4 W

Adenine a-Amanitin P-Amanitin Anabaseine Antimycin A Arecoline Benzoylaconitine 2,3’-Bipyridyl Caffeine

Calcimycin

Mouse Rat Cat Man Mouse Rat Rat Mouse Mouse Mouse Mouse Mouse Dog Rat Mouse Agelaius Mouse Hamster Rabbit Rat Mouse

LDIMi.p. 23-30 mglkg LD, i.p. 42-44 mglkg, S.C. 68-75 mg/kg LD, i.p. 55(m)-67(f) mg/kg, i.v. 17(m)-20(f) mglkg, p.0. 350(m)-510(f) mglkg LD,, p.0. 450 mg/kg Lethal dose i.v. 20-30 mglkg Lethal dose i.v. 50-70 mglkg Lethal dose i.v. 40-50 mglkg LD, i.v. 0.166 mg/kg, i.p. 0.328 mg/kg, p.0. - 1 mg/kg LD, i.v. 0.08-0.14 mglkg LD, i.v. 0.07-0.13 mg/kg Lethal dose p.0. 1.5-5 mg LD, i.v. 800 mglkg LD, i.v. I550 mglkg LD, p.0. 745 mg/kg LD, i.p. 0.1 mg/kg LD, i.p. 0.4 mg/kg LD, i.v. 84 pg/kg LD, i.p. 1.8 mg/kg, S.C. 1.6 mg/kg LD, S.C. 100 mglkg LD, S.C. 5 mg/kg LD, i.v. 27 mg/kg LDWi.v. 3500 pg/kg LD, i.p. 316 mglkg LD, p.0. 127(m)-137(f) mglkg LD, p.0. 230(m)-249(f) mg/kg LD, p.0. 246(m)-224(f) mg/kg LD, p.0. 200 mg/kg LDw .. i.p. . 10 mg/kg - -

268

269 270 271 272 272 272 149 259 259 259 149 149 149 149 149 230 149 149 149 259 230 149 149 149 149 259 149 (continued)

TABLE I1 (Continued) Alkaloid

Test System

LD

Ref. ____

s

Carubicin Carzinophilin Coniine Cycloheximide Damascenine Daunorubicin Delphinine

Epinephrine (adrenaline) Glomerine H ypaconitine Lappaconitine Lycoctonine Maitotoxin (algaelfish) Maytansine Mesaconitine

Mouse Mouse Agelaius

Guinea pig Mouse Mouse Mouse Frog Rabbit Mouse Mouse Mouse Mouse Mouse Mouse Rat Mouse

LD, LD, LD, LD,, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD,, LD, LD,

p.0. 7.3 mglkg, i.v. 1.3 mglkg i.v. 150 pglkg p.0. 56 mglkg p.0. 150 mglkg, S.C. 40 mg/kg i.v. 150 mg/kg p.0. 1800 mglkg i.v. 26 mg/kg i.p. 0.05-0.1 mglkg i.p. 1.5-3.0 mg/kg i.p. 4 mglkg p.0. 17-34 mg/kg S.C. 1.2 mglkg i.v. 6.9 mg/kg, p.0. 20 mglkg i.p. 350 mglkg i.p. 0.17 pg/kg S.C. 0.48 mg/kg S.C. 0.2 mglkg

149 149 175 259 149 149 149 267 267 149 259 259 149 267 259 149 259

Methyl-lycaconitine Mitomycin Muscimol Nemertilline Nereistoxin Nicotine

Nornicotine

W

Frog Mouse Mouse Rat Mouse Mouse Agelaius Starling Mouse

Ochratoxin Palytoxin Pellertierine Ricinine Saxitoxin

Rat Rabbit Rat Mouse Rabbit Agelaius Mouse

Tetrodotoxin

Guinea pig Mouse

Theobromine

Rat

LD, i.p. 3.0-3.5 mglkg LD, i.p. 18 mglkg LD, i.v. 5-10 mglkg LD, p.0. 45 mg/kg LD, i.v. 500 pg/kg LDlw S.C. 38 &kg LD, p.0. 17.8 mglkg LD, p.0. 42 mg/kg LD, i.v. 0.3 mglkg, i.p. 9.5 mglkg, p.0. 230 mg/kg LD, i.p. 23.5 mglkg LD, i.v. 3 mg/kg LD, p.0. 20-22 mglkg LD, i.v. 0.45 pglkg, i.p. 0.05-0.15 pg/kg LD, i.v. 40 mglkg LD, p.0. 42 mg/kg LD, i.p. 10 p g / k g , i.v. 3.4 mg/kg, p.0. 263 mglkg LD, P.O. 135 pglkg LD, i.p. 10 pglkg, S.C. 8 p g / k g , p.0. 0.3 mg/kg LD, p.0. 950 mg/kg

267 267 149 266 230

221 175 175 149 149 149 149 149 149 259 149 259 149,259 259

TABLE I11 CYTOTOXIC ACTIVITY OF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Annomontine Apparicine Bisnordihydrotoxiferine Boldine Brevicolline Camptothecine

W h)

Canthin-Gone Cinchonidine Cinchonine Conoduramine Conodurine Coronoaridine Ellipticine 1GEpi-(Z)-isositsirikine 9-Epivoacarine Gabunamine Gabunine Harmaline Harman Harmine Harmol 20-H ydrox yvoacamidine

Isovoacangine Leurosidine Methoxyannomontine

Effect Antiamebic Cytotoxic to P388 cells Inhibition of sarcoma 180 Inhibition of human epidermoid carcinoma of larynx Photogenotoxic in CHO cells Antitumor properties, L1210 Walker sarcoma Cytotoxic to KB and P388 cells Photogenotoxic in CHO cells Growth inhibition of Plasmodium falciparum Growth inhibition of Plasmodium falciparum Inhibition of P388 leukemia cells Inhibition of P388 leukemia cells Cytotoxic to P388 cells Antitumor agent in L1210 cells Antineoplastic to KB and P388 cells Cytotoxic to P388 cells Inhibition of P388 leukemia cells Inhibition of P388 leukemia cells Growth inhibition of Trypanosoma cruzi Photogenotoxic to CHO cells Growth inhibition of Trypanosoma cruzi Growth inhibition of Trypanosoma cruzi Growth inhibition of Trypanosoma cruzi Antineoplastic Inhibition of P388 leukemia cells Antitumor activity Antiamebic

ED54 50 pg/ml

18 mg/ kg 0.17-0.53 pg/ml

200 ng/ml 27-130 ng/ml 20 pg/ml 26 pg/ml 0.43pglml

-

1.2 p g h l I .7 pg/ml 1.3 pg/ml 3.2 pg/ml

18 pglrnl

Ref. 257 283 284 285 57 286 283 57 287 287 281 281 283 288 280 281 281 281 289 57 289 289 289 282 281 282

2v

Antitumor activity in L1210, P388 Inhibition of Eagle carcinoma of nasopharynx 9-Methox yellipticine Cytotoxic Growth inhibition of Trypanosoma cruzi and Crirhidia Olivacine Tumor inhibition in L1210 cells Cytotoxic to KB cells Inhibition of P388 leukemia cells Pericyclivine Inhibition of P388 leukemia cells Perivine Relefolonium Inhibition of animal/human cells Growth inhibition of Plasmodium falciparum Quinidine Growth inhibition of Plasmodium berghei Quinine Growth inhibition of Trypanosoma cruzi Growth inhibition of Plasmodium falciparum Cytotoxic to Walker 256 carcinosarcoma Reserpine Inhibition of P388 leukemia cells Tabernamine Cytotoxic to P388 cells Tubotaiwine N4-oxide Tubulosine Inhibition of leukemia and carcinoma cells Amebicidal Cytotoxic to KB and P388 cells Vallesiachotamine Growth inhibition of Trypanosoma cruzi Vinblastine Antitumor activity in Hodgkin’s disease, testicular cancer Antitumor activity in childhood leukemia, Wilm’s Vincristine tumour, lymphomas Vinleurosine Antitumor activity Vinrosidine Antitumor activity Voacamine Cytotoxic to P388 cells Alkaloids derived from phenylalanine/tyrosine Antioquine Growth inhibition of Leishmania Aristolochic acid Antitumor activity Cytotoxic to KB cells Armepavine N-oxide 9-Methox ycamptothecine

1-Methoxycanthin-Gone

W

0.4 pg/ml 13 pg/ml 20 pg/ml 10 pM

22-80 nglml 50 mg/kg

-

45-280 ng/ml

-

2.1 pg/ml 1.8 pg/ml 0.01-0.oooO1 pg/ml

-

1.1-3.5 pg/ml

283 279 282 290 291 292 281 281 256 287 293 289 287 282 281 281 174 304 280 289 286 286

2.6 pg/ml

282 282 281 294 282 295 (continued)

TABLE I11 (Continued) Alkaloid

Effect

Ref.

~

Berbamine Berberine

W P

Berbermbine Capnoidine Chelerythrine Chelidonine Chondrodendrine Cissamparein Claviculine Cocsuline Colchicine Coptisine Coralyne Corpaine Corydine Curin Cycleacurine Cycleadrine Cycleanine Cycleanorine Cycleapeltine Daphnandrine Dehydroemetine Demecolcine Dicentrine N-oxide

Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Inhibition of Plasmodium fakiparum Cytotoxic properties Antitumoral Growth inhibition of Trypanosoma brucei Antitumor activity Cytotoxic Growth inhibition of Leishmania Active against nasopharyngal carcinoma Growth inhibition of Plasmodium berghei Growth inhibition of Leishmania Cytotoxic activities Cytotoxic activity Antileukemic to L1210, P388 cells Antitumor Growth inhibition of Trypanosoma brucei Cytotoxic activity Active against nasopharyngal carcinoma Cancerostatic Cancerostatic Growth inhibition of Leishmnnia Cancerostatic Cancerostatic Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Low anticancer activity Cytotoxic activity Cytotoxic to KB cells

294 289 2% 282 297 293 298 282 299 282 293 299 282 297 300 297 301 282 282 282 282 299 282 282 299 299 302 282 295

Emetine Fagaronine Fangchinoline Glaziovine Gyrocarpine Isochondrodendrine Isocorypalmine Jatrorrhizine Krukovine Limacine Liriodenine Lycorine 0-Methylatheroline Nitidine Obaberine Oxodicentrine Oxoglaucine 0x0-0-methylbulbocapnine Oxopurpureine Oxoxylopine Palmatine Penduline Pheantine Protopine Pseudo1ycorine

Growth inhibition of Trypanosoma cruzi Weak anticancer activity Cytotoxic to KB cells, leukemia L1210, P388 cells Active against nasopharyngal carcinoma Cytotoxic Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Active against nasopharyngal carcinoma Antitumoral Inhibition of Plasmodium falciparum Growth inhibition of Leishmania Growth inhibition of Leishmania Active against nasopharyngal tumors Cytotoxic to A-549, HCT-8, KB, P388 cells Toxic to Rauscher virus NIH13T3 cells Cytotoxic Antileukemic to mouse, L1210, P388 cells Antitrypanosomal Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Cytotoxic to A-549, HCT-8, P388 cells Cytotoxic to HCT-8, KB cells Cytotoxic to A-549, HCT-8 cells Cytotoxic Cytotoxic to A-549, HCT-8, KB, P388 cells Antitumoral Inhibition of Plasmodium falciparum Cytostatic Growth inhibition of Leishmania Cytotoxic Toxic to Rauscher virus NIH/3T3 cells

289 303 88,286 282 282 299 294 282 297 2% 299 299 282 295 147 282 300

-

1.O pglml

299 294 295 295 295 282 295 297 2% 282 299 282 147 (continued)

TABLE 111 (Continued) Alkaloid Sanguinarine Tetrandrine Thalfoeditine Thalicarpine (=thaliblastine) Thalidasine Xylopine Acndone alkaloids Acronycine Atalaphillidine Atalaphillinine W

QI

Citpressine I Citracidone I Citrusinine I Dercitine (sponge)

Des-N-methylnoracronycine Dimethoxyacronycine Glandisine Glycobismine A Glycocitrine I Glyfoline Grandisine

5-Hydroxy-N-methylseverifoline 5-H ydroxynoracronycine

Effect

ED%

Ref.

Antitumor activity Active against Walker carcinoma cells Active against carcinoma 256 in rats Antileukemic to Walker S, TLX-5 cells Active against carcinosarcoma 256 in rats Cytotoxic to A-549, HCT-8, KB, P388 cells

298 286,305 282 306 282 295

Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Active against P388, HCT-8 cells Growth inhibition of Plasmodium yoelii Active against some leukemia L1210 cells Growth inhibition of Plasmodium yoelii Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelli Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii

145 307 145 307 145 307 145 145 145 144 307 145 307 307 145 307 I45 I45 307 145 145 307

10 Fg/ml 10 pglml 10 pg/ml

-

10 pg/ml 10 pg/ml

10 pg/ml

Melicopine 5-Methox yacronycine N-Methylatalaphilline

1.3-0-Methyl-N-methylacridone Normelicopidine Steroidal alkaloids Solamargine &Solamarine Solasodine Solasoninelsolamargine Pyrrolizidine alkaloids Echinatine-N-oxide Europine N-oxide Fulvine Heliotrine Heliotrine N-oxide Indicine N-oxide Lasiocarpine Monocrotaline Senecionine Senecionine N-oxide Spectabiline Supinine Quinolizidine alkaloids Matrine Oxymatrine Miscellaneous alkaloids Arecoline

Antitumor activity Active against mouse leukemia LIZ10 cells Growth inhibition of Plasmodium yorlii Active against mouse leukemia L1210 cells Growth inhibition of Plasmxfirrm yorlii Growth inhibition of Plasniodirtm yoelii Antitumor activity

282 145

Cytotoxic to PLC. PRF cells Antitumor activity Cytotoxic to PLC, PRF cells Inhibition of skin cancer

310

Active against P388 mouse leukemia Active against P388 mouse leukemia Antitumor activity Antitumor activity Antitumor activity Active against P388 mouse leukemia Antitumor activity Antileukemic effects Antitumor activity Antitumor activity Antitumor activity Antitumor activity

311 31 I

31 I

Antitumor activity in Ehrlich ascites tumor Antitumor activity in mouse sarcoma 180 Antitumor activity in mouse sarcoma 180

31 I 31 I 311

Growth inhibition of Tryprrnosornu crrczi .. Inhibition of intestinal cestodes and nematodes

312

145 145

307 307 282

282

310 309

282 282 282 282 286 282 282 282 282

289

(continued)

TABLE I11 (Continued) W

00

Alkaloid Aristolactam Atropine Cephalomannine Crinamine Cryptopleurine Demethyltylophorinine Deoxyhaningtonine Didemnins trans-Dihydronarciclasine Diplamine Ecteinascidins (tunicate) Emarginatine B Febrifugine Haemanthamine Haningtonine

Effect Antitumoral in lung cells, colon tumors Growth inhibition of Trypanosoma cruzi Antileukemic agent Active against KB cells Toxic to Rauscher virus NIHl3T3 cells Active against KB carcinoma cells Antitumor activity Active against lymphocytic leukemia Antitumor activity in L1210 cells Active against P388 mouse leukemia Cytotoxic toward L1210 leukemia cells Active in P388 mouse leukemia, L1210 cells Cytotoxic in KB cells Antitumor activity Toxic to Rauscher virus NIHl3T3 cells Active against lymphocytic leukemia

EDs

Ref.

-

313 289 314 315 147 133 282 316.317 109 323 189 109 318 282 147 316,317

-

0.38 pglml 0.2 pglml

0.01-0.005 pglml 0.003 pglml 0.002 pglml 0.0001-0.08 pglml 0.4 pglml

-

0.2 pglml

-

'

Homohaningtonine 6-Hydrox ycrinamine

Isohaningtonine Jatropham Maytansine Narciclasine Odorinol F’ancratistatin Patellamid A (tunicate) Pilocarpine F’recriwelline Pretazettine

W

Sesbanimide Solapalmitenine Solapalmitine Tylocrepine Tylophorine Ungeremine

Active against lymphocytic leukemia Toxic to Rauscher virus NIH/3T3 cells Active against lymphocytic leukemia Active in P388 mouse leukemia Antileukemic agent Toxic to Rauscher virus NIH/3T3 cells Antileukemic agent Antineoplastic Antileukemic agent Antitumor activity Toxic to Rauscher virus NIH/3T3 cells Antileukemic agent Toxic to Rauscher virus NIH/3T3 cells Antileukemic agent Antitumor activity Antitumor activity Antitumor activity Antitumor activity Cytotoxic to S180 tumor cells

0.2 pg/ml 0.005 pg/ml

-

2-4 pglml

-

0.05 pg/ml

0.05 pg/ml

-

316,317 147 316,317 282 319 147 320 321 320 282 147 322 147 320 282 282 282 282 114

TABLE IV MOLECULAR TARGETS OF ALKALOIDS: PROTEINS, NUCLEIC ACIDS,BIOMEMBRANES, A N D ELECTRON CHAINS Alkaloid Indole and quinoline alkaloids Acronycine Anonaine BoIdine Brucine

Camptothecine P-Carboline-1-propionicacid Dictamnine Ellipticine

g Ergot alkaloids Ervatamine Eseridine Eserine (physostigmine) 1-Ethyl-P-carboline Gelsemine Gramine Harmaline Harman Harmine Harmol Isoboldine

Effect

Ref.

Inhibition of nucleoside transport Inhibition of adenylate cyclase Quenching of singlet oxygen Quenching of singlet oxygen Inhibition of muscle lactate dehydrogenase Binding to glycine receptor Inhibition of 45 S rRNA transcription Inhibition of cAMP phosphodiesterase Monofunctional photoaddition to DNA Intercalation with DNA Inhibition of mitochondria1 respiration Inhibition of cytochrome c oxidase, interaction with phospholipids Interaction with dopamine, serotonin, and norepinephrine receptors Inhibition of Na+ channels Cholinergic Inhibition of acetylcholinesterase Inhibition of cAMP phosphodiesterase Modulation of glycine neurochemical activity Uncoupling of photophosphorylation Inhibition of Na+,K+-ATPase, Na+ transport, and monoamine oxidase A Interaction with insect synapses Binding to DNA Inhibition of monoamine oxidase Interaction with insect synapses Binding to DNA Interaction with insect synapses Inhibition of adenylate cyclase

360 361 362 362 363 364 365,366 357 367 368 369 358 370,371 372 149 259,373 357 364 374 375,376 377 166 376 377 378 377 36 I

Melinone F 9-Methoxyellipticine Norharman Normelinone F Pseudanel pseudene Quinine

Reserpine Serotonin Skimmianine Strychnine

P Vincristine Tetrahydro-/3-carboline Toxiferine Tryptamine Tubocurarine Vinblastine Vincamine Yohimbine

Binding to DNA Inhibition of cytochrome c oxidase, interaction with phospholipids DNA intercalation Binding to DNA Binding to DNA Inhibition of mitochondria1 electron transport Intercalation with DNA Modulation of ion channels Inhibition of glucose response in chemosensory cells Quenching of singlet oxygen Inhibition of noradrenaline transport Interaction with endogenous neurotransmitter, inhibition of pyridoxal kinase, aromatic amino acid decarboxylase, histamine methyltransferase Intercalation in DNA, photoaddition Binding to glycine receptor Quenching of singlet oxygen Inhibition of muscle lactate dehydrogenase Binding and dimerization of tubulin Inhibition of protein biosynthesis and DNA-dependent RNA polymerase Inhibition of intracellular transport Inhibition of biogenic amine uptake Inhibition of monoamine oxidase Binding to acetylcholine receptor Inhibition of pyridoxal kinase, tyrosine-tRNA ligase Binding to acetylcholine receptor Binding and dimerization of tubulin Inhibition of protein biosynthesis and DNA-dependent RNA polymerase Inhibition of intracellular transport Quenching of singlet oxygen Adrenergic blocking agent

379 358 359 166 379 380 381 382 383 362 312 221,376 57 364 362 363 384-386 387 388 389 389 390 376 391 384-386 387 388 36 1 312 (continued)

TABLE IV (Continued) Alkaloid Alkaloids derived from phenylalanine/tyrosine Alpinigenin Avicine Berbamine Berberine

N P

Bicuculline Bulbocapnine Canadine Cepharanthine Chelerythrine Chelidonine Chelilutine Colchicine

Columbamine Coptisine

Effect

Ref.

Inhibition of mitochondria1 respiratory chain Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Interaction with plasma membranes Inhibition of reverse transcriptase Intercalation with DNA Inhibition of aldose reductase Inhibition of acetylcholinesterase, alcohol dehydrogenase, aldehyde reductase, diamine oxidase, tyrosine decarboxylase, RNA synthesis Modulation of GABA neurochemical activity Inhibition of peripheral dopamine receptors Inhibition of aldose reductase Interaction with plasma membranes Intercalation with DNA Inhibition of reverse transcriptase, alanine and aspartate aminotransferases Inhibition of reverse transcriptase Inhibition of microsomal monooxygenase Inhibition of DNA polymerase Depolarization of microtubules, inhibition of urate-ribonucleotide phosphorylase Binding to tubulin, inhibition of microtubule polymerization Inhibition of intracellular transport Inhibition of RNA synthesis Inhibition of butrylcholinesterase Intercalation with DNA Inhibition of acetylcholinesterase, alcohol dehydrogenase

392 393 393 394 395 3%-398 399 297 364 149 399 394 400 259,401 401 402 393 376,441,442 384,448 388 12 297 3% 297

Coralyne

Corlumine Corysamine Demethylpapaverine Dihydrochelerythrine Dihydrosanguinarine Domesticine Emetine Ephedrine Fagaronine P W

Galanthamine Glaucine Isoboldine Jatrorrhizine Laudanosine 0-Methylfagaronine 13-Methylpalmatine Nandazurine Nantenine Nitidine

Nuciferine

Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Inhibition of catechol 0-methyltransferase, alcohol dehydrogenase Inhibition of acetylcholinesterase, RNA polymerase, tRNA methyltransferase Modulation of a-aminobutryric acid (GABA) neurochemical activity Inhibition of alcohol dehydrogenase Inhibition of aldose reductase Inhibition of reverse transcriptase Inhibition of reverse transcriptase Inhibition of aldose reductase Inhibition of protein biosynthesis Modulation of noradrenaline release and noradrenaline receptors Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Inhibition of acetylcholinesterase Quenching of singlet oxygen Inhibition of aldose reductase Inhibition of butyrylcholinesterase Modulation of glycine neurochemical activity Inhibition of reverse transcriptase Inhibition of reverse transcriptase Inhibition of aldose reductase Inhibition of aldose reductase Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Inhibition of tRNA methyltransferase Inhibition of Na+, K+-ATPase Blocking of receptors for neurotransmitters (glutamate, aspartate, acetylcholine)

386 403 298 297 364 297 399 401 401 399 404 12,312 88,400 403,404 405 361 399 297 364 403 297 399 399 400 403 298 298 260 (continued)

TABLE IV (Continued) Alkaloid Palmatine Papaverine

Salsolinol Sanguinarine

Stepholidine Tetrah ydroberberine Tetrah ydroisoquinoline Tetrahydropalmatine Tetrandrine Thebaine Tubulosine Tyramine Polyhydroxy alkaloids Alexine

Effect

Ref.

Inhibition of reverse transcriptase Inhibition of aldose reductase Inhibition of acetylcholinesterase Inhibition of aldose reductase Inhibition of GABA response in chemosensory cells Inhibition of glucose response in chemosensory cells Inhibition of phosphodiesterase Inhibition of monoamine oxidase Inhibition of biogenic amine uptake Uncoupler of respiration and oxidative phosphorylation in mitochondria Inhibition of photosynthetic phosphorylation Inhibition of reverse transcriptase Inhibition of Na+, K+-ATPase Intercalation with DNA Inhibition of catecholamine uptake Inhibition of adenylate cyclase Inhibition of catechol 0-methyltransferase Inhibition of uptake of biogenic amines Inhibition of catecholamine uptake Inhibition of respiratory chain in mitochondria Inhibition of aldose reductase Interaction with plasma membrane Inhibition of acetylcholinesterase Inhibition of protein biosynthesis nhibition of tyrosine-tRNA ligase odulation of noradrenaline release

395 399 297 297 383 383 406 389 389 143 407 401 259,408 400,409 297 297 389 389 297 392 399 243 260 404 3 76 I2

Inhibition of myrosinase/glucosinate hydrolysis at 64-860 pM

212,410,4/1

L

Castanospermine

Deoxynojirimycin I -Deoxynojirimycin 1J-Dideox y- I ,5-imino-D-mannitol

2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine 6-Epicastanospermine Homonojirimycin

2

Nojirimycin Swainsonine Purine alkaloids Caffeine Theophylline Quinolizidine alkaloids Angustifoline Cytisine 13-Hydroxylupanine Lupanine Matrine

Inhibition of glucosidases Inhibition of myrosinase Inhibition of insect disaccharidases Inhibition of myrosinase/glucosinate hydrolysis Inhibition of glucosidase Inhibition of myrosinase/glucosinate hydrolysis Inhibition of a-mannosidase, trehalase Inhibition of myrosinaselglucosinate hydrolysis Inhibition of glucosidase Inhibition of trehalase, invertase Inhibition of a-glucosidase Inhibition of myrosinase/glucosinate hydrolysis Inhibition of glucosidase Inhibition of a-amylase, P-fructofuranosidase, a-glucosidase Inhibition of a-mannosidase, mannosidase I1

150 412 197 212,410,41I 150,212 212,410 212,410 212,410 150,212 212 411 212,410,411 413 376 376,414

Inhibition of cAMP phosphodiesterase, dATP(dGTP)-DNA purinetransferase Inhibition of cAMP phosphodiesterase

202,376

Inhibition of Phe-tRNA binding to ribosomes Inhibition of Phe-tRNA binding and elongation Inhibition of Phe-tRNA binding Inhibition of in uitro translation (wheat germ) Inhibition of Phe-tRNA binding to ribosomes Inhibition of in uitro translation (wheat germ) Inhibition of Phe-tRNA binding and elongation Inhibition of Phe-tRNA binding to ribosomes Inhibition of Phe-tRNA binding and elongation Inhibition of in uitro translation (wheat germ) Inhibition of neural glutamate action

417 99,422 56 56 417 56 99,422 41 7 99,422 56 420

202,415

(continued)

TABLE IV (Continued) Alkaloid 17-Oxosparteine Sparteine

13-Tigloylox ylupanine

Effect

Ref.

Inhibition of Phe-tRNA binding Inhibition of in uitro translation (wheat germ) Modulation of Kt channels Inhibition of Phe-tRNA binding to ribosomes Inhibition of GABA response in chemosensory cells Increase in insulin release in /3 cells Inhibition of aminoacyl-tRNA synthase Inhibition of Phe-tRNA binding and elongation Inhibition of in uitro translation (wheat germ) Inhibition of Phe-tRNA binding Inhibition of in uitro translation (wheat germ)

56 56 416,418 417 383 419 421 99,422 56 56 56

Alkylation of DNA and proteins Inhibition of acetylcholinesterase Modulation of pulmonary Naf/K+ pumps

425,426 424 423

Activation of Nat channels Depolarizes membranes Disruption of biomembranes by cholesterol binding Inhibition of acetylcholinesterase Inhibition of acetylcholinesterase Inhibition of acetylcholinesterase Blocking of action potential Blocking of action potential Inhibition of inactivation of Nat channels, depolarization of membranes Inhibition of cholesterol biosynthesis Disruption of biomembranes Binding of cholesterol, hemolysis Inhibition of acetylcholinesterase

427,428 234,429 430,433 431,432 432 432 234 234 234,259 434 435 435 431

Pyrrolizidine alkaloids 01 4

2.3-Deh ydropyrrolizidines

Heliotrine Monocrotaline Steroidal alkaloids Batrachotoxin (frog) Cevadine Chaconine Commersonine Demissine Isorubijervine Muldamine Protoveratrines A,B Solacongestidine Solamargine

Solanine Solanidine Solasonine Tomatine Veratramine Veratridine Tropane alkaloids Atropine

3

Cocaine Miscellaneous alkaloids Aconitine Amanitin Anabaseine Arecoline Batrachotoxin Capsaicine Cassaine Cryptopleurine Cycasin (=methylazoxymethanoI) Dendrobine DIMBOA/MBOA

Complexing with sterols, membrane disruption Inhibition of acetylcholinesterase Inhibition of GABA response in chemosensory cells Inhibition of acetylcholinesterase Synergistic with solarnargine Binding of cholesterol Inhibition of GABA response in chemosensory cells Blocking of action potential Activation of Na+ channels

430,433 432 383 432 435 435 383 234 234,427

Quenching of singlet oxygen Binding to muscarinergic acetylcholine receptor Binding/inhibition of dopamine uptake carrier

361 312 12,436

Activation of Na+ channels, no repolarization Inhibition of RNA polymerases I1 and I11 (transcription) Modulation of acetylcholine receptor Binding to acetylcholine receptor Increase of Na' permeability Inhibition of Na+,K+-ATPase,glucose transport Inhibition of mitochondria1 electron transport Inhibition of Na+,K+-ATPase Inhibition of protein biosynthesis Alkylation of DNA Modulation of glycine neurochemical activity Inhibition of energy transfer in mitochondria Inhibition of energy transfer in chloroplasts Binding to auxin receptors in plants Inhibition of ATPase Inactivation of SH groups Inactivation of amino groups

259,427 376 230 437 234,388 439 440 408 404,444 343 364 445 106 106 106 446,447 446,448 (continued)

TABLE 1V (Continued) Alkaloid Gephyrotoxin Haningtonine Homoharringtonine Hemanthamine Hippeastrine Histrionicotoxin Irehdiamine Isoharringtonine Lycorine

pm

Maitoxin Malouetine Maytansine Maytansinine Methyl1ycaconitine C15-2,bmethylpiperidine Muscarine Narciclasine Nicotine

Ochratoxin Olivacine Palytoxin Pilocarpine Pretazettine Pseudo1ycorine

Effect Inhibition of acetylcholine receptor Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of DNA polymerase Inhibition of K+ channels Disturbance of membrane permeability Inhibition of protein biosynthesis Inhibition of DNA polymerase Inhibition of protein biosynthesis, binding to 60 S subunit Activation of CaZ+channels Disturbance of membrane permeability Binding to microtubules Inhibition of cell division Cholinergic agonist (insect nicotine receptor) Inhibition of mitochondria1 electron transport Inhibition of Na',K+-ATPase Binding to acetylcholine receptor Inhibition of protein biosynthesis Activation of acetylcholine receptor Inhibition of carotenoid biosynthesis Induction of vacuole formation in Puccinia Quenching of singlet oxygen Inhibition of glucose transport Intercalation with DNA Increase of Na+/K' permeability, hemolysis Binding to muscarinic acetylcholine receptor Inhibition of protein biosynthesis Inhibition of protein biosynthesis

Ref. 428 449 390 390 148 428 390 390 148 259,390 259 390 390 450 200 228 438 312 451 200,312 452 453 361 259 454 259 259 390 390

\o P

Psilocin/psiloc ybin F’umiliotoxin B Pumiliotoxin C Saxitoxin Solenopsine Streptonigrine Taxol Tetrodotoxin Trigonelline Tylocrebrine Tylocrepine Tylophorine Xestoaminol A, C Antibiotics Actinobolin Actinomycin Amphotericin B Bacitracin Bleomycin Calcirnycin Calichem ycin Cephalosporin Cephamycin Chloramphenicol Cycloheximide Cytochalasin B Daunom bicin Demecloc yclin

Interaction with serotonin receptor (hallucinogen) Inhibition of Ca2+channels Inhibition of acetylcholine receptor Inhibition of Na+ channels Inhibition of Nat,Kt-ATPase and mitochondria1 respiratory chain Inhibition of reverse transcriptase Promotion of polymerization of tubulin, polyploidization Inhibition of Na+ channels Promotion of cell arrest in G2of cell cycle in plants Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of reverse transcriptase

312 428 428 234,259 259 455 443 259,388 456,457 390 404 444 I I2

Inhibition of protein biosynthesis Intercalation in DNA, inhibition of RNA synthesis Interaction with membrane sterols, formation of membrane channels Inhibition of dolichol metabolism, geranyltransferase DNA binding and cleavage Inhibition of DNA polymerase, RNA polymerase, protein-glutamine y-glutamyltransferase CaZt ionophore in mitochondria DNA binding and cleavage Inhibition of transpeptidase Inhibition of transpeptidase Inhibition of translation Inhibition of translation Inhibition of glucose transport, blocking of contractile rnicrofilaments Inhibition of RNA polymerase, procollagen-proline,2-oxoglutarate 4-dioxygenase, intercalation with DNA Inhibition of translation

149 437 312 376 437 376 149 437 312 312 312 312 149,376,388 312,376 312 (continued)

TABLE IV (Continued) Alkaloid Doxorubicin Erythromycin Esparamycin Gentamycin Gramicidin Josamycin Kanamycin Lincomycin Mitomycin C Neomycin Novobiocin Nystatin A Oxytetracyclin Penicillins and p-lactam derivatives Polymyxins A-E Rifampicin Rifamycin Spectinomycin Spiramycin Streptomycin Tetracyclin Tobramycin Tyrothricin Vancomycin

Effect Inhibition of RNA polymerase, intercalation into DNA Inhibition of translation DNA binding and cleavage Inhibition of translation Formation of ion channels (Na', K+,H') in plasma membrane Inhibition of translation Inhibition of translation Inhibition of translation Alkylation of DNA, inhibition of replication phosphodiesterase Inhibition of I-phosphatidylinositol-4,5-biphosphate Inhibition of translation Inhibition of DNA topoisomerase Interaction with membrane sterols, formation of membrane channels Inhibition of translation Inhibition of transpeptidase (murein formation) Inhibition of protein kinase C, increase of membrane permeability Inhibition of DNA polymerase Inhibition of RNA and DNA polymerases Inhibition of translation Inhibition of translation Inhibition of translation Inhibition of translation Inhibition of translation Modulation of membrane permeability Inhibition of peptidoglycan biosynthesis

Ref. 312,376 312 437 312 312 312 312 312 312.437 312,376 376 312 312 312 312,376 376 376 312 312 312 312 312 312 312

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

51

a. Cellular Targets Nucleic Acids. DNA, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. It is not surprising that a number of secondary metabolites have been selected during evolution which interact with DNA or DNAprocessing enzymes. Some alkaloids bind to or intercalate with DNA/RNA (Table IV) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (Table V): 9-methoxyellipticine,dictamnine, ellipticine, harmane alkaloids, melinone F, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (PAS), cycasin, olivacine, etc. Many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of AT and GC base pairs. Other alkaloids act at the level of DNA and RNA polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. Whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (Table V). Besides basic data obtained in Salmonella or Drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. Anagyrine, anabasine, and coniine cause “crooked calf disease” if pregnant cows or sheep feed on these alkaloids during the first period of gestation (329,341,348,349,351,352).The offspring born show strong malformation of the legs. Some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by Veratrum species, cause the formation of a central large cyclopean eye (329-330, an observation that was probably made by the ancient Greeks and thus led to the mythical figure of the cyclops. It is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. In effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores. Another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (Tables IV and V). The alkaloid aristolochic acid, which is produced by plants of the genus Aristolochia, is carcinogenic. The mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine (344,345),because of its NO, group. Pyrrolizidine alkaloids and their N-oxides, which are abundantly produced by members of the Asteraceae and Boraginaceae but also occur in the families Apocynaceae, Celestraceae, Elaeocarpaceae, Euphorbiaceae, Fabaceae, Orchidaceae, Poaceae, Ranunculaceae, Rhizo-

TABLE V MUTAGENIC OR CARCINOGENIC ACTIVITYOF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Vinblastine/vincristine Vaocristine Quinoline alkaloids Dictamnine

Effect Fetal malformation in hamster Skeletal, ocular, and CNS malformations in man Mutagenic in yeast

Induction of revertants in Salmonella typhimurium (ST) Frameshift induction in E. coli Induction of revertants in ST Evolitrine Induction of revertants in ST Fagarine Induction of sister-chromatid exchanges Induction of revertants in ST Flindersiamine Induction of revertants in ST Kokusaginine Maculine Induction of revertants in ST Maculosidine Induction of revertants in ST Induction of revertants in ST Pteleine Induction of revertants in ST Skimmianine Alkaloids derived from phenylalanine/tyrosine Carcinogenic, mutagenic Aristolochic acid Mutagenic in ST Berberine Mutagenic Mutagenic in Lolium Colchicine Thebaine Teratogenic in hamster, congenital malformations Steroidal alkaloids 1 I-Deoxojervine (cyclopamine) Teratogenic, cyclopian malformation Jervine Teratogenic, cyclopian malformation Solanine Teratogenic in chick embryo, rumplessness

EDXI

50-100 pg/ml 5-20 &plate

-

5-20 pg/plate 5-20 &plate

5-20 5-20 5-20 5-20 5-20 5-20

pg/plate pg/plate pg/plate pglplate pglplate &plate

Ref. 324 325 284 326 327 326 326 328 326 326 326 326 326 326 344,345 346 297 347 260 329,330 329,330 261

Solasodine Veratrosine Pyrrolizidine alkaloids 7-Acet ylintermedine 7-Acety llycopsamine

Heliotrine Indicine Integenimine Intermedine Jacoline Lasiocarpine Lycopsamine Monocrotaline W 111

Retrorsine Senecionine Seneciphylline Senkirkine

Symphytine PAS general Quinolizidine alkaloids Anagyrine Cytisine

330.33I 329,330

Teratogenic, malformations in hamster embryos Teratogenic, cyclopian malformation Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Abdominal abnormalities in Drosophila Mutagenic in Drosophila Chromosome damage in mouse bone marrow cells Teratogenicity, mutagenicity Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in ST Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Chromosome breakagehearrangements in root tips Chromosome breakage in leukocytes Teratogenic, congenital malformations in calves (“crooked calf disease”) Teratogenic in chicks and rabbits

Minimal 0.01 mM Minimal 0.025 mM Minimal 0.025 mM 10 pM

Minimal 1 mM 18-38 mg/kg

Minimal 0.5 mM Minimal 0.1 mM

-

Minimal 1 mM 1 mM Minimal 0.0025 mM Minimal 0.05 mM Minimal 0.005 mM >I0 pM >10 pM

Minimal 0.005 mM Minimal 0.1 mM

333 333 332,333 334 333 336 336 337 333 333 338 333 332 333 333 333 335 335 333 333 339 340 329,341 341 (continued)

TABLE V Alkaloid Miscellaneous alkaloids Anabasine Arecaidine Caffeine Capsaicin Coniceine Coniine Cryptopleurine Cycasin DIBOA, DIMBOA Theobromine

(Continued)

Effect Teratogenic, crooked calf disease Chromatid exchanges in bone marrow cells Chromatid exchanges Mutagenic Teratogenic, congenital skeletal malformation in pigs Teratogenic, crooked calf disease Chromosome breaks in Drosophilu Mutagenic, carcinogenic Mutagenic in ST Genotoxicity Chromatid exchanges

ED%

Ref.

-

351 ,352 356 354 355 350 348,349 332 342,343 106 353 354

-

-

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

55

phoraceae, Santalaceae, Sapotaceae, and Scrophulariaceae (502)(-3% of higher plants produce these alkaloids), have mutagenic 2nd carcinogenic properties, provided the molecules have the 1,Zdehydro- l-hydroxymethyl-pyrrolizidine structure and are esterified (425,426).After oral intake, the N-oxides are reduced by bacteria in the gut. The lipophilic alkaloid base is resorbed and transported to the liver, where it is “detoxified” by microsomal enzymes. As a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. The alkaloid can then cross-link DNA and RNA and thus cause mutagenic or carcinogenic effects (especially in the liver) (502).Thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. The PA story is very intriguing, since it shows how ingenious Nature was in the “arms race.” The herbivores invented detoxifying enzymes, and Nature the compound which is activated by this process. A herbivore feeding on PA-containing plants will eventually die, usually without reproducing properly. Only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. The protection due to PA can easily be seen on meadows, where Senecio and other PA-containing plants are usually not taken by cows and sheep, at least as long other food is available. Protein biosynthesis. Protein biosynthesis is essential for all cells and thus another important target. Indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (Table IV), such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. For lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyltRNA with amino acids, as well as the elongation step. The inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected (503). Electron chains. The respiratory chain and ATP synthesis in mitochondria demand the controlled flux of electrons. This target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, CH,-(CH2),,-2,6-methyl-piperidines, capsaicin, the hydroxamic acid DIMBOA, and solenopsine. As mentioned before, however, only a few alkaloids have been evaluated in this context (Table V). Biomembranes and transport processes. A cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmenta-

56

MICHAEL WINK

tion that provides separated reaction chambers. Because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. The controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems. These complex systems are also targets of many natural products (Table IV). Disturbance of membrane stability is achieved by 9-methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. Steroidal alkaloids, such as solanine and tomatine, which are present in many members of the Solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky. Cells carefully control the homeostasis of their ion concentrations by the action of ion channels (Na+,K+,Ca2+channels) and through Na+,K+ATPase and Ca2+-ATPase.These channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. Inhibition of transport processes (ion channels, carriers) is achieved by (Table IV) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin. A special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (NA), serotonin, dopamine, glycine, and acetylcholine (ACH). We can distinguish two types. Type 1 is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ACH-receptor. In Type 2 the receptor is an integral protein. When a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent Gprotein molecule, which consists of three subunits. The a subunit then activates the enzyme adenylate cyclase, which in turn produces cAMP from ATP. The cAMP molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ACH receptor). A number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. Targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. Alkaloids relevant here include (Table IV) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, ga-

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

57

lanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine,anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. A number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly. Cytoskeleton. Many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. A number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (Table IV), namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. Colchicine, the major alkaloid of Colchicum autumnale (Liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. As a consequence, chromosomes are no longer separated, leading to polyploidy . Whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. Because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. The derivative colcemide is less toxic and can be employed in the treatment of certain cancers (312).Also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. For normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity. Colchicum plants are not attacked by herbivores to any substantial degree (185). Another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from Catharanthus roseus (Apocynaceae). These alkaloids also bind to tubulin (312).Both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias. From Taxus baccata (Taxaceae) the alkaloid taxol has been isolated. Taxol also affects the architecture of microtubules in inhibiting their disassembly (322). Nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin (312).In conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule. Enzyme inhibition. The inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see Table IV). Briefly, inhibition of CAMP metabolism (which is important for signal transduction I

58

MICHAEL WINK

and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by 1-ethyl-P-carboline, P-carboline-1-propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. Inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (Table IV). Castanospermine, swainsonine, and other polyhydroxyalkaloids are examples. b. Action at Organ Level. Whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too. Central nervous system and neuromuscular junction. A remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (Table IV). The cellular interactions have been discussed above. Disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects. A herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in Nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. Thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. Some individuals of Homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically. The activity of muscles is controlled by ACH and NA. It is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. In the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis. Alkaloids which activate neuromuscular action (so-called parasympathomimetics)include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. Inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) (312). Skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. The compounds are usually considered to be strong poisons, and it is obvious that

1. ALLELOCHEMICAL PROPERTIES OF

ALKALOIDS

59

they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare LD,, values in Table 11). Inhibition of digestive processes. Food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. The next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. The ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms (312). Another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. Relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (Table IV). Modulation of liver and kidney function. Nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. In the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. The liver is also the main site for detoxification of xenobiotics. Lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids (312).These conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination. Both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (Tables IV and V). The alkaloids are activated during the detoxification process, and this can lead to liver cancer. Also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins. Many alkaloids and other allelochemicals are known for their diuretic activity (312).For an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. Since Na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially. Disturbance of reproduction. Quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. Antihormonal effects could be achieved by mimicking the structure of sexual hormones. These effects are not known for alkaloids yet, but have been confirmed for other natural products. Estrogenic properties have been reported for coumarins, which di-

60

MICHAEL WINK

merize to dicoumarols, and isoflavones (4,17). Insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern Polypodium uulgare, or azadirachtin from the neem tree (4,17). Juvenile hormone is mimicked by a number of terpenes, present in some Coniferae. Spermatogenesis is reduced by gossypol from cottonseed oil (17). The next target is the gestation process itself. As outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo (Table V). The last step would be the premature abortion of the embryo. This dramatic activity has been reported for a number of allelochemicals, such as mono- and sesquiterpenes and alkaloids. Some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids (312). The antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. Nevertheless, they are defense strategies with long-term consequences. Blood and circulatory system. All animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. Inhibitors of the driving force for this process, the heart muscle, have already been discussed. However, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine (312). Some allelochemicals have hemolytic properties, such as saponins. If resorbed, these compounds complex membrane sterols and make the cells leaky. Steroidal alkaloids from Solanum or Veratrum species display this sort of activity as well as influencing ion channels (Table IV). Allergenic effects. A number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. Common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds (4,17,312). Activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. The function of alkaloids in this context is hardly known. This selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. However, most of the potential targets for the 10,000 alkaloids known at present remain to be established. If no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property.

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

61

Summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly. B. PLANT-MICROBE INTERACTIONS Dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay (7). How is this achieved? The aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products (4,7). This cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. In perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues. The only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. At the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas (4,17). In addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. Secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. These studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. It may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. In the pharmaceutical context we search for very active compounds which can be employed at low concentrations. Therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. These precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids. Secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono- and disesquiterpenes, and last but not least, alkaloids (4,17,42,149,322). In Table VI 183 alkaloids are tabulated for which antibacterial activities have been detected. The alkaloids usually affect more gram-positive than gram-negative bacteria. Especially well represented are alkaloids which

TABLE VI WITH ANTIBACTERIAL PROPERTIES ALKALOIDS Active against Alkaloid Alkaloids derived from tryptophan Athisine Ajmalicine Apparicine Aspidospermine Bisnordihydrotoxiferine

3

Bisnordihydrotoxiferine N-oxide Borreverine Brevicolline 5-Bromo-N,N-dimethylaminoethyltryptamine Brucine Bufotenine Canthin-Gone Caracurine V Caracurine V di-N-oxide 1-Carbomethoxy-P-carboline Catharanthine Cinchonine Cinchophylline Conoduramine Conodurine Cryptolepine 1GDecarbomethoxytetrahydrosecamine 18,19-Dehydro-ochrolifuanineF

Gram (+)

Gram (-)

Test

+

+ + + + + +

AL AD AD AL AD AL AD AL

+ + + + + + + +

+ +

-

+ + + + + + + + + + + + + +

+ + + + + + -

+

AD AD LD LD AD AD AD AD SP AD AD AD AD AD

Concentration tested (pg/m) I 15 12 1

MIC (mg/ml)

ED, (mdml)

lo00

lo00 270-3000 1-100

2 2-400

5 1 12-100 2 10- 1400 2 50 1 16-32 15-400 4-400

Ref. 50 51 52 50 53 54 53 55 57 72 53 56 113 50,58 53 53 41 51 56 69 59-60 59.60 61 42,43 69

Dehydropteleatinium Dictamnine Dihydrocinchonine 18,19-DihydrocinchophyIline Dihydrocorynantheol 4,5-Dimethoxycanthin-6-one 10,IO'-Dimethoxy-Nmethyltetrahydrousambarensine Diploceline Fagarine Glycozolidol Gramine Harmaline

W QI

+ + + +

+ + + + + + + +

Harmalol Harman Harmine

+ +

Harmol 3-Hydrox yconoduramine 3-Hydroxyconodurine 3-H ydroxyconopharyngine 3-H ydroxy isovoacangine 3'-Hydroxy-p-demethylervahanine B 3'Hydroxy-N"-demethyItabernamine 19-Hydroxy-18,19-dihydrocinchophylline 9-Hydroxyellipticine 3-Hydroxy-(19R)-heyneanine 5-Hydroxy-4-methoxycanthin-6-one

+ + + + + + + + + + +

+

AL

50-100 10

BG

-

94 95

AL AD BG

50 69 42,62,63 41 69

AD

+ + + + + + + + + + + + + +

BG AD LD PD SP SP PD SP

500-2000 20 200

4

10 (light)

I 4 10 (light)

< 100 SP AD AD AL AL BG BG AD AD BG

10 (light)

8-170 14-750 60-140 50-500

1-250

64 95 65 113 67 66 56 67 66 68 66 68 45 45 47 45 44 44 69 48,49 44 41 ~

(continued)

TABLE VI (Continued) Active against Alkaloid

10'-Hydroxy-10-methoxy-Nmethyltetrahydrousambarensine 3'-Hydroxytabernamine 16Hydroxytetrahydrosecamine 10-Hydroxyusambarensine 3-Hydrox yvoacamine

2

Ibogaine Ibogamine Iboxygaine Isoraunescine Isovoacangine Melicopicine 6-Methox ytecleanthine

11-Methoxytubotaiwine Mimosamycin Norharmane Ochrolifuanine A Ochrolifuanine E Ochrolifuanine F Perivine Pteleatinium Ptelefolonium Renierol Reserpine Stemmadine Strychnine

Gram ( + )

+ + + + + + + + + + + + + +

+ + + + + + + +

-

+

Gram (-)

+ +

-

+ + + + -

+ +

Test

Concentration tested (pg/m)

MIC (mg/ml)

ED, (mg/ml)

Ref.

AD

69

BG BG AD BG AD AL AL AL AL AD AD AD

44 43,43 69 45 46 50 50 50 50 97 97 42.43 93 66 43,69 69 69 51 94,96 42 93 51 70 53 56

50 1 1 1

1

lo00 lo00 lo00 lo00 >200 >200 100

SP BG AF AF AD AL

10 (light) 32 32 15 100-lo00 100

AD

SP AD SP

37 1-7 5

1

c

Tabernaemontanine Tabernanthine Tchibangensine Tecleanthine Tetrahydroalstonine Tetrahydrosecamine Tetrahydrousambarensine Usambarensine Vindoline Vindolinine Voacamine Vobparicine Vobparicine N-oxide Woodinine Yuehchukene Alkaloids derived from phenylalanine/tyrosine Actinodaphnine Anhydroushinsunine Anolobine Anonaine Berbamine Berberine Berberrubine Bulbocapnine Cassameridine Cepharanthine Chelerythrine

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

+

+ -

+ + + + +

AD AL AD AD AD AD AD AD AD AD AD AD BG AD

38 1

51 50

lo00 64 >200

69 97 51 62.63 69 69 51 51 60 45 45 72 71

54 110 32 38 70 20-400 50- 100

20-25

+ + + + + + -

+ +

AL AL AL AL AL AL AL SP AL AL AL AL

50-300 50 6-200 3-100 50 125-1000 1000 3-100 1

100 1000 25-50 8-1000 6-100

74 74 80,81 80,81 74 73 50 89 56

90 50

82 73 50,84,85 (continued)

TABLE VI (Continued) Active against Alkaloid Chelidonine

Chelidonine N-oxide Columbamine Corytuberine I-Curine Dehatrine Dehydroglaucine Dihydroberberine Fagaronine Funiferine Glaucine Hernandezine Isoboldine Isotetrandine Isotrilobine Liriodenine Lysicamine Magnoflorine N-Desmethylthalidezine N-Desmethylthalistyline N-Methylactinodaphnine Nornantenine Nuciferine

Gram (+)

Gram (-1

Test AL AL AL AL AL AL AL AL AL AL AL

Concentration tested (pglm)

MIC (mg/ml) 1000 1000- 10,000 20-50 30-500 1000-10,000 100 1000 300 25 1000

SP AL AL AL AL AL AL

SP AL AL AL AL AL AL AL

100 300 25-100

100-200 8-500 1-100 0.4-3 12-26 50-1000 100-1000 50-300 3-100

loo0

ED, (mglml)

Ref. 50 86 87 42 86 79 80,81 50 74 83 50 88 50 74 75 80,81 76 73 80,81 83 82 78,79 75 75 74 80,81 50

0-Methyldauricine 0-Methylthalibrine 0-Methylthalmethine Obamegine Oxonantenine Oxyacanthine Palmatine Papaverine Pennsylvanine Protopine Protothalipine Sanguinarine

3

Tetrandrine Thalibrine Thalicarpine Thalicerbine Thalidasine Thalidezine Thaliglucinone Thalistine Thalistyline Thalmelatine Thalmirabine Thalphenine Thalrugosaminine Thalrugosidine Thalrugosine Tubocurarine

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

-

+ + + -

+ + -

+ + + +

-

-

+ +

250- lo00

AL AL

AL AL

AL

SP AL AL AL AL AL

AL SP

100 100

50-200 6-25 62-100 lo00

I lo00 100

300 lo00 13-100 1-5

84 0.01

15-1000

AL AL AL AL AL AL AL AL AL AL AL AL AL SP

73 77 75 76 82 73 50 56 75 87 74 79

lo00 100-1000 250- I 000

25-200 100

25-200 100

50 100 100 1000 50-100

100-200 100-200 1

87 56 73 75 42,78 73 76 75 79 77 75 42,78 77 78,79 78,79 76 76 56 (continued)

TABLE VI (Continued) Active against Alkaloid Xylopine Steroidal alkaloids Conessine Samandarone Samandarine Solacasine Solanidine Solanocapsine Quinolizidine alkaloids Angustifoline 13-Hydroxylupanine Lupanine Sparteine 13-Tigloyloxylupanine Pyrrolizidine alkaloids Lasicarpine Miscellaneous alkaloids Antofine

Gram ( + )

+ + + + + + + + + + + + + + +

Gram (-)

Test

Concentration tested (pg/m)

MIC (mg/ml)

AL

50-100

AL LD LD AL AL AL AL

100-1Ooo

ED, (mg/ml)

74 50 113 113 50 50 91 91

3-13 loo0 3 100 50 mM 50 mM 50 mM

SP AD

<0.5-10 mM 5 mM 50

BG

Ref.

99 99 99 98 98

100 115

%

Benzoxazolinone (BOA) Chaksine Dihydrookolasine Dihydrowisanine DIMBOA/MBOA Diplamine Ecteinascidins Ficuseptine Hydroxyrutacridone epoxide GMethylspinaceamine Rutacridone epoxide Scutlianins A-E Spinaceamine Tryptanthrine Tuberin Ungeremine Wisanine Xestoaminol A (I

+

+ + + + + + + + +

-

AL AL AL

100 128 128

+

+ + + + + + + +

BG TLC LD TLC

0.1-10 0.2-5

1

LD AL

SP AL AD

i05 101

103 103 106 111 109 115 95 113 95

I I0 3-6 0.1-1 128

113 104 107,108 114 102

112

+. active; -, no activity observed in the concentration range tested (many alkaloids were only assayed in low concentrations as microbial antibiotics); AD,

agar diffusion, AL, agar dilution; BG, biogram; LD, liquid culture; MIC,minimal inhibitory concentration; PD, paper disk; SP,suspension; TLC, TLC disk test according to Wolters and Eilert (95). If more than one value is given, the data refer to different bacterial species tested.

70

MICHAEL W I N K

derive from tryptophan (indole alkaloids) and phenylalaninehyrosine, which may be due to the fact that these alkaloids have obtained considerable scientific attention since the discovery of many medicinally important compounds within these groups (42,50,59,60,63,68,75-84).Some of these alkaloids are highly antibiotic, with similar activities as fungal antibiotics, namely, cinchophylline (69), dictamnine ( 9 3 , fagarine (95), stemmadine (70),yuehchukene (71), liriodenine ( 8 3 , lysicamine (82), oxonantenine (82),sanguinarine (87), solacasine (50,92),rutacridone epoxide (95),tryptanthrine ( I @ # ) , and tuberin (107,108) (Table VI). In many instances, when alkaloids are assessed for their antibacterial activity, they are often also tested for antifungal properties. Usually yeasts and Candidu are used as test organisms (Table VII). Table VII lists 117 alkaloids with antifungal activity. Besides indole, quinoline, and isoquinoline alkaloids, the group of steroidal alkaloids shows significant activities. Especially active compounds include dictamnine ( 9 3 , skimmianine ( 9 3 , anolobine (80,81), berberine (89,f20), cassameridine (82), chelerythrine (119), chelidonine (120,121), dehydroglaucine ( 8 3 , liriodenine (83,118), lysicamine (82), sanguinarine ( I19,121), thaliglucinone (79), demissidine (126,127),solacasine (92),soladulcidine (126,127),solasodine (26,127)tidine (126,127),tomatine (42,126),verazine (124),cryptopleurine (133) hydroxyrutacridone epoxide ( 9 3 , tryptanthrine (104), and tuberin (107). Whereas the mode of action and targets of antibiotics of fungal and bacterial origin have been elucidated in many instances (see Table IV), relevant information for plant-derived compounds is scant. However, the molecular targets of some alkaloids have been determined at the general level, but not specifically for bacterial or fungal systems (Table IV) that may be responsible for the antibiotic effects observed. The following interactions of alkaloids having antimicrobial properties with molecular targets of bacterial or fungal cells are likely (compare Tables VI and VII with Tables IV and V). Protein biosynthesis in ribosomes is affected by sparteine (56,423, lupanine, angustifoline, 13-tigloyloxylupanine,and 13hydroxylupanine (56,98,99,417,421,422).Intercalation or binding to DNA is influenced by fagaronine, dictamnine (367),harman alkaloids (376,378) [binding to DNA is light dependent (66)],berberine (396-3981, chelerythrine (400), and sanguinarine (400,409);these compounds may thus inhibit important processes such as DNA replication and RNA transcription that are also vital for microorganisms. The stability of biomembranes may be disturbed by cepharanthine, tetrandrine, and steroidal alkaloids such as solamargine ( 4 3 3 , solanine (430,432,433), and solasonine (435), thus leading to an uncontrolled flux of metabolites and ions into microbial cells. Inhibition of metabolically important enzymes is affected by berberine (399),chelerythrine (259,401),chelidonine (402),palmatine (399),sanguinarine (143,259),solacongestidine (434), and papaverine.

1. ALLELOCHEMICAL

PROPERTIES OF ALKALOIDS

71

In contrast to antibiotics of microbial origin that could be classified as alkaloids from a chemical point of view in many instances, and which often interfere with the biosynthesis or maintenance of the cell wall (murein) (Table IV), such an interaction has not been described for plantderived compounds. Since this topic has not been studied in detail it remains open whether this complex is another target for alkaloids. We can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de now. Inducing agents, which have been termed “elicitors” by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents (4,17,22-24).The induced compounds are called “phytoalexins,” which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. In another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below). Many of the antimicrobial alkaloids found are constitutively expressed and accumulated, that is, they are already present before an infection. Using plant cell cultures, it was observed that some cultures start to produce new secondary metabolites when challenged with bacterial or fungal cell walls, culture fluids, or other chemical factors (4,17,22-24). Among the compounds found to be inducible are alkaloids such as sanguinarine and hydroxyrutacridone epoxide (see Table XI). Quinolizidine alkaloids display some antimicrobial properties, besides their main role in antiherbivore defense (503) (see Table I). On wounding, QA production is enhanced, thus increasing the already high alkaloid concentration in the plant; in other words, the antimicrobial and herbivoral effect is further amplified (Table XI) (2,184,503). The reactions leading to the induction and accumulation of phytoalexins with phenolic structures have been studied in molecular detail (4,17,22-24).These studies revealed that plants can detect and react rapidly to environmental problems, such as wounding or infection: Within 20 min of elicitation, mRNAs coding for enzymes that catalyze the reactions leading to the respective defense compounds are increasingly generated, leading to the accumulation of the respective enzymes and consequently the production of the secondary metabolites (4,17,22-24). Similar processes are likely for alkaloids, but so far the mechanisms have not been elucidated. We assume that a substantial number of the 10,000alkaloidshave antimicrobial properties (which remain to be tested in most cases) that are directed against the ubiquitous and generalist microbes which have not

TABLE VII ANTIFUNGAL ACTIVITY OF ALKALOIDS ~~

~

A1kaloid Alkaloids derived from tryptophan Minisine Ajrnalicine Apparicine Bisnordihydrotoxiferine

N 4

Active against

Yeast Fungi Fungi Yeast Yeast, fungi Ph ytopathogens Brevicolline Fungi Canthin-6-one Fungi Carcurine V Yeast Catharanthine Fungi Dihydrocinchonine Yeast Dihydropteleatinium Yeast Fungi Gramine Yeast, fungi Erysiphe graminis Harmine Yeast Harmol Yeast Ibogamine Yeast Isatin (2,3-indolinedione) Lagenidium Reserpine Fungi Tetrahydroalstonine Fungi Vindoline Fungi Alkaloids derived from phenylalanine/tyrosine Actinodaphnine Candida Anhydroushinsunine Candida Anolobine Yeast

Test AL AD AD AD AL AL AD AD AL AL TLC AD AL

Concentration tested (mg/ml) 1

EDXI (mg/ml)

1000

I5 12 270-3000 40-100 40- 100

2 10- 1400 50 1 50-100 20-100 <2 mM

AL

1

AD AD AD

31 54 38

AL AL AL

MIC (Fglml)

1000

250-1000 125-1000 6-200

Ref. 50 51 5132 53 54 54 57 57 53 51 42,128 94,131 95 113 132 68 68 50 117 51 51 51 74 74 80,81

Anonaine Berbenne

Boldine Bulbocapnine Cassameridine Chelerythrine Chelidonine

I .

w

Chelidonine N-oxide Coclaurine Columbamine Dehatrine Dehydroglaucine N-Desmethylthalidezine Dihydroberbenne Glaucine Hernandezine Jatrorrhizine Laudanosine Laurotanine Liriodenine

Ly sicamine N-Methylactinodaphnine

Yeast Candida Yeast Yeast, fungi Yeast, fungi Candida Yeast Yeast, fungi Yeast Yeast Yeast, fungi Yeast, fungi Fungi Yeast, fungi Candida Candida Candida Yeast Yeast Yeast Fungi Candida Candida Yeast Candida Candida Yeast, fungi Yeast, fungi Candida Yeast, fungi Candida

AL AL AL AL AL AL AL AL AL

SP AL AL TLC AL AL AL AL AL AL AL CT AL AL CT AL AL AL AL AL AL AL

3-100 62-259 lo00 3-100 15-500 250 lo00 25-50 6-100 10 1o00- 10,000 15-125

25 lo00-10,OOo lo00 100 lo00 25-50 lo00 lo00 25 1000 50 250 500-1000 1000 6-100 6

3 12-26 125-1000

80,81

74 50 89 120 74 50 82 50 119 42,50,86 120 121 86 74 79 74 83 75 50 121 74 75 122,123 74 74 80,81 83 118 82 74

(continued)

TABLE V11 (Continued) Alkaloid 0-Methylbulbocapnine 0-Meth ylthalibrine Nornantenine Oxonantenine Oxyacanthine Palmatine Sanguinarine

2

Thalibrine Thalicarpine Thalidezine Thaliglucinone Thalphenine Xylopine Steroidal alkaloids Cevadine Conessine Demissidine Isorubijervine Jervine

Active against Candida Candida

Yeast Yeast, fungi Yeast Yeast Fungi Yeast Yeast Fungi Yeast, fungi Candida Candida

Yeast Candida Candida Candida

Fungi Fungi Yeast Fungi Fungi Fungi Yeast, fungi Fungi Yeast, fungi

Test

Concentration tested (mglml)

AL AL AL AL AL AL CT AL SP CT AL AL AL AL AL AL AL

MIC (pglml) 500-1000 500 3-100

6-25 lo00 1000 250 12-100 10

5-25 2-250 1000 1000 100 50 1000 250

CT CT AL CT TLC CT

1

CT

0.1

30-250 100-1Ooo

5 4-20 150 72-200 9-120

ED, (mglml)

Ref. 74 77 80.81 82 50 50 122,123 50 119 121 120 42,78 42.78 75 79 42,78 74 125 126,127 128 126.127 126,127 125 129,130 125 129.130

Protoveratrine A Protoveratrine B Pseudojervine Rubijervine Samandarone Samandarine Samandaridine Solacasine Solacongestidine Soladulcidine Soladulidininetatraosid Solatloridine Solamargine

G! Solanidine Solanine Solanocapsine Solasodine

Solasonine Tomatidenol Tomatidine Tomatillidine

Fungi Fungi Fungi Yeast, fungi Fungi Yeast, fungi Yeast, fungi Yeast, fungi Yeast Yeast, fungi Fungi Fungi Fungi Yeast, fungi Fungi Fungi Yeast Fungi Fungi Yeast Fungi Fungi Yeast Fungi Fungi Yeast Fungi Fungi Fungi Fungi Yeast, fungi

CT CT CT CT

AL AL CT TLC CT AL CT TLC AL CT TLC AL CT TLC AL CT TLC AL CT CT CT TLC AL

19-54 I50 0.34 mM

3-13 0.8-1 15

20 10-50 6-100

40 >80

lo00 5 20-40 lo00 40 10

100 15 20 >I00 40 1-40 2-22 15

>I00

125 125 125 129,130 125 113,529 113,529 113,529 121 124 126,127 126 126,127 124 126,127 42 128 126,127 121 128 126,127 121 121 126,127 121 124 126,127 121,126,127 126,127 126 124 (continued)

TABLE VII (Continued) Alkaloid Tomatine

rn 4

Veratramine Veratridine Veratrobasine Verazine Quinolizidine alkaloids Lupanine Sparteine 13-Tigloyloxylupanine Miscellaneous alkaloids Antofine Benzoxazolinone (BOA) Cryptopleurine Dictamnine 6,6’-Dihydroxythiobinupharidine DIMBOAlMBOA 3,4-Dimethoxy-(piperid2-yl)-acetophenone

Active against

Test

Fungi Fungi Yeast, fungi Fungi Yeast, fungi Yeast, fungi

CT TLC

Etysiphe graminis Etysiphe graminis Fungi Erysiphe graminis

AL AL AL AL

Fungi Fungi Candida, fungi Fungi Fungi Phytopathogenic fungi Candida

CT AL

Concentration tested (mdml)

MIC (pg/ml)

EDXI (mg/ml)

2-40 5 72-200

126,127 42,126 129,130 125 129,130 124

1

72-200 3-12 2 mM <2 mM 5-50 m M

AL TLC AL

0.1

AL

3

10-100

0.I

Ref.

132 132 98 132 113 105 133 95 134 106 133

Eupolauridine Ficuseptine Hydroxyrutacridone epoxide Julandine Lasiocarpine Melicopicine 6-Methoxytecleanthine Onychine Papuamine Phidolopine

I I .

Pteleatinium Rutacridone epoxide Scutianins A-E Skimmianine Stemmadine Supinine Tecleanthine Tryptanthrine Tuberin Xestoaminol A

Candida Fungi Yeast, fungi Candida Candidal Aspergillus Cladosporium Cladosporium Candida Trichophyton Helmithosporium, Rhizoctonia Yeast Yeast, fungi Pythium Fungi Candida Candida Cladosporium Yeast Saccharomyces Candida , Trichophyron

TLC

1.5

135

0.1-10

113 95

12.5

AL

50

133 100

3.1 10 70

97 97 135 136 137

TLC TLC

100-lo00 0.2-5

AL TLC 1

TLC SP

>loo

37

50 TLC AL

3-6 0.1

94,131 95 110 95 70 100 97 104 107 137

* CT, Channel test according to Wolters ( I 16); other abbreviations are as in Table VI. If a range is given, the first value gives a 10% inhibition, the second value a 100% inhibition.

78

MICHAEL WINK

specialized on a particular host plant. However, alkaloid production does not necessarily have to be involved with antimicrobial defense. For example, Phytophthora or Fusarium will attack alkaloid-rich plants of Nicotiana, Solanum esculentum, and S . tuberosum. Cladosporium and Fusarium can develop in nutrient-containing media enriched with alkaloids, and Aspergillus niger can utilize alkaloids as a nitrogen source (506). In addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. In these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. We suggest that these specialists have adapted to the chemistry of their host plants. Mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). These mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. Some associations between plants and fungi are symbiotic in nature, such as Rhizobia in root nodules of legumes or microrhizal fungi in many species. In lupines, nitrogen-fixing Rhizobia are present both in alkaloid-rich and alkaloid-free plants. They must therefore be able to tolerate the alkaloids, which are also present in the root. Alkaloid production in lupines is more or less unaffected whether or not the plants harbor Rhizobia (185,506). An ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. Graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi (4). A similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity. From the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (Tables I1 and 111). Since many alkaloids are antibacterial and antifungal (Tables VI and VII) and are present in plants at relatively high concentrations (Section IILA), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. It should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes (2-4,7). A cooperative, or even synergistic, process could thus be operating.

1. ALLELOCHEMICAL PROPERTIES

OF ALKALOIDS

79

C. ANTIVIRALPROPERTIES Plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (Heteroptera). Resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place. The assessment of antiviral activity is relatively difficult. As a result, only a few investigators have studied the influence of alkaloids on virus multiplication. Nevertheless, at least 45 alkaloids have been reported with antiviral properties (Table VIII). Only sparteine (527) and cinchonidine (142) have been tested for antiviral activities against a plant virus, the potato X virus. All other evidence for antiviral activities (Table VIII) of alkaloids comes from experiments with animal viruses. Because viral life strategies are related in plants and animals, we suggest that a wider number of plant viruses may be controlled by alkaloids in Nature than the limited data imply. Viral multiplication can be controlled at the level of replication, transcription, protein biosynthesis, and posttranslational protein modification. The number of molecular targets is thus quite restricted for antiviral activities (compare Tables IV and VIII). The processing of DNA and RNA is extremely important for viruses, and it is not surprising that this area (intercalation in DNA, binding to DNA, inhibition of RNA and DNA polymerases) is probably one of the potential targets of alkaloids, for example, camptothecine (365,366),quinine, p-carboline alkaloids (1381, and acridone alkaloids (145). Other alkaloids could inhibit protein biosynthesis or posttranslational protein modifications. Examples include polyhydroxy alkaloids (150,212,410-414), cryptopleurine (404,444), haemanthamine (390),hippeastrine (148), narciclasine (451), pretazettine (390), sparteine and other QAs, and pseudolycorine (390). Because retroviruses rely on reverse transcriptase, inhibition of this enzyme by alkaloids would have a dramatic effect. However, plant viruses are not retroviruses, and the significance of the anti-reverse transcriptase effects of the alkaloids listed in Table VIII are difficult to interpret at present. Polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum- and Golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins. Because alkaloids often deter the feeding of insects, such as aphids and bugs (Table I), viral infection rates may be reduced in alkaloid-rich plants. Such a correlation exists for alkaloid-rich lupines (so-called bitter

TABLE VIII ANTIVIRAL ACTIVITY OF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Apparicine Camptothecine Cinchonidine

Dimethoxy-1-vinyl-P-carboline Eudistomins C, E, K, L (tunicates) Harman Harmine 7-Methoxy-I-methyl-P-carboline Norharman Alkaloids derived from phenylalanineltyrosine Fagaronine Acridone alkaloids Acronycine Atalaphillidine Atalaphillinine Citpressine I Citracridone I Citrusinine I Dercitine (sponge) Dimethox yacronycine Glycocitrine I GIyfoline Grandisine

ED, (dnl)

Activity

Ref.

Anti-polio I11 activity Inhibition of herpes and other virus Inhibition of potato X virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition of murine cytomegalovirus, Sindbis virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus

-

-

141 140 142 138 109 138 139 138 138

Inhibition of reverse transcriptase of oncorna virus

-

143

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition

3.3 0.7 0.8 0.6 1.3 0.7 1-5

145 145 145 145 145 145 144 145 145 145 145

of of of of of of of of of of of

herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes

simplex virus simplex virus simplex virus simplex virus simplex virus simplex virus simplex virus, murine corona virus simplex virus simplex virus simplex virus simplex virus

-

6.5 5

>20 10

5-Hydrox y-N-methylseverifoline 5-H ydroxynoracronycine 5-Methox yacronycine N-Methylatalaphilline Miscellaneous alkaloids Abikoviromycin Ageliferin Crinamine Cryptopleurine Sceptrin Didemnin Haemant hamine Hippeas trine 6-H ydroxycrinamine Ly corine

May tansine Narciclasine Oxysceptrine Precriwelline Pretazettine Pseudo1ycorine Sparteine Polyhydroxy alkaloids Castanospermine Deox ynorjirimycin Dihydroxymethyl-dihydrox ypyrrolidine

" MAD, Minimal active dose.

Inhibition of Inhibition of Inhibition of Inhibition of

herpes simplex virus herpes simplex virus herpes simplex virus herpes simplex virus

2.0 5 5.5 8.4

145 145

Antiviral activities Inhibition of herpes simplex virus Inhibition of Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition of murine sarcoma virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition of potato x virus

-

149 109 147 141 109 109 147 148 147 147 148 146 147 109 147 147 148 147 148 150

Inhibition of cytomegalovirus, retroviruses Inhibition of cytomegalovirus, retroviruses Inhibition of cytomegalovirus, retroviruses

0.8 mM 1.0 mM 1.8 mM

M A P 0.2 pg/ml

-

MAD 0.2 pg/ml

-

MAD 0.2 pg/ml MAD 0.2 pg/ml -

MAD 0.005 pg/ml

-

MAD 0.05 pg/ml

-

MAD 1.0 pg/ml

-

i45 145

150 150 150

82

MICHAEL W I N K

lupines) and low-alkaloid varieties (the so-called sweet lupines) (see Table XII).

D. ALLELOPATHIC PROPERTIES Plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. This phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense (4,17,498-500). A number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated. It was observed by Molisch in 1937 (497) that plants can also influence each other by their constituent natural products, and he coined the term “allelopathy” for this process. Secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain (4,17). Both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. Allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments (4,17,19,497-500),but how it operates in ecosystems is still often a matter of controversy. It is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. Thus allelochemicals might never reach concentrations high enough to be allelopathic. Allelopathic natural products have been recorded in all classes of secondary metabolites. Few research groups have studied the effect of alkaloids in this context, but at least 50 alkaloids have been reported with allelopathic properties (Table IX). As can be seen from Table IX, allelopathic activities can be found within nearly all structural types of alkaloids. At higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. More sensitive, however, is the growth of the radicle and hypocotyl. They respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. In species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in Colchicum autumnale (503,506). It is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms.

TABLE IX ALLELOPATHIC ACTIVITY OF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Quinine Cinchonidine Cinchonine Ergometrine Ergotamine Grarnine

Harmaline Hordenine 5-H ydroxytryptophan

Physostigmine Quinidine

Strychnine

Activity Toxic to Cinchona cells Toxic for Lemna Toxic to Cinchona cells Toxic for Lemna Toxic to Cinchona cells Reduction of radicle length in Lepidium, Lacruca Reduction of radicle length in Lepidium Reduction of radicle length in Lepidium Reduction of radicle length in barley Growth inhibition of Stellaria, Capsella. Nicotiana Reduction of radicle length in Lepidium, Lactuca Reduction of radicle length in Lepidium Toxic for Lemna Reduction of radicle length in barley Growth inhibition Toxic for Lemna Inhibition of germination Toxic to Cinchona cells Reduction of radicle length in Lepidium, Lactuca Toxic for Lemna Reduction of radicle length in Lepidium Toxic for Lemna Toxic for Lemna

Yohimbine Alkaloids derived from phenylalanine/tyrosine Reduction of radicle length in Lepidium. Lactiica Berberine Growth inhibition in plant cell cultures

Ref. 0.04% 0.04% -

0.4%

244 56 244 56 244 56 56 56 239 240 56 56 56 239 238 56 24 I 244 56 56 56 56 56

0.01% -

56 243

0.01% 0.1% 0.1%

-

0. I% 0.01% 0.04%

0.4% 0.1-0.0 1% 0.4% 0.1% 0.4%

(continued)

TABLE IX (Continued) Alkaloid Boldine Chelidonine Colchicine Emetine Ephedrine Morphine Narcotine Papaverine Salsoline Sanguinarine

g

Tropane alkaloids Cocaine H yoscyamine

Scopolamine

Quinolizidine alkaloids Cytisine Lupanine Sparteine

13-Tigloyloxylupanine

Activity

ED54

Toxic for Lemna Reduction of radicle length in Lepidium Reduction of radicle length in Lepidium Toxic for Lemna Reduction of radicle length in Lepidium Reduction of root growth, induction of polyploidy in Allium Inhibition of germination Reduction of root growth, induction of polyploidy in Allium Reduction of radicle length in Lepidium Reduction of radicle length in Lepidium, Lactuca Toxic for Lemna

0.04% 0.1% 0.01% 0.4% 0.1%

Inhibition of germination Inhibition of germination, radicle growth in Linum Toxic for Lemna Inhibition of germination Inhibition of germination, radicle growth in Linum, wheat Reduction of radicle growth in Lactuca Inhibition of germination

0.4% -

Reduction of radicle length in Lepidium Inhibition of seed germination in Lactuca Inhibition of seed germination in Lacruca Reduction of radicle length in Lepidium, Lactuca Inhibition of seed germination in Lactuca Inhibition of radicle growth in Raphanus Inhibition of radicle growth in Sinapis Inhibition of seed germination in Lactuca

0. I% 6 mM
0.01% 0.1-0.01%

0.4%

0.01%

-

0.01%


Ref. 56 56 56 56 56 242 241 242 56 56 56 241 245 56 241 245,246 56 241 56 56,247 247 56 56,247 185 185 247

Miscellaneous alkaloids Aconitine Balfourodinium Caffeine

Castanospermine Coniine Delcosine Delsoline DIMBOA and other hydroxamic acids 'A oc

Lobeline Mimosine Nicotine

8-Oxyquinoline Paraxanthine Piperine Ptelefolonium

Theobromine Theophylline Trigonelline a-Tripiperideine

Reduction of radicle growth in Lepidium Reduction of seedlings growth in Poaceae Reduction of cell growth in topinambour Autotoxicity in coffee seedlings Growth inhibition of lettuce seedlings and various species Reduction of radicle length in Lepidiurn Inhibition of root length elongation Toxic for Lemna Reduction of cambial growth, gibberellic acid (GA) antagonism Reduction of cambial growth, GA antagonism Inhibition of germination and seedling growth in Abutilon, Lepidium,and other plants Inhibition of Auena fatua growth Reduction of radicle length in Lepidium Toxic for Lemna Allelopathic Reduction of radicle length in Lepidium Toxic for Lemna Toxic to Trifolium Reduction of radicle length in Lepidium Growth inhibition of lettuce seedlings Reduction of radicle length in Lepidiurn Reduction of seedling growth in Poaceae Reduction of seedling growth in Solanum esculenfurn Reduction of seedling growth in topinambour, vigne-vierge Growth inhibition of lettuce seedlings Growth inhibition of lettuce seedlings Arrest of cell cycle in Haplopappus roots Toxic to Trifolium Toxic for Lemna

0.1% 0.1 mM 40 p M

-

56 256 256 249 249,250 56 253 56 249,252

-

249,252 106

-

0.1%

-

0.04%

0.1% 0.04%

0.1% 0.4%

0.01%

0.01% 10 pM

-

1 FM

-

0.4%

255 56 56 248 56 56 254 56 249 56 256 256 256 249 249 25 I 254 56

86

MICHAEL W I N K

The mechanisms of alkaloid toxicity toward other plants have not been elucidated yet, but it is likely that the following targets are involved: DNA binding o r intercalation [e.g., quinine and other quinoline alkaloids (381), harman alkaloids (56,166,378),berberine (396-398), sanguinarine (400,409) and Veratrum alkaloids]; inhibition of protein biosynthesis [e.g., emetine (404) and quinolizidine alkaloids (56,99,416-418,422)];inhibition of microtubules [e.g., colchicine (376,441,442)];inhibition of metabolically important enzymes [e.g., papaverine (297,4061, colchicine (376,441,442L chelidonine (402), castanospermine (253), caffeine (202,376), and DIMBOA (106,446-448)l; uncoupling of electron chains [e.g., gramine (374), sanguinarine (143,407), and DIMBOA (106,445)l;and interference with growth factors [e.g., delcosine (249,252), delsoline (249, 252), DIMBOA (106), nicotine, and trigonelline (456,457)] (compare Tables IV and IX). The inhibitory action of quinolizidine alkaloids should be explained in this context (184,503).They are very abundant in lupine seeds (up to 3-8% dry weight). During germination, 13-hydroxylupanine is converted to ester alkaloids, such as 13-tigloyloxylupanine. The latter compound is predominantly excreted via the roots of young seedlings and in germination assays proved to be the most allelopathic QA. These alkaloids influence only heterologous systems, not the germination of lupine seeds themselves. When lupine and Lepidium seeds were grown together in the same pot, growth of the Lepidium seedlings was much reduced and inhibited, indicating that QAs may also be relevant in the ecological context (184). Although the number of alkaloids with known allelopathic properties is not large, owing to the limited number of studies conducted, it is clear from Table IX that alkaloids can be toxic to plants, probably by interfering with basic metabolic o r molecular processes.

111. Raison d’Etre of Alkaloids

Although comparably few alkaloids have been studied for their biological activities in detail, and considering that our data collection (Tables I-IX) is far from complete, we can safely state that alkaloids have potent deterrent o r poisonous properties in herbivorous animals, and also affect bacteria, fungi, viruses, and plants. The next question will be whether all the adverse activities of alkaloids, which are often assayed in in uitro systems only, are meaningful in Nature.

I.

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87

A. CONCENTRATIONS I N PLANTS A N D ALLELOCHEMICAL ACTIVITIES

Because most of the allelochemical activities are dose dependent (others may be synergistic, additive, etc.), the question is whether the amounts of alkaloids produced and stored in plants are high enough to be ecologically meaningful. It is difficult, and also dangerous, to make a general statement concerning alkaloid levels in plants. We must remember that alkaloid composition and levels are often tissue or organ specific (4,25,38).They may vary during the day [a diurnal cycle has been observed for QAs and tropane alkaloids (185,503,506)lor during the vegetation period (39. 505,506). Furthermore, as in all biological systems, there are differences at the level of individual plants and between populations and subspecies. Unfortunately, many phytochemical reports do not contain any quantitative information, or these data are given for the whole plant without realizing the above-mentioned variables. In addition, concentrations are usually given on a dry weight basis, which is appropriate in the chemical or pharmaceutical context. However, herbivores or pathogens do not feed on the dry plant in general, but on the “wet” fresh material. In the context of chemical ecology we urgently need data on a fresh weight basis. As an approximation, in this chapter we use a conversion factor of 10 to convert dry weight to fresh weight data if only the dry weight data are available. Summarizing the relevant phytochemical literature, we find that alkaloid levels are between 0.1 and 15% (dry weight), which is equivalent to O.OI-lS%fresh weight, or 0.1-15 mg/gfresh weight. For plantscontaining quinolizidine alkaloids, actual alkaloid contents are given for a number organs or parts (Table X ) , which fall in the range deduced before. We have evaluated the situation for quinolizidine alkaloids and found that the actual concentrations of alkaloids in the plant are usually much higher than the concentrations needed to inhibit, deter, or poison a microorganism or herbivore (2,184,503,527).This means that plants obviously play safe and have stored more defense chemicals than actually needed. If we look at the ED,, and LD,, values given in Tables 1 through IX, it is likely that the situation is similar for other alkaloid-producing plants, but these correlations need to be experimentally established in most instances. It seems trivial that plants not only synthesize but also store their secondary products, which makes sense only in view of their ecological functions as defense compounds, since they can fulfil these functions only if the amounts stored are appropriate. Achieving and maintaining the high levels of a defense compound are very demanding from the point of view of physiology and biochemistry. Most allelochemicals would probably

88

MICHAEL WINK

TABLE X ORGAN-SPECIFIC CONCENTRATIONS OF QUINOLlZIDlNE ALKALOIDS IN LEGUMESPECIES Species Cytisus scoparius

Laburnum anagyroides

Lupinus albus

L . angustifolius L . consentinii L . luteus L . mutabilis L . polyphyllus

Organ tissue Stem epidermis Shoots Leaves Seeds Roots Leaves Twigs Bark Wood Flower Fruit Seed Endosperm Testa Stem epidermis Phloem sap Leaves Stem Flower Fruit Seed Roots Phloem sap Xylem sap Phloem sap Xylem Stem epidermis Stem epidermis Petiole epidermis Stem epidermis Leaves Stems Flower Pollen Carpels Petals Fruits Seeds Roots

Total alkaloids (per g fresh weight)

SELECTED

Ref.

46 mg/g; 200 mM 2 mg 0.2-1 mg 2 mg dry wt 0.03 mg 0.3 mg

486 487,488 487,488 487,488 487,488 184,487,492

11.1 mg 0.5 mg 0.4 mg 0.5 mg 10-30 mg dry wt 21 mg 2 mg 6.3 mg 0.5-1.2 mg/ml 2.8 mg 0.7 mg 4.1 mg 3.1 mg 43.0 mg dry wt 0.8 mg/ml 0.05 mg/ml 5 mg/ml 0.05 mg/rnl 0.6 mg 5.3 mg 1.7-10 mg 6.3 mg 1-4 mg 1-2 mg

184,487,492 184,487,492 184,487,492 184,487,492 184,487,492 492 492 489 491 184,487,490,491 184,487,490,491 184,487,490,491 184,487,490.491 184,487,490,491 184,487,490,491 46 I 46 I 46 I 46 I 489 489 486,487,489 489 184,487,490 184,487,490

1.8 mg 1.3 mg 0.4 mg 1.6 mg 30-40 mg dry wt 0.2 rng

184,487,490 184,487,490 184,487,490 184,487,490 184,487,490 184,487,490

0.5 mg

I.

ALLELOCHEMICAL PROPERTIES O F ALKALOIDS

89

interfere with the metabolism of the producing plant if they would accumulate in the compartments where they are made (25).Whereas biosynthesis takes place in the cytoplasm, or in vesicles (berberine) or organelles such as chloroplasts (QAs, coniine), the site of accumulation of water-soluble alkaloids is the central vacuole, and that of lipophilic compounds includes latex, resin ducts, or glandular hairs (e.g., nicotine) (4,25). In this context it should be recalled that many alkaloids are charged molecules at cellular pH and do not diffuse across biomembranes easily. During recent years, evidence has been obtained that at least some alkaloids pass the tonoplast with the aid of a carrier system. The next problem is determining how the uphill transport, that is, the accumulation against a concentration gradient, is achieved. Proton-alkaloid antiport mechanisms and ion trap and chemical trap mechanisms have been postulated and partially proved experimentally (503,510,512).Thus, the sequestration of high amounts of alkaloids in the vacuole is a complex and energy-requiring task, which would certainly have been lost during evolution were it not important for fitness. As a rule of thumb, we can assume that all parts of an alkaloidal plant contain alkaloids, although the site of synthesis is often restricted to a particular organ, such as the roots or leaves. Translocation via the phloem, xylem, or apoplastically must have therefore occurred. Phloem transport has been demonstrated for quinolizidine, pyrrolizidine, and indolizidine alkaloids, and xylem transport for nicotine and tropane alkaloids (36,39,511).

B. PRESENCE OF ALKALOIDS AT

THE

RIGHTSITEA N D RIGHTTIME

If the plant relies on alkaloids as a defense compound, these molecules have to be present at the right place and at the right time. Alkaloids are often stored in specific cell layers, which can differ from the site of biosynthesis (25,38,39). In lupines, but also in other species (486,4891, alkaloids are preferentially accumulated in epidermal and subepidermal cell layers, reaching local concentrations between 20 and 200 mM (Table X), which seems advantageous from the point of view of chemical ecology, since a pathogen or small herbivore encounters a high alkaloid barrier when trying to invade a lupine. The accumulation of many alkaloids in the root or stem bark, such as berberine, cinchonine, and quinine, can be interpreted in a similar way. A number of plants produce laticifers filled with latex. For example, isoquinoline alkaloids in the family Papaveraceae are abundant in the latex (39), where they are sequestered in many small latex vesicles. In latex vesicles of Chelidonium mujus the concentration of protoberberine and

YO

MICHAEL WINK

benzophenanthridine alkaloids can be in the range of 0.6-1.2 M, which is achieved by their complexation with equal amounts of chelidonic acid (512). If a herbivore wounds such a plant, the latex spills out immediately. Besides gluing the mandibles of an insect, the high concentration of deterrent and toxic alkaloids will usually do the rest, and, indeed, Chelidonium plants are hardly attacked by herbivores. In addition, as these alkaloids are also highly antimicrobial (Table IV), the site of wounding is quickly sealed and impregnated with natural antibiotics. Other well-known plants that have biologically active alkaloids in their latex belong to the families Papaveraceae (genera Papauer, Macleya, and Sanguinaria)and Campanulaceae (genus Lobelia) (39). It is intuitively plausible that a valuable plant organ must be more protected than others. Alkaloid levels are usually highest during the time of flowering and fruit/seed formation. In annual species actively growing young tissue, leaves, flowers, and seeds are often alkaloid-rich, whereas in perennial ones, like shrubs and trees, we find alkaloid-rich stem and root barks in addition. All these plant parts and organs have in common that they are important for the actual fitness or for the reproduction and thus the long-term survival of the species. Spiny species, which invest in mechanical defense, accumulate fewer alkaloids than soft-bodied ones (15); examples are isoquinoline alkaloids in cacti or QAs in legumes (184). If a plant produces few and large seeds, their alkaloid levels tend to be higher than in species with many and small seeds (15,184);thus. a plant with few and big seeds is generally a rich source of alkaloids, which makes sense in view of the defense hypothesis. These few examples show that accumulation and storage of alkaloids have been optimized in such a way that they are present at strategically important sites where they can ward off an intruder at the first instance of attack. Thus, specialized locations must be regarded as adaptive. Alkaloid concentrations can fluctuate during the vegetation period, or even during a day (36,506).but in biochemical terms their biosynthesis and accumulation are constitutive processes. This ensures that a certain level of defensive compounds is present at any time. Furthermore, continuous turnover is a common theme for molecules of the cells whose integrity is important, such as proteins, nucleic acids, and signal molecules. The same seems to be true for a defense compound. An alkaloid which mimics a neurotransmitter, such as hyoscyamine, nicotine, or sparteine, could be oxidized or hydrolyzed in the cell by chance, and thus would be automatically inactivated. Only by replacing these molecules continuously can the presence of the active compounds be guaranteed. For example, it was suggested that nicotine has a half-life of 24 hr in Nicotiana plants, and that more than 10% of the CO, fixed passes through this alkaloid (505).

I.

91

A L L E L O C H E M I C A L P R O P E R T I E S OF A L K A L O I D S

In other groups of natural products it was possible to show that plants can react to infection by microbes or to wounding by herbivores by inducing the production of new defense compounds. These compounds are termed “phytoalexins” in phytopathology (22-24). Classic examples of phytoalexins include isoflavones, phenolics, terpenes. protease inhibitors, coumarins, and furanocoumarins. Using plant cell cultures it could be shown that a similar process can be observed with some alkaloidal plants, which start to produce alkaloids with antimicrobial properties (e.g., sanguinarine, canthin-6-one, rutacridone alkaloids)when challenged with elicitors from bacterial or fungal cell walls (Table XI). But what is the situation after herbivory? When plants are eaten by large herbivores, a de nouo synthesis would be almost useless for a plant (except maybe trees), since this would not be quick enough. The situation is different, however for small herbivores such as insects or worms, which may feed on a particular plant for days or weeks. Here the de nouo production of an allelochemical would be worthwhile. There are indeed some preliminary experimental data that support this view. In Liriodendron rirlipifera several aporphine alkaloids accumulate after wounding, which are otherwise not present (506). In tobacco the producTABLE XI INDUCTION OF ALKALOID BIOSYNTHESIS AFTER WOUNDING OR ELICITATION Alkaloid

Plant species

Alkaloids derived from tryptophan Catharanthus Ajmalicinel catharanthine Canthin-bone Ailunthiis I-Methoxycanthin-6-one

Ailiinrhus

Indole alkaloids Cathurunthus roseus Alkaloids derived from phenylalanine Sanguinarine Papauer bracteaturn Pupauer sornnferurn Eschscholtzia Other types Atropine Afropa Harringtonia alkaloids Lupanine and other quinolizidine alkaloids Methylxanthines Nicotine

Cephuloruxus hurringtoniu Lupinus

Rutacridone alkaloids

Rura graueolens

Coffeu Nicotiana

“ CC, Cell culture; PL, plant

Stimulus

System“

Ref.

Fungal elicitor

cc

4 73

Yeast /fungal elicitor Yeast/fungal elicitor Fungal elicitor

cc

477

cc

477

cc

472

Fungal elicitor Fungal elicitor Fungal elicitor

cc cc cc

466.467 468.469 470.471

Wounding, herbivory Fungal elicitor Wounding Chemical elicitors NaCl Wounding, herbivory Fungal elicitors

PL

48 I

cc

PL

476 482.493 483.485 478 479.480

cc

474.47s

PL

cc cc

92

MICHAEL W I N K

tion of nicotine, in lupines that of QAs, and in Atropci belleidonnu that of hyoscyamine are induced by wounding, thus increasing the already high levels of alkaloids by up to a factor of 5 . Whereas the response was seen after 2-4 hr in lupines, it took days in Nicotiunu and in Atropei (Table XI). We suggest that the wound-induced stimulation of alkaloid formation is not an isolated phenomenon, but rather an integral part of the chemical defense system. The induced antimicrobial and antiherbivoral responses show that plants can detect environmental stress and that secondary metabolism is flexible and incorporated in the overall defense reactions. Many details on how a plant perceives and transmits information remain to be disclosed, but this will surely be a stimulating area of research in the future. Although the physiology and metabolism of most alkaloids are extremely intricate ( 3 8 3 9 ) and often not known, the available data suggest that they are organized and regulated in such a way that alkaloids can fulfill their ecological defense function. In other words, the alkaloids are present at the right time, the right place, and the right concentration.

c. IMPORTANCE O F ALKALOIDS FOR FITNESS OF P L A N T S The aforementioned arguments strongly support the hypothesis that alkaloids serve as defense compounds for plants. Besides circumstantial evidence, we would welcome critical experiments which clearly prove that alkaloids are indeed important for the fitness and survival of the plants producing them. We suggest that if a plant species which normally produces alkaloids is rendered alkaloid-free, it should have a reduced fitness because it is much more molested by microorganims and herbivores than its alkaloid-producing counterpart. For one group of alkaloids, the quinolizidine alkaloids, these experiments have already been performed (2,184,484,503,527).As mentioned before, QAs constitute the main secondary products of many members of the Leguminosae, especially in the genera Lirpinus, Genistu, Cyfisiis, Bccptisiu, Thrrmopsis, Sophoru, Ormosici, and others (503). Lupines have relatively large seeds which contain up to 40-50% protein, up to 20% lipids, and 2-8% alkaloids. To use lupine seed for animal or human nutrition, Homo scipiens, for several thousand years, used to cook the seeds and leach out the alkaloids in running water. This habit has been reported for the Egyptians and Greeks in the Old World, and for the Indians and Incas of the New World. The resulting seeds taste sweet, in contrast to the alkaloid-rich ones which are very bitter. In Mediterranean countries people still process lupines in the old way, and sometimes the

1 . ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

93

seeds are salted afterward and served as an appetizer, comparable to peanuts. At the turn of the twentieth century, German plant breeders set out to grow alkaloid-free lupines, the so-called sweet lupines. Although sweet lupines are extremely rare in Nature ( 1 in >100.000), the efforts were largely successful, and at present, sweet varieties with an alkaloid content lower than 0.01% exist for Lupinus albus, L. mutabilis, L . luteus, L . angustifolius, and L . polyphyllus. As far as we know, the sweet varieties differ from the original bitter wild forms only in the degree of alkaloid accumulation. This offers the chance to test experimentally whether bitter lupines have a higher fitness than sweet ones with regard to microorganisms and herbivores. The results of these experiments were clearcut (2,184503,506,527)(Table XU). In the greenhouse, where plants are protected from herbivores or pathogens, no clear advantage was seen. When lupines were planted in the field, without being fenced in and without man-made chemical protection, however, a dramatic effect was regularly encountered, especially with regard to herbivores (2,184,503,527).Rabbits (Cuniculus europaeus) and hares (Lepus europaeus) clearly prefer the sweet plants and leave the bitter plants almost untouched, at least as long as there was an alternative food source. Before dying rabbits will certainly try to eat bitter lupines. A similar picture was seen for a number of insect species, such as aphids, beetles, thrips, and leaf-mining flies (Table XII), namely, the sweet forms were attacked, whereas the alkaloid-rich ones were largely protected. The alkaloid-poor variety of L . luteus also became a host of Acyrthosiphon pisii (506).In Poland, where the sweet yellow lupine is one of the more important fodder plants, the invasion of the aphids became a serious problem not only because the aphid enfeebles the plants by sucking its phloem sap, but also because it transfers a viral disease. The disease, known as lupine narrow leafness, decreases seed production in infected plants, and the infection takes place early, that is, prior to the plants’ blossoming. Thus, a mixed population of sweet and bitter lupines can, after a few generations, lose all sweet forms. Infestation by the aphid and the following viral infection accelerate the elimination of alkaloid-poor plants, which, even without infection, are already inferior in seed production (506).This observation again stresses the importance of alkaloids for the fitness of lupines. Plant breeders have also observed that bacterial, fungal, and viral diseases are more abundant in the sweet forms, but this effect has not been documented in necessary detail.

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MICHAEL WINK

TABLE XI1

BITTER(ALKALOID-RICH) VERSUS Lupine species

Species Nonadapted herbivores Vertebrates Sheep

S W E E T (LOW-ALKALOID) LUPINES

Alkaloid content

n.i."

n.i.

Lepus europaeus

n.1.

n.i.

Oryrolagus europaeus

L. olbtrs

0.01 mgig 2.0 mg/g

L . alhrts

0.01 mg/g

Insects Agrom yzidae

Sirona linearus

L . ulhrts L . mttrabilis

Myzus sps

L. lulrlts

Acyrrhosiphon pisum

Litpinus

Aphis faboe

L . polyph~llus

Frunkliniello r r i d c i

Lrtpinus

F. bispinoso

Lupinus

2.0 mgig 2.2 mgig <0.02 mg/g 1500 mg/g 2500 mg/g 0.01 mgig >0.7 mglg Sweet Bitter Sweet Bitter Sweet Bitter Sweet Bitter

Effect

Sweet lupines are preferred bitter discriminated Sweet lupines are preferred bitter discriminated Herbivory almost 100% Herbivory
Ref

458 459.460 2,184,527

2.527

460 460 46 I 460.462 463 464 464

Adapted herbivores Macrosiphum albifrons

L . albus

0.01 mgig

2.0 mgig 2.2 mg/g

L . polvphvllrr.~ L. ungustifolirrs L . mrrrabilis

>I I .5 2.5

mg/g mg/g mg/g

Infestation Infestation Infestation Infestation Infestation Infestation

< 10%

465

100%)

100%

80% 100% 30%

465 465 465

mi., No information

These experiments and observations clearly prove the importance of QAs for lupines, but it should not be forgotten that other secondary metabolites, such as phenolics, isoflavones, terpenes, saponins, stachyose, erucic acid, and phytic acid, are also present in lupines and may exert additional or even synergistic effects. The lupine example also tells us about the standard philosophy and problems of plant breeding. With our present knowledge on the ecological importance of QAs for the fitness of lupines, it seems doubtful whether the selection of sweet lupines was a wise decision. In order to grow them

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

95

we have had to build fences and, worse, to employ man-made chemical pesticides, which have a number of well-documented disadvantages. It can be assumed that similar strategies, namely, breeding away unwanted chemical traits, have been followed with our other agricultural crops, with the consequence that the overall fitness was much reduced (2). We can easily observe the reduced fitness by trying to leave crop species to themselves in the wild: they will quickly disappear and not colonize new habitats. There are, however, alternatives. Taking lupines as an example, we could devise large-scale technological procedures to remove alkaloids from the seeds after harvest (similar to sugar raffination from sugar beets). At present a few companies are actively exploring these possibilities. One idea is to produce pure protein, lipids, dietary fibers from bitter seeds. A spin-off product would be alkaloids, which could be used either in medicine (sparteine is exploited as a drug to treat heart arrhythmia) or in agriculture as a natural plant protective, that is, as an insecticide (185,503). It is evident, however, that each plant has developed its own strategy for survival. If all plants would follow the same strategy, it would be an easy life for herbivores and pathogens, since being adapted to one species would mean adapted to all species. This specialization becomes evident if we analyze the qualitative patterns of secondary metabolite profiles present in the plant. We regularly see one to five main alkaloids in a plant, but also several (up to 80) minor alkaloids. This qualitative pattern is not constant, but differs among organs, developmental stages, individuals, populations, and species. Normally, we classify the compounds as belonging to one or two chemical groups. This does not mean, however, that their biological activities are identical. On the contrary, the addition of a lipophilic side chain to a molecule seems to be a small and insignificant variation from the chemical point of view, but this may render the compound more lipophilic, and thus more resorbable. In consequence, its toxicity may be higher (see QAs in Table I). Thus, a herbivore or pathogen has to adapt not only to one group of chemicals but to the individual compounds present. As the composition of these chemicals changes, it is even more difficult for them to cope. Therefore, we suggest that structural diversity and continuous variation are means by which Nature counteracts the adaptation of specialists. In medicine, we do a similar thing if we want to control microbial diseases. To overcome or to prevent resistance of bacteria toward a particular antibiotic, very often mixtures of structurally different antibiotics are applied, whose molecular targets often differ. If only one antibiotic were given to all patients, the development of resistance would be much favored.

96

MICHAEL W I N K

It has been argued that alkaloids cannot have a significant role in plants because not all plant species produce alkaloids (only 30% of all plants do). These authors, such as Robinson ( 5 0 3 , have overlooked the fact that if all plants would produce one single alkaloid, even a very toxic alkaloid such as colchicine, it could be certain that nearly all herbivores would have developed a resistance toward this alkaloid. Only the variation of secondary metabolites, and thus of the targets which they affect, provides a means to develop efficient defense compounds. The arguments of Robinson would be correct if there were higher plants without any secondary metabolites, which, nevertheless, would thrive in Nature; however, these plants are not known. From an evolutionary perspective it is not important whether the defense chemical is an alkaloid or a terpene; it is only essential that it affect certain and important targets in herbivores or pathogens. Although the biological activities of many alkaloids have not yet been studied and their ecological functions remain to be elucidated or proved, we can nevertheless safely say that alkaloids are neither waste nor functionless molecules, but rather they are important fitness factors, probably mostly antiherbivore compounds. Since Nature obviously favored multitasking, additional activities, such as allelopathic or antimicrobial activities, are plausible. For quinolizidine and pyrrolizidine alkaloids, these multiple functions are already well documented (Tables I-X).

D. EXCEPTIONS TO THE RULE: ROLE OF ADAPTED SPECIALISTS 1 . Microorganisms

Plants that defend themselves effectively constitute an ecological niche almost devoid of herbivores and pathogens. It is not surprising that during evolution a number of organisms evolved which have specialized on a particular host plant species and found ways to tolerate, or even to exploit, the defense chemistry of their hosts (4,10-22). As compared to the huge number of potential enemies, the number of adapted specialists is usually small, and in general a “status quo” or equilibrium can be observed between the specialists (or parasites) and their hosts. A specialist is not well advised to kill its host, since this would destroy its own resources; a mutualism is more productive for survival. Host plant-specific specialists occur within bacteria, fungi, and herbivores. The interaction of the former two groups is a central topic for plant pathologists. They often find that susceptible and nonsusceptible microbe strains exist. In most cases, it is not known how these microbial specialists achieved a relationship with the host plant chemistry, for example, whether they degrade secondary metabolites or whether they simply toler-

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

97

ate them. Many phytopathogenic bacteria and fungi produce their own secondary metabolites, which are often toxic to plants. It is assumed that these phytotoxins serve to weaken the host plants’ defense, but may be this is not the whole story. Many grasses are infected with fungi that produce ergot alkaloids. It has been assumed that these fungi (e.g., Clauiceps) are proper parasites. In recent years, however, experimental evidence suggests that the relationship between grasses and ergot may be of a symbiotic nature (513). Ergot alkaloids are strong vertebrate toxins (Tables I-IV); they mimic the activity of several neurotransmitters, such as dopamine, serotonin, and noradrenaline (Table IV). In fact, the impact of herbivores on populations which were highly infected by fungi was more reduced than those without. This means that the fungi exploit the nutrients of their host plants and supply them with strong poisons, which are not produced by the plants themselves. Since the fungi do not kill their hosts, this close interrelationship seems to be of mutual interest. We expect that similar relationships are likely to be detected in the future. 2. Insect Herbivores As mentioned earlier, a large number of mono- and oligophagous insects exist which have adapted to their host plants and the respective defense chemistry in complex fashions. In general, we can see the following main schemes (4,15,17,32,507,508). In Type 1 adaptations, a species “learns” (or, as we should say, during evolution variants have been selected by natural selection which can tolerate a noxious defense compound) (a) by finding a way to avoid its resorption in the gut; (b) if resorption cannot prevented, by eliminating the toxin quickly via the Malpighian tubules or degrading it by detoxifying microsomal and other enzymes; and (c) by developing a target site that is resistant to the toxin, such as a receptor which no longer bind the exogenous ligand. Alternatively, in Type 2 strategies a species not only tolerates a plants’ defense compound, but exploits it for its own defense or for other purposes, such as pheromones (4,I7,494496,506). Examples of Type 1 include Manduca sexra, whose larvae live on Nicoriana and other solanaceous plants. The alkaloids present in these plants, such as nicotine or hyoscyamine, are not stored but are degraded or directly eliminated with the feces (182). In addition, it has been postulated that nicotine may either not diffuse into nerve cells or that the acetylcholine recpetor no longer binds nicotine as in “normal” animals (17). The potato beetle (Leptinotarsa decernlineata) lives on Solanurn species containing steroid alkaloids, which are tolerated, but not stored, by this species, The bruchid beetle Callosohruchus fasciarus predates

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seeds of QA-rich plants, such as Laburnum anagyroides; this beetle eliminates most of the dietary cytisine with the feces (492). Examples of Type 2 are to some degree more interesting. In a number of plants alkaloids are translocated via the phloem (511).When aphids live on these plants they are in direct contact with the alkaloids present. A number of examples are known at present which show that adapted aphids can store the dietary alkaloids. Examples are the quinolizidines in Aphis cytisorum, A. genistae, and Macrosiphum albifrons, the pyrrolizidines in Aphis jacobaea, A . cacaliaster, and aconitine in Aphis aconiti (185,511). For alkaloid-storing M . albifrons it was shown experimentally that the QAs stored provide protection against carnivorous beetles, such as Carabus problematicus or Coccinella septempunctata (465,503). Acyrthosiphon spartii prefers sparteine-rich Cytisus scoparius plants (506);although it is likely that this species also stores QAs, it has not been demonstrated to do so. Larvae of the pyralid moth Uresiphita reversalis live on QA-producing plants, such as Teline monspessulana. The larvae store some of the dietary alkaloids, especially in the integument and also the silk glands. The uptake is both specific and selective and is achieved by a carrier mechanism. Whereas alkaloids of the 10-oxosparteine type dominate in the plant, it is the more toxic cytisine that is accumulated by the larvae, with the 10oxosparteines being eliminated with the feces (503,514).The larvae gain some protection from storing QAs, as was shown in experiments with predatory ants and wasps. When the larvae pupate, most of the alkaloids stored are used to impregnate the silk of the cocoon, thereby providing defense for this critical developmental stage (503,514).The emerging moth lives cryptically, has no aposematic coloring, and does not contain alkaloids. In contrast the alkaloid-rich larvae are aposematically colored and live openly on the plants (503,514). The larvae of the blue butterfly (Plebejus icaroides) feed only on lupines, rich in alkaloids. As far as we know, the larvae do not sequester or store the dietary alkaloids (506). Helopeltis feeds on Cinchona bark, which is rich in cinchonine-like alkaloids; it stores and uses them for its own defense (506).Larvae of the butterflies Pachlioptera aristolochiae, Zerynthia polyxena, Ornithoptera priamus, and Battus philenor live on Arisrolochia plants and were shown to take up and sequester aristolochic acid, a carcinogenic alkaloid discussed earlier, as an effective defense compound (4,28,236). The best-studied group of acquired alkaloids are the pyrrolizidines, which are produced by plants, especially in the families Asteraceae and Boraginaceae (502). Some arctiid larvae of Tyria jacobaea, Cycnia mendica, Amphicallia bellafrix, Arginia cribaria, and Arctia caja were shown

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to store the dietary PAS and exploit them for their own defense (4,17,28,31,222-224,237).In Tyria jacobaea, Arctia caja, Diacrisia sannio, Phragmatobia fuligonosa, and Callimorpha dominula PAS are taken up and stored in the integument (523). Monarch butterflies (e.g., Danaus plexipus) combine two sets of natural compounds. Larvae feed on plants rich in cardiac glycosides and use them as chemical defense compounds. Adult butterflies visit plants with PAS, where they collect PAS that are converted to pheromones or transferred to their eggs (4,f7,31,33,36f,515).A similar PA utilization scheme was observed with larvae of the moth Utetheisa ornatrix (367,516), where the compounds were shown to be deterrent for spiders and birds (225, 525). The chrysomelid beetle Oreina feeds on PA-containing plants, such as Adenostyles, and stores the dietary PAS in the defense fluid (463,524). In the arctiid Creatonotos transiens was observed an advanced exploitation of PAS (31,33,429,517-521). The alkaloids are phagostimulants for larvae, which are endowed with specific alkaloid receptors. Dietary pyrrolizidine N-oxides are resorbed by carrier-mediated transport. After resorption, free PAS are converted to the respective N-oxides and (7S)-heliotrine to (7R)-heliotrine. The latter form is later converted to a male pheromone, (7R)-hydroxydanaidal. PAS are stored in the integument, where they serve as defense compounds and are not lost during metamorphosis. In the adult moth, however, the PAS are mobilized. In the female adult, PAS are translocated into the ovary and subsequently into the eggs. In the male, PASare necessary for the induction of abdominal scent organs and concomitantly for the biosynthesis of PA-derived pheromones, which are dissipated from these coremata. In addition, PAS are transferred into the spermatophore and thus donated to the female. A significant amount of PAS is further transferred to the eggs, which thus obtain chemical protection from the PAS previously acquired by both male and female larvae. Marine dinoflagellates produce a number of toxins, such as saxitoxin, surugatoxin, tetrodotoxin, and gonyautoxin, that affect ion channels (Table IV). These algae are eaten by some copepods, fish, and molluscs that also store these neurotoxins (4,17,28,29,494,495).As a consequence, these animals have acquired chemical defense compounds, which they can use against predators. This discussion is not meant to be complete, but should illustrate that a number of insect herbivores exploit the chemistry of their food plants. These insects are adapted and have evolved a number of molecular and biochemical traits that can be considered as prerequisites. However, many of the respective plant-insect interactions have not yet been studied, and

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it is therefore likely that the acquisition of dietary defense compounds is even more widely distributed in Nature than anticipated. 3. Vertebrate Herbivores

Whereas insect herbivores are often highly host plant specific, vertebrate herbivores tend to be more of the polyphagous type, although some specialization may occur. For example, grouse (Lagopus lagopus) or capercaillies (Tetra0 urogallus) prefer plants of the families of Ericaceae or Coniferae, and crossbills seeds of Picea and Abies species, which are rich in terpenes. The Australian koala is oligophagous and prefers terpene-rich species of the genus Eucalyptus. For approximately 65 million years, the only true herbivorous vertebrates have been the mammals. The Mesozoic reptiles disappeared following the mesophytic flora. Birds, though a few species feed on seeds and berries, seldom eat leaves (except geese and grouse), and they frequently use insects, in addition to plant parts, as a food source (18). Although a single plant can be a host for hundreds of insect larvae, hundreds of plants comprise a daily menu for a larger mammal. The strategies of the polyphagous species include the following. 1. Avoidance of plants with very toxic vertebrate poisons (these species are usually labeled toxic o r poisonous by man) by olfaction or taste discrimination. Often such compounds may be described as bitter, pungent, bad smelling, o r in some other way repellent. 2. Sampling of food from a wide variety of sources and thus minimizing the ingestion of high amounts of a single toxin. 3. Detoxification of dietary alleochemicals, which can be achieved by symbiotic bacteria or protozoa living in the rumen or intestines, or by liver enzymes which are specialized for the chemical modification of xenobiotics. This evolutionary trait is very helpful for Homo sapiens, since it endowed us with a means to cope with our man-made chemicals which pollute the environment. Carnivorous animals, such as cats, are known to be much more sensitive toward plant poisons (505). It was suggested that these animals, which d o not face the problem of toxic food normally, are thus not adapted to the handling of allelochemicals. 4. Some animals, such as monkeys, parrots, or geese, ingest soil. For geese (185) it was shown that the ingested soil binds dietary allelochemicals, especially alkaloids (185). This procedure would reduce the allelochemical content available for resorption. 5. Animals are intelligent and can learn. The role of learning in food and toxin avoidance should not be underestimated, but it has not been studied in most species.

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For most vertebrate herbivores, the ways they manage to avoid, tolerate, or detoxify their dietary allelochemicals have not been explored. Sometimes, only domesticated animals were used in experiments, but they tend to make more mistakes in food choice than the wild animals. More evidence on this subject is available for Homo sapiens, who has evolved a number of “tricks,” some of them obviously not anticipated by evolution. First, man tends to avoid food with bitter, pungent, or strongly scented ingredients. As a prerequisite he needs corresponding receptors in the nose or on the tongue which evolved during the long run of evolution as a means to avoid intoxication. Second, our liver still contains a set of detoxifying enzymes which can handle most xenobiotics. Furthermore, some of these enzymes, such as cytochrome P.450 oxidase, is inducible by dietary xenobiotics. Third, besides these biological adaptations, man has also used his brain to avoid plant allelochemicals. (a) Many fruits or vegetables are peeled. As many alkaloids and other compounds are stored in the epidermis, for example, steroid alkaloids in potato tubers or cucurbitacins in cucurbits, peeling eliminates some of these compounds from consumption. (b) Most food is boiled in water. This leads to the thermal destruction of a number of toxic allelochemicals, such as phytohaemagglutinins, protease inhibitors, and some esters and glycosides. Many watersoluble compounds are leached out into the cooking water and are discarded after cooking (e.g., lupines or potatoes). (c) South American Indians ingest clay when alkaloid-rich potato tubers are on the menu. Since clay binds steroidal alkaloids, geophagy is thus an ingenious way to detoxify potential toxins in the diet (522).(d) Man has modified the composition of allelochemicals in his crop plants, in that unpleasant taste components have been reduced by plant breeding. From the point of view of avoidance, this strategy is plausible, but, as was discussed earlier, it is deleterious from the point of view of chemical ecology. These plants often lose their resistance against herbivores and pathogens, which then has to be replaced by man-made pesticides. In general, only a few plants are exploited by man as food, as compared to the 300,000 species present on our planet. This means that even Homo sapiens with all his ingenuity has achieved only a rather small success, indicating the importance and power of chemical plant defenses. 4. Alkaloid Production by Animals

In this context, it is worth recalling that a number of animals are able to synthesize their own defense compounds, among them several alkaloids (4,17,28,494-496). These animals have the common feature that they are usually slow-moving, soft-bodied organisms. Marine animals, such as mol-

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luscs, sponges, zooanthids, and fishes, have been shown to contain a variety of alkaloids, such as acrylcholine, neosaxitoxin, murexin, pahutoxin, palytoxin, petrosine, and tetramine, that are toxic to other animals (4.17,28,29,221,226,229,232,233,234,495). A number of nemertine worms, such as Amphiporus or Nereis, produce alkaloids such as 2,3-bipyridyl, anabaseine, nemertelline, or nereistoxin, which are toxic to predators such as crayfish ( 4 1 7,28,230,226,). Arthropod-made alkaloids include glomerine and homoglomerine in Glomerus (215), adaline in Adalia (227),coccinelline, euphococcinine, and derivatives in Coccinella, Epilachna, and other coccinellid beetles (28,226,227,235),and stenusine in Stenus (215),which are considered to be antipredatory compounds (4,17,28,494-496). Solenopsis ants produce piperidine alkaloids which resemble the plant alkaloid coniine. These alkaloids are strong deterrents and inhibit several cellular processes, such as electron transport chains (Table IV) (28,494). Many insects indicate the content of toxic natural products by warning colors (aposematism) or by the production of malodorous pyrazines (4,17,231,494). Not only are lower animals able to synthesize alkaloids, but also vertebrates, especially in the class Amphibia. Tree frogs of the genus Dendrobates accumulate steroidal alkaloids, such as batrachotoxin, pumiliotoxins A-C, gephyrotoxin, and histrionicotoxin, in their skin, which are strong neurotoxins (Table IV) (4,17,28).Natives have used the alkaloids as arrow poisons. Similar alkaloids (i.e., homobatrachotoxin) have recently been detected in passerine birds of the genus Pitohui (528).Salamanders, Salamandra maculosa, which are aposematically colored, produce the toxic salamandrine and derivatives, alkaloids of the steroidal group (4,17,28). Salamandrine is both an animal toxic (paralytic) and an antibiotic. Toads (Bufonidae) produce in their skin cardiac glycosides of the bufadienolide type, but also a set of alkaloids, such adrenaline, noradrenaline, adenine, bufotenine, or bufotoxin (4,17,28).Except for bufotoxin, the other chemicals are, or mimic, neurotransmitters. These examples show that alkaloids found in animals can either be derived from dietary sources (see Section 111,D,2) or be made endogenously. Common to both origins is their use as chemical defense compounds, analogous to the situation found in plants. In animals we can observe the trend that sessile species, such as sponges and bryozoans, or slow-moving species without armor, such as worms, nudibranchs, frogs, toads, and salamanders, produce active allelochemicals (28,29,494,495), but not so those with weapons, armor, or the possibility for an immediate flight. Plants merely developed a similar strategy as these “unprotected”

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animal species. In this context it seems amazing that hardly anybody has doubted the defensive role of alkaloids in animals, whereas people did, and still do, where alkaloids in plants are concerned.

IV. Conclusions

Evidence is presented in this overview that alkaloids are not waste products or functionless molecules as formerly assumed (34,35),but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. These molecules were obviously developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells (i.e. they are specific inhibitors or modulators), which can clearly be seen in molecules that mimic endogenous neurotransmitters (Table IV; Section II,A,3,a). On the other hand, microorganisms and herbivores rely on plants as a food source. Since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. Many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. In addition, many herbivores have potent mechanisms to detoxify xenobiotics, which allows the exploitation of at least the less toxic plants. In insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. Alkaloids obviously function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. The overall picture of alkaloids and their function in plants and animals seems to be clear, but we need substantially more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants.

Acknowledgments The work of the author was supported by the Deutsche Forschungsgemeinschaft. I thank Dr. Th. Twardowski for reading an earlier draft of the manuscript.

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-CHAPTER 2-

MAMMALIAN ALKALOIDS 11” ARNOLDBROSSI Depurtment of Chemistry Georgetown University Washington, D.C. 20057

I. Introduction ......................................................................................

11. Mammalian Indole Alkaloids ...................................

111.

IV. V. VI. VII.

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122 A. P-Carbolines ................................................................................. 136 B. Other Indole Alkaloids Found in Mammals ........................................ Mammalian lsoquinoline Alkaloids ...... 141 A. Tetrahydroisoquinolines and 1-Carb 141 B. Pyridoxal-Derived Isoquinolines ....................................................... 160 Mammalian Morphine ....... A. Introduction ............... B. Biosynthetic Pathways t Alkaloid Formation in Mammals as a Therapeutic Concept ....................... 168 Addendum ........................................................................................ 169 Conclusions ......................................................... ............. 172 References ........................................................................................ 173

I. Introduction

Alkaloids formed endogenously in mammals and in man are named “mammalian alkaloids.” These substances are closely related to, and sometimes identical with, alkaloids found in plants. The alkaloid harman, which occurs in rat brain (I), also is present in the bark of tropical trees ( 2 3 and in marine organisms (4). Chemical structures of mammalian and plant alkaloids illustrating such similarities are shown in Fig. 1. Mammalian alkaloids were reviewed in Vol. 21 of this treatise ( 5 2 ) ; they have been referred to in connection with reviews on indole and * This chapter is dedicated to Professor Oskar Jeger from the Laboratory of Organic Chemistry at the Swiss Federal Institute of Technology in Zurich, Switzerland, in honor of his 75th birthday. Professor Jeger, called Oskar by his friends, was a master in the teaching and exploration of the chemistry of natural products. Oskar inspired many of us who later devoted their careers to this discipline. I19

THE A L K A L O I D S . V O L 43 Copyright 0 IYY3 by Academic Press. Inc All rights of reproduction in any form reserved

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ARNOLD BROSSl

(SMALSOLINOL [MAMMALS)

(S)-ISOSALSOLINE (PLANTS)

[S)-6HYDROXYTETRAHYDRO-~~ HARMALINE (PLANTS) CARBOLINE (MAMMALS) FIG. 1. Structural comparison of mammalian and plant alkaloids.

isoquinoline alkaloids (5b-f) and have been discussed on other occasions (6a-h). This chapter discusses the chemistry of mammalian alkaloids, their occurrence, analytical methods for their detection and quantitation, and their biological properties, and it updates our present knowledge of this somewhat obscure group of natural products. Minute amounts of these alkaloids are found in human tissues and fluids, in platelets, and as urinary or fecal excretion products. Enhanced amounts of these alkaloids are produced in medical disorders such as phenylketonuria, as a result of drug regimens such as L-dopa therapy, on intake of excessive amounts of L-tryptophan and possibly other amino acids, and on excessive ethanol consumption. The presently known mammalian alkaloids are either isoquinolines originating from amino acids and amines derived from Ldopa and dopamine, or indoles similarly derived from L-tryptophan and tryptamine. Alkaloids related to L-histidine and histamine that occur in shark liver (7) and in frog skin (8,9) have so far not been detected in humans. The mammalian alkaloids are formed from aromatic amino acids and their metabolically derived amines by reaction with carbonyl substrates at physiological pH. The reaction is catalyzed by acid and is commonly referred to as the Pictet-Spengler cyclization (fOa,b,If). Winterstein and Trier in 1910 proposed that the Pictet-Spengler reaction might be of significance in the biosynthesis of benzylisoquinoline alkaloids in plants (5a). The carbonyl compounds participating in the Pictet-Spengler synthesis of mammalian alkaloids are aldehydes and a-keto acids, which are produced

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MAMMALIAN ALKALOIDS I1

121

endogenously during amine and amino acid metabolism. Acetaldehyde, often mentioned as a carbonyl substrate, is believed to be derived from ethanol and to be the origin of the 1-methyl group in P-carbolines. The 1methyl group in these alkaloids, however, and as discussed later, more likely originates from an initial condensation of amines o r amino acids with pyruvic acid. The Pictet-Spengler cyclization proceeds in uitro as in Fig. 2, which shows a hypothetical example in the isoquinoline series, via several distinct intermediates which cannot be isolated in the laboratory (6c,106). Addition of acetaldehyde to a phenethylamine activated by a phenolic hydroxy group first affords a carbinolamine, which, under the influence of acid, dehydrates to a Schiff base that is most likely present as the more stable E isomer, affording on protonation directly the racemic tetrahydroisoquinoline [( *)-TIQ] (IOa,b).In attempting to explain the formation of optically active mammalian isoquinolines in uiuo, we cannot disregard the possibility that the phenolic group of the phenethylamine substituent is

L

SCHIFF BASE

-

FIG.2. Pictet-Spengler cyclization to tetrahydroisoquinolines.

122

ARNOLD BROSSI

sufficiently acidic to promote an intramolecular cyclization of a chiral carbinolamine, or its activated bioequivalent, to afford optically active TIQ in a SN2 type reaction (6c,10b).

11. Mammalian Indole Alkaloids A. p-CARBOLINES

The occurrence of mammalian p-carbolines, their formation, and their biological properties have been reviewed (5a,6c-h). The nomenclature used for mammalian p-carbolines, as illustrated in Fig. 3, is quite confusing. It became particularly complicated with the unnecessary introduction of the name “tryptoline” for 1,2,3,4-tetrahydr0-/3-carbolineand the use of a different numbering. We use here the p-carboline nomenclature and its numbering of carbon and nitrogen atoms (12). We have also adopted the following abbreviations: BC, p-carboline; DBC, 3,4-dihydro-p-carboline; and TBC, 1,2,3,4-tetrahydro-p-carboIine.A lowercase a following the numbers will be used to mark the ( S )absolute configuration of the alkaloid, and b similarly marks the ( R ) enantiomer.

5

4

CH3

Tetrahydroharmine l-Methyl-7-methoxy-l.2.3.4tetrahydro-bet-carboline 1.2.3.4Tetrahydro-7-methoxy-l-methyl-9H-pyridd3,~blindole 6Methoxy-Smethykryptoline

Harmine. Banisterine 7-Methoxy-l-methyl-9H-pyridd3.4blindole 1-Methyl-7-methoxy-beta-carboline

Beta-Carboline Norharman

Harmaline 4,SDihydro-7-methoxy-l-methyl~3H-pyridd3,4b~indol~ 1-Methyl-7-methoxy-3,4dihydro-beta-carboline Harmalol methyl ether

FIG.3. Nomenclature used for P-carbolines and analogs.

2. MAMMALIAN

ALKALOIDS I1

123

1. Chemistry

The chemistry of P-carbolines of plant origin (5b-f) as well as the history and ethnopharmacological background of these alkaloids have been reviewed ( 5 4 . The chemistry of the mammalian p-carbolines relevant to synthesis and further chemical transformation, however, has never been presented. With the information detailed in Figs. 4-7, and in Fig. 8 for the preparation of optically active representatives, this gap has now been filled. There is now good evidence that the Pictet-Spengler reaction in the formation of TBC proceeds through spiro intermediates, and details of the reaction sequence were recently published (5c). The condensation of tryptamine with an aldehyde in the presence of acid generates mainly the (E)-iminium cation, in which the bulkiest group displays a trans relationship. Nucleophilic attack at the iminium ion can take place with the -NH-CH=CHmoiety from either below or above the plane of the indole ring, affording on migration TBC (Fig. 4). The classic chemical synthesis for the preparation of TBC, DBC, and BC is summarized in Fig. 5 . Acylation of tryptamines (1)affords indolylethylamides (2), which on Bischler-Napieralski cyclization give DBCs (3)

R

FIG.4. Pictet-Spengler cyclization of Schiff bases derived from tryptamine.

124

ARNOLD BROSSI

5

8

FIG.5. Synthesis of tetrahydro-,B-carbolines,dihydro-P-carbolines,and P-carbolines.

(13-16). Chemical reduction of DBCs (3) with sodium borohydride in alcohol readily yields TBCs 4 (17,18). Heating DBCs (3) over palladium catalyst to high temperatures leads to BCs (5) (13), but the fully aromatic compounds are more easily available by dehydrogenation of TBCs 4 with platinum catalyst in refluxing toluene (18).The p-carboline lactams (7) are accessible by Fischer indole synthesis (19), and are converted to TBCs by reduction with lithium aluminum hydride in carefully selected solvents, such as benzene. Reduction of lactams (7) that are substituted in the aromatic ring by halogen affords halogen-substituted TBCs (20). The Pictet-Spengler cyclization of tryptamines with glyoxylic acid or with pyruvic acid yields TBCs 6 (R'= H and CH,, respectively) (20,21). These TBCs are substituted at C-1 by a carboxy group and have been investigated by Hahn and colleagues in great detail (22a,b). They decarboxylate on heating in the presence of mineral acids, and practical procedures for this process are available (21). Alkylation of the indole nitrogen in TBCs 4 with methyl iodide in liquid ammonia in the presence of sodium amide was accomplished after protection of the basic nitrogen with an acetyl group, affording, after acid hydrolysis, the TBC 8 (15). The Pictet-Spengler reaction of tryptamines or tryptophans with aldehydes (23) often proceeds under so-called physiological conditions (pH 6-7), and it has proved to be the most efficient route to TBCs.

2.

125

MAMMALIAN ALKALOIDS I1

There are several chemical reactions which are relevant to the chemistry of mammalian 0-carbolines, some of which are summarized in Figs. 6 and 7. N-Acetyltryptophan ethyl ester on reaction with Lawesson’s reagent afforded the thioamide 9, which with methyl iodide in acetone yielded the methylthioiminium salt 10 which cyclized spontaneously to the quaternary salt 11 (24). BC 12 on condensation with benzaldehyde gave stilbene l3, which on oxidation with potassium permanganate afforded

I H

CH3

H H3C SCH3

9

10

H

CH3 11

H

CH3

H

12

CH=CH-Ph 13

H

I COOH 14

FIG.6

126

ARNOLD BROSSI

CH3O 0 y Q N T C H 3

-

CH30 QYQN-CHs

CH3 CH3 17 18 FIG.7. Interesting reactions of tetrahydro-picarbolinesand p-carbolines.

carboxylic acid 14 (25). Oxidation of the N-acetylated TBC 15 with air led to a compound which, on the basis of UV spectral analysis, is most likely the quinonimine 16 (26). Conversion of the quaternary salt 17 to the anhydronium base 18 by abstraction of the indole proton with base was investigated many years ago in connection with the chemistry of Harmala alkaloids (27), and more recently by Potier and co-workers for a different purpose (28).

2 . Optically Active Tetrahydro-p-carbolines Tetrahydro-p-carbolines substituted at C- 1 by a methyl group occur in mammalian tissues and fluids as optical isomers, and they are often present in unequal proportions of the enantiomers (29,30).There are now several practical methods available to prepare optically active TBC, as summarized in Fig. 8. Pictet-Spengler cyclization of L-tryptophan with formaldehyde afforded the monochiral carboxylic acids 20a,b, whereas cyclization with acetaldehyde yielded the diastereomeric carboxylic acids 21a,b (23). Acids 20a,b with a hydrogen at C-1 are enantiomers, but acids 21a,b are diastereomers; the cis isomer 21a was the major reaction product when the cyclization of L-tryptophan with acetaldehyde was carried out in the presence of sulfuric acid. Direct removal of the carboxy group in these acids is difficult, but it can be accomplished in several steps: dehydration of the amides prepared from the acids with phosphorus oxychloride affords nitriles, and the nitrile group can be removed by reduction with sodium borohydride in pyridine-ethanol (31). Chemical resolution of tetrahydroharmine (4 in Fig. 8) with camphorsulfonic acid afforded the optically pure enantiomers (4a,b) (32). Fragmen-

2.

127

MAMMALIAN ALKALOIDS I1

Q Q c C C 0 O H H

19

3

2oa 21a

R

R=H R=CHs

i

20b 21b

4c

FIG.8. Preparation of chiral tetrahydro-P-carbolines.

tation of the phenylethyl-substituted urea diastereomer 22 in refluxing butanol in the presence of sodium butoxide, on the other hand, gave an optically impure isomer (33).It was reported that the optically active tetrahydroharmines (4a,b) racemized on standing in chloroform solution or on heating in aqueous solution in the presence of acid (32). This behavior might explain the different optical rotations which were reported for the optically active alkaloids (33,34).The asymmetric reduction of the imine (3) obtained by Bischler-Napieralski synthesis with chiral acyloxy-borohydrides, prepared from (S)-N-acylprolines, afforded the @)-tetrahydroharman (4c) of good optical purity (35).The Yamada method is at the moment the easiest route to optically active TBCs. An interesting optically active TBC, which has almost gone unnoticed, is the dicarboxylic acid 23a prepared from L-tryptophan and pyruvic acid (36) (Fig. 9). Acid 23a was obtained as the major product of the two diastereomers formed, and on hydrolysis with 25% sulfuric acid at 80°C it afforded several products. Four of these (3, 5, 21a, and Ma), which are shown in Fig. 9, were identical with products obtained by acid hydrolysis of casein. It is believed that acid 23a originates from a condensation of tryptophan with pyruvic acid derived from an amino acid precursor, and that 23a is the intermediate in the formation of the other carbolines. Although no configurational assignments were made for 23a, it can be assumed that it has the (lS,3S) absolute configuration as shown. This is

128

ARNOLD BROSSI

AH3

3

5

Q)--yooH q i i c o o H /

21a 24a FIG.9. Acid hydrolysis of tetrahydro-P-carboline (1S,3S)-dicarboxylic acid (23a).

based on the identity of its physical data with those reported for 23a obtained by synthesis (23). [mp 296°C; [aID - 115" (pyridine)]. 3. Occurrence of Mammalian p-Carbolines The mammalian p-carbolines shown in Fig. 10were detected by GC-MS techniques, often supplemented by comparison with synthetic standards and with derivatives obtained on reaction with analytical reagents. The presence of optically active TBCs, often occurring in unequal amounts of (S)and ( R ) enantiomers, was detected by chromatographic techniques using chiral support systems. In no instance were mammalian TBCs isolated in optically active form, and confirmation of their presence in tissues and fluids rests entirely on spectroscopic evidence. TBC 25 occurs in rat brain and urine (37-45),as well as in human platelets (43). The 6-hydroxy analog (TBC 26) was identified in rat brain homogenates (41,42)and in the urine of rats and humans (44). The methyl ether analog (TBC 27) was detected in rat and mice tissues (45-48), and in the retina of several animal

2. MAMMALIAN

28 1471

25 R = H I37431 26 R = OH I41.42441 27 R=OCH3145481

I29

ALKALOIDS I1

NH

29 R = H 137,41.~-5n 30 R=OH I58621 31 R = OCH3 158.631

OH

CH3

32 I64661

33 [661

35 R=HI441 36 R = CH3 I46,54,68,691

37 m 1

34 I671

H ~ Ccoon

H3C COOH

38 1711

FIG. 10. Presently known mammalian p-carboline alkaloids.

species (49,50). N-Methylated TBC 28, the only tertiary amine of the TBC group of mammalian P-carbolines found so far, occurs in rat tissue (47). The origin of mammalian alkaloids is obscured by their presence in alcoholic beverages (511, particularly in beer (52), and in fruits, such as bananas and plums (53). TBC 29 was found to be present in rat tissues (37423 - 5 6 ) , and in increased amounts after alcohol consumption (57), and it is also a constituent of human urine (58). Occurrence and formation of TBC 30, the condensation product of serotonin with acetaldehyde, has received special attention by the Swedish group of Beck and colleagues. They found TBC 30 to be present in human urine and in animal tissues, in which it is often excreted in conjugated form (59). Analysis of tissue extracts using GC-MS techniques and chiral columns showed the compound to be present in rat brain and to be excreted in the urine of different animal species in unequal proportions of the enantiomers (60,61).Unequal proportions of deuterated TBC 30 enantiomers were found when rats were fed trideuterated ethanol (30). TBC 30 occurs in the urine of controls and alcoholics in comparable amounts and is excreted mostly in conjugated form (62). The methyl ether TBC 31 is a

130

ARNOLD BROSSI

normal constituent of human urine (58), and it is formed in rat brain when animals are injected with 5-methoxytryptaminetogether with acetaldehyde (63). TBC 32 is present in human and cat urine, occurring in cat urine predominantly as the ( S ) enantiomer (64). TBC 32 is also found to be a major metabolite of the carboxylic acid 38 in rats, and it is excreted predominantly as the ( S ) enantiomer (65,66).Administration of TBC 29 to rats in the presence of 3-methyl-cholanthrene, which is an inducer of cytochrome P-450enzymes, led to the formation of the novel metabolite TBC 33 with a hydroxy group at C-5 (66). DBC 34 (harmalan) is a rare mammalian alkaloid present in goat urine (67). The fully aromatic BC 35 (norharman), on the other hand, is an endogenous metabolite in man (44), and BC 36 (harman) was found to be present in tissues of rat and man (1,46,54,68,69).Carboxylic acid 37 was detected in cerebral fluids of monkeys (70),and analog 38 was detected in bovine milk and urine (71). 4 . P-Carboline-3-Carboxylic Acid Esters

The ethyl ester 42 (72)and the n-butyl ester 43 (73)occur in human urine and were isolated and fully characterized. Reports that these esters may represent endogenous ligands of the benzodiazepine receptor (74)and that they play a role in the function of endogenous benzodiazepines (75)started a flurry of research activity. Both esters 42 and 43, but not the acid 39, have pharmacological properties that are mediated by the benzodiazepine receptor, but they are completely opposite to those generally associated with benzodiazepines; they are convulsants (76)and show anxiogenic effects (77).The term “inverse agonists” has been introduced to distinguish them from the classic agonists (78). Both esters, 42 and 43, were found useful in experimental animals to treat anxiety (79)and to enhance memory (80). Because ethanol was used in the extraction of 42 from animal tissue, there are doubts whether 42 is indeed an endogenous ligand or an artifact produced during isolation (72). The n butyl ester 43, on the other hand, extracted from the gray matter of bovine cerebral cortex and fully characterized by chromatographic comparison with a synthetic standard, is believed to be an endogenous compound (73). Synthesis of 42 devised by Braestrup et al. (72)proceeds as shown in Fig. 1 1. L-Tryptophan (19) was cyclized with formaldehyde in a Pictet-Spengler reaction to afford the known carboxylic acid 39 (23).Esterification of 39 with ethanol, saturated with hydrogen chloride (81), yielded the ester 40. Oxidation of 40 to TBC 42 was accomplished with chloranil in refluxing benzene, with sulfur in dimethyl sulfoxide (DMSO) (82), or with lead tetraacetate in acetic acid (83).

2.

MAMMALIAN ALKALOIDS I1

H

131

H 19

39 R = H 40 R=CzH5 41 R=C4H9

H 42 R=CzH5 43 R=C,Hg FIG.

1 1 . P-Carboline-3-carboxylatesas ligands of the benzodiazepine receptor.

5 . Metabolic Transformations

The formation of p-carbolines in vivo can be enhanced by injecting rats intraventricularly with tryptamine and pyruvic acid to produce significant brain levels of the 1-carboxylicacid TBC 38 (84). Injection of TBC 38 into rats resulted in the formation of DBC 34, TBC 29a, and BC 36 as the major metabolites (85). It was suggested that DBC 34 resulted from TBC 38 by oxidative decarboxylation and that TBC 29a was formed from DBC 34 by asymmetric reduction, with BC 36 occurring via further oxidation. The nonenzymatic decarboxylation of TBC 38 was greatly increased on addition of pydridoxal phosphate (86). The incubation of human platelets with tryptamine or serotonin afforded TBC 25 (87). These compounds were named tryptolines, which are identical with TBC, and were fully characterized by GC-MS techniques, and by comparison with synthetic standards and trifluoroacylated derivatives. The one-carbon unit required for the formation of the p-carboline tricycle is derived from 5-methyltetrahydrofolic acid, a cofactor present in platelets (88). This finding led to a diagnostic assay which allows for the measurement of the enzyme activity of human platelets in terms of their ability to produce, on addition of tryptamine, TBC 25. This alkaloid, when given to rats, led to two metabolites hydroxylated in the aromatic ring at C-6 and C-7, respectively (89). The formation of these metabolites may proceed via an arene oxide. An increase in brain levels of 5-hydroxytryptamine after i.p. injection of TBC 27 into mice and rats was noted, with 72% of the radiolabeled drug

HO

R

0TQNH H

30a,33a

CH3

FIG. 12. Metabolic pathways to mammalian p-carbolines. A, Aldehyde Pictet-Spengler; P, pyruvic acid Pictet-Spengler;-+, preferred routes.

2.

MAMMALIAN ALKALOIDS I1

133

being excreted in the urine and 9% in the feces within 72 hr (90).Racemic TBC 29 was hydroxylated in the aromatic ring, affording the (S)configurated TBC 30 in a 9-fold enantiomeric excess, whereas the 7-hydroxylated analog TBC 32 was present in slight excess of the (R) enantiomer (65). Several investigators reported that the levels of 1-methyl-substitutedTBC 29 and BC 36 (harman) were consistently increased in platelets (54), in plasma (57),and in urine (91-94a-c) after alcohol consumption, but other investigators could not corroborate these findings (55). A recent report on the presence of elevated levels of BC 35 (norharman) in alcoholics disputes the theory that acetaldehyde formed by oxidation of ethanol would be required to produce biologically active P-carbolines (94e).It seems difficult at this time to delineate a biosynthetic pathway to optically active TBC, and to define clearly whether they originate by the aldehyde or by the pyruvic acid route, although the latter seems to be preferred (Fig. 12). No 3-carboxy-substituted TBCs, derived from L-tryptophan by the Pictet-Spengler route, have yet been isolated from mammalian tissues. The same is also true for the dicarboxylic acid 23a derived from the condensation of L-tryptophan with pyruvic acid (36). The 1-carboxy-substituted TBCs 37 and 38, on the other hand, occur in mammalian systems (70,72) and are metabolically decarboxylated (65,85).Whether a direct enzymatic decarboxylation of racemic material, occurring with the (S) and (R)enantiomers at a different rate, could account for the formation of unequal amounts of the enantiomers of TBC has not been investigated so far. The pyruvic acid route to optically active TBC (Fig. 12) leading from TBC 38a to TBC 29a via DBC 34 is at the moment the preferred pathway (85,86,89), although the enzymes involved in the asymmetric reduction leading to TBC 29a and the hydroxylated metabolites TBCs 30a and 33a have been neither isolated nor characterized. 6 . Analytical Methods

TLC separation of Harmala alkaloids was achieved on silica gel plates using Dragendorff’s reagent for detection (95). Separation of N-acylated TBCs using a two-dimensional TLC system was also reported (37).TBCs were also successfully separated on HPLC using methanol-water as a solvent system, measuring fluorescence between 355 and 425 nm with a fluorimetric detector (96), and by using reversed-phase HPLC with fluorimetric detection (97).Electron impact (EI) MS and chemical ionization (CI) MS of tryptamines and p-carbolines were reported (19). Routine GC-MS techniques used to characterize and to quantitate TBCs in tissue extracts (51-56) were later replaced by tandem MS-MS techniques, measuring molecular ions and fragments (41,98a). The UV spectra of the

134

ARNOLD B R O W

Harmine

Harmaline

Tatrahydroharmine

241 (4.61)

218 (4.27)

224 (4.72)

301 (4.21)

260 (3.90)

269 (3.98)

338 (3.89)

376 (4.02)

294 (4.00)

FIG. 13. UV maxima (nm) of Harmala alkaloids in methanol, with log E values.

Harmala plant alkaloids harmine, harmaline, and tetrahydroharmine, tabulated in Fig. 13, also are typical for mammalian BCs, DBCs, and TBCs (98b,c). Specifically labeled p-carbolines were prepared with pentafluoropropionic anhydride as the N-acylating agent in order to measure endogenous levels of the drug (55,99). A serious pitfall disqualifying the analytical methods used for the determination of p-carbolines in tissues and in fluids is the presence of formaldehyde in the solvents used for the extraction, or introduced from endogenous sources during the workup (100,101). Formaldehyde reacts instantaneously with tryptamines, particularly serotonin, to afford TBC in a Pictet-Spengler reaction. The removal of formaldehyde from solvents used for the extraction was accomplished with semicarbazide(ZOO), but can be better achieved with 5-methoxytryptamine (101). Glyoxylic acid reacts with tryptamines to form highly fluorescent compounds that are useful for the detection and quantitation of these amines in biological systems (102). The fluorescent material formed from serotonin and glyoxylic acid is the betain 47 (Fig. 14). It originates from the carboxylic acid 45 by addition of another molecule of glyoxylic acid to afford the dicarboxylic acid 46, which dehydrates and decarboxylates on heating (103).

7. Pharmacological Effects of Mammalian p-Carbolines P-Carbolines exert a variety of pharmacological effects, including sedation, catalepsy, inhibition of convulsion, hallucination, and inhibition of monoamine oxidases (MAO) and of monoamine uptake (104a,b). Extensively investigated was the inhibition of M A 0 by p-carbolines, which is probably responsible for their antidepressant effects in man ( 5 4 . pCarbolines inhibit the oxidative deamination of serotonin at micromolar

2.

H 44

135

MAMMALIAN ALKALOIDS I1

7

H 47

COOH

COOH

46

FIG. 14. Fluorescent betaine from serotonin and glyoxylic acid.

concentration, and they inhibit phenethylamine deamination at millimolar levels. They also show significant reduction of type A, but not type B, MA0 activity (105). The M A 0 inhibitory effect was generally greater with compounds not substituted in the aromatic ring, and 6-hydroxy-substituted congeners were better inhibitors of serotonin uptake (106,107) than 6methoxy-substituted analogs, which showed both effects (108,109).Both BC 35 (norharman) and BC 36 (harman) are potent inhibitors of type A M A 0 (110), and they inhibit significantly the binding of labeled flunitrazepam to benzodiazepine receptors (68). Similar inhibitory activity was also reported for other P-carbolines (111,112). The binding of P-carbolines to the benzodiazepine receptor was calculated by a Free and Wilson analysis (113), which showed the recognition site to be a planar cleft. Structural changes which destroyed planarity also hindered binding. It is speculated that good binding of the P-carboline esters 41 and 42 makes the ester carbonyl group an important structural feature, possibly mediating the interaction with the receptor through a hydrogen bond. BC 35 also is a potent inhibitor of indolamine-2,3-dioxygenase and tryptophan-2,3-dioxygenase,both heme-containing enzymes which catalyze the cleavage of the pyrrole ring in these alkaloids ( 1 14). Inhibition of dopamine uptake (1 15,116) and tryptamine uptake by P-carbolines was reported (117). It was implicated that p-carbolines are part of a cellular aging phenomenon (1 18),manifested by the isolation of various fluorescent substances which were isolated from high molecular weight protein aggregates from aging human lenses and absent in the lenses of young calfs. Some of these substances were isolated and identified by comparison with standard compounds by TLC, UV, and fluorescent spectral data and are shown in Fig. 15.

136

ARNOLD BROSSI

34

23a

24a

FIG. IS. P-Carbolines in aging human lens tissue.

B. OTHERINDOLEALKALOIDS FOUND I N MAMMALS

I . 3-Amino-P-Carbolines Although not mammalian alkaloids by definition, but rather formed as artifacts, 3-arnino-P-carbolines, represented by BC 48 (Fig. 16), are present in small amounts in human plasma, bile, and cataract lenses (119). BC 48 is produced from L-tryptophan and from proteins on heating and is a potent mutagen (120). BC 48 and chemically related compounds are carcinogenic in experimental animals (121) and produce tumors when administered to newborn mice (122). These amines also have potent anticonvulsant properties and act as antagonists of y-aminobutyric acid (GABA) receptors by suppressing GABA-induced C1- currents (123). In uitro, they inhibit catecholamine metabolism, MAO, and tryptophan hydroxylase, the enzyme involved in the biosynthesis of serotonin (124). BC 48 is metabolized to the hydroxylamine BC 49, a directly acting mutagen causing DNA strand cleavage (125).

2 . 1,l'-Ethylidenebis(tryptophan),Peak E The amino acid L-tryptophan (19), used as a food supplement and in insomnia, is manufactured in Japan by a novel biotechnological process that leads to the contaminant bisindole 50 (peak E) (126), for which earlier an aminal structure was proposed (127). An outbreak of an eosinophilia

CHI

48 R=NHz 49 R=NHOH

FIG. 16. 3-Amino-P-carbolines present in human tissues and fluids.

2.

137

MAMMALIAN ALKALOIDS I1

syndrome (EMS) was associated with tryptophan intake (128) and traced to supplies of L-tryptophan which showed the presence of "peak E." The structure of 50 was deduced from MS and NMR data, then proved to be correct by reduction of 50 with sodium borohydride to yield 1ethyltryptophan besides tryptophan (129). Although bisindole 50 is not a mammalian alkaloid per se, and either is an artifact of the technical synthesis or formed in the workup, its relationship to TBCs became evident with the formation of TBCs 21a and 21b on acid hydrolysis (Fig. 17). It can be speculated that protonation of 50 will afford the carbinolamine 51 besides L-tryptophan. Carbinolamine 51 is a masked acetaldehyde, and it will transfer this moiety to the more basic amino group on L-tryptophan to afford the protonated Schiff base 52,already encountered as an intermediate in the Pictet-Spengler cyclization to TBCs (Fig. 4). Spiroannelation of 52 will ultimately lead to TBCs 21a and 21b which both were found to be present in the products obtained in the acid hydrolysis of 50 (127). It is questionable whether TBCs 21a and/or 21b could account for the eosinophilia syndrome, since both compounds prepared by total synthesis were well tolerated in mice at doses up to 100 mg/kg (23).

I H - C-CHJ I

H ,CH-CH3

Ho

19

51

so

q

C

O

O

H

+

QJ-

CH3 218

FIG. 17. Acid hydrolysis of peak E.

CH3 21 b

138

ARNOLD BROSSI

3. Condensation Products of Tryptamine and L-Tryptophan with Chloral Hydrate Convinced that many novel mammalian alkaloids of possible biological significance may be formed on intake of carbonyl compounds in uiuo, Bringmann and group investigated the condensation products obtained from tryptamine and L-tryptophan with chloral hydrate (6h). Chloral hydrate has hypnotic and sedative properties and is still in use in veterinary medicine. The reaction of chloral hydrate with tryptamine in water afforded racemic TBC 54, which was fully characterized. TBC 54 was converted in the presence of liver microsomal enzymes to the 6- and 7-hydroxy analogs of TBC 55. A similar Pictet-Spengler reaction of chloral hydrate with L-tryptophan, as shown in Fig. 18, afforded the cis-acid 56 as the major product and the trans-acid 57 as a minor constituent, which were difficult to separate. A similar Pictet-Spengler reaction with the methyl ester 53 afforded ester 58 and its diastereomer (not shown), which were also difficult to separate on a preparative scale. The N-benzylated TBC 59, however, was obtained in the condensation of N-benzyltryptophan methyl ester with chloral hydrate as a single product; it will serve as an intermediate in future work.

-"H

H

i cc13

/ qcoo 1 R=H

19 R=COOH 53 R=COOCHs

59

H

54

CClJ

CClJ

56 58

HO

55

+

CCI,

R=H R = CHj

cc13

FIG. 18. Chloral-derived p-carbolines.

57

2. MAMMALIAN

ALKALOIDS 11

I39

4 . Isatin, a New Endogenous Factor

Isatin (59), having the structure shown in Fig. 19, is present in normal human urine, and it was fully identified in extracts by direct probe mass spectrometry and by GC-MS methods using 5-methylisatin as an internal standard (130). There is a distinct distribution of isatin in brain tissue, with the highest level found in the hippocampus, suggesting that isatin has a biological function. The concentration in some brain tissue is as high as that of serotonin. Although nothing is known about its biosynthesis, or its metabolism, it was found that isatin is metabolized in rat liver. There is some evidence that isatin is produced in the gut by gut flora, acting possibly on tryptophan (131). The anxiolytic agent pentylenetetrazole, when given to rabbits, increased the isatin level in the brain (132). Isatin given to rats i.p. in 1520 mg/kg doses was found to have an antianxiolytic effect (133). Isatin is a potent inhibitor of type B MAO, with an IC,, of about 3 mM; higher concentrations also inhibit several other enzymes, such as alkaline phosphatase (130). It was reported that isatin acts in rodents as an antiseizure agent (134), and its in uiuo activities were summarized (131). It was concluded that isatin, and possibly its metabolites, are in part responsible for the activity associated with a factor named tribulin (133). The relationship of isatin with tribulin, an endogenous factor which shows MA0 inhibitory activity and is found in human urine (135), however, remains unclear. Tribulin is widely distributed in rat tissue (136) and is also present in tissues of other animal species (137). The production of tribulin is greatly increased under conditions of stress and anxiety, and its output is suppressed in a dose-related fashion by benzodiazepines. Tribulin is not a peptide, but is of low molecular weight; however, attempts to identify it fully have remained unsuccessful (138). Several facts militate against the claim that tribulin is identical with isatin as has been reported (135). Isatin is a selective inhibitor of type B of MAO, whereas tribulin is equally potent against type A MA0 (139). Isatin was also found to be a less potent inhibitor of the benzodiazepine receptor (140), leaving in question what tribulin is and how it is chemically related to isatin. Evidence is accumulating that isatin, and possibly its

ao I

0

H FIG. 19. Isatin (59).

140

ARNOLD BROSSI

metabolites, are endogenous compounds with a possible role in stress and anxiety. It has been suggested that these effects are not caused by its inhibition of type B M A 0 as such, but are possibly related to a protection of monoamines in times of stress by inhibiting the enzymes responsible for their further degradation (131). 5 . Novel Mammalian Metabolites

Rommelsbacher, in following the concept of Holtz that mammalian alkaloids are formed from chemically reactive biogenic amines and amino acids by reaction with endogenously formed ketonic substrates under physiological conditions, has extended this idea to include indole-3acetaldehyde. It was found that tryptamine, on incubation with pig brain and bovine brain, but not with liver tissue, formed the thiazolidine carboxylic acid 60 shown in Fig. 20 (141). Compound 60 is a mixture of diastereomers, and the structures were proposed on the basis of spectral data and proved to be correct by synthesis from indole-3-acetaldehyde and L-cysteine. It was demonstrated that tryptamine is first converted by M A 0 to indole-3-acetaldehyde, which spontaneously cyclizes with free L-cysteine present in brain tissues (142). The reaction was strongly inhibited by addition of the M A 0 inhibitor pargyline, indicating participation of the enzyme in the formation of the aldehyde, but not in the condensation with L-cysteine, which was found to be pHdependent and probably nonenzymatic (143). It seems possible that compound 60 is identical with material obtained during metabolic degradation of tryptamine in brain tissue (144,145).

H Tryptamine

-2..

Indole-3-acetaldehyde

dH

NH

COOH

* N- I

CH2SH

60

L-cysteine

FIG. 20. Formation of (4R)-2-(3-indolylmethyl)-I ,3-thiazolidine-4-carboxylic acid (60) from tryptamine.

2.

MAMMALIAN ALKALOIDS I1

141

111. Mammalian Isoquinoline Alkaloids

A. TETRAHYDROISOQUINOLINES A N D I-CARBOXY CONGENERS Mammalian isoquinoline alkaloids include simple tetrahydroisoquinolines, catecholic congeners such as salsolinol and its methyl ether analogs, 1-carboxy-tetrahydroisoquinolineswith a methyl o r benzyl group at C- 1 , and benzylisoquinolines related to reticulines. The latter compounds are intermediates in the biosynthesis of morphine in the opium poppy, and they may also play a role in the biosynthesis of mammalian morphine, which is discussed in Section IV. For naming the mammalian isoquinolines, and for numbering the carbon and nitrogen atoms, we have adopted the nomenclature used for plant isoquinoline alkaloids in the classic texts of Kametani (146a,b)and Shamma (147a,b) and in the reviews of simple isoquinoline alkaloids by Lundstrom (148), and Menachery et al. (149). The following abbreviations are used throughout this chapter: TIQ, 1,2,3,4-tetrahydroisoquinoline;bIQ, 3,4-dihydroisoquinoline;and IQ, isoquinoline. A lowercase a marks the molecules with (S) absolute configuration at C-1, and b is used for the enantiomers with (R) absolute configuration. 1 . Chemistry of Mammalian Isoquinolines

Several syntheses of racemic salsolinol(64) by Pictet-Spengler cyclization of dopamine with acetaldehyde have been reported (IOa,150).The synthesis of the optically active (S) enantiomer TIQ 64a from the methyl ether analogs TIQs 62a and 68a by acid hydrolysis correlates the chemistry of mammalian alkaloids with that of plant isoquinolines (146-148). The chemical reactions used to prepare mammalian isoquinolines, shown in Fig. 21, are as follows. Condensation of dopamine methyl ether (61) with acetaldehyde afforded racemic salsoline (62) (151).The enantiomers of TIQ 62 were obtained by chemical resolution of the benzyl ether analogs followed by deprotection (152). Aromatization of the benzyl ether of TIQ 62 with Pd catalyst in refluxing toluene gave IQ 63 directly (153). Homoveratrylamine (65) on acetylation yielded acetamide 66, and its Bischler-Napieralski cyclization gave DIQ 67. This compound has been frequently used in the synthesis of isoquinoline plant alkaloids, and a practical procedure for its large-scale preparation has been reported (154). Reduction of DIQ 67 gave racemic salsolidine (68) which was resolved into its optical isomers (155).The latter compounds on treatment with concentrated hydrobromic acid afforded (S)-salsolinol (64a) and its ( R ) enantiomer (not shown) (156). Ether cleavage of DIQ 67, effected with

142

ARNOLD BROSSI

65R=H

66 R = COCH3

CH3

HO

CH3 69 -

0

CH2

71

72 R = COCH, 73R=H

FIG. 21. Synthesis and chemical transformations of simple mammalian isoquinolines.

concentrated mineral acids, afforded DIQ 69 as the salts. The free base, prepared from aqueous solutions of DIQ 69 with ammonium hydroxide or sodium bicarbonate, is the quinone methide 70 (157). Compounds 69 and 70 exist in a pH-dependent equilibrium. Treatment of DIQ 67 with acetic anhydride gave the N-acetyl enamine 71 with an exocyclic double bond (154). Oxidation of 71 with ozone (158), perfected in another series of compounds with periodate in the presence of ruthenium chloride (159), afforded the lactam 72, and its hydrolysis with hydrochloric acid gave corydaline (73)(158).

2.

MAMMALIAN ALKALOIDS I 1

I43

The benzylisoquinolines represent one of the largest group of plant alkaloids (146,147), and catecholic representatives occur in mammalian tissues and fluids. The best known is tetrahydropapaveroline, shown in Fig. 22 as the (S) enantiomer TIQ 75a. Racemic material is often referred to as “THP” (160,161). The synthesis of TIQ 75a as well as that of the plant alkaloid (S)-N4’-bisnorreticuline(77a) is shown in Fig. 22. ( S ) Tetrahydropapaverine (74a), on treatment with concentrated hydroiodic acid at 125”C, afforded TIQ 75a, which was fully characterized as its hydrochloride (156).TIQ 77a, possibly an intermediate in the plant biosynthesis of (S)-norreticuline (78a) and derived TIQs, and possibly mammalian morphine as well, was prepared from the benzyl-protected TIQ 76a. Deblocking was achieved over Pd catalyst and hydrogen in the presence of hydrochloric acid, leading directly to the hydrochloride salt of TIQ 77a (162).The O-methylation of TIQ 77a with S-adenosyl-L-methionine (SAM) in the presence of mammalian catechol O-methyltransferase (COMT) gave (S)-norreticuline (78a), besides other products. The 4-hydroxy substituted TIQ 79, shown in Fig. 23, was detected as a metabolite of 1,2,3,4-tetrahydroisoquinoIine(117) when treated with rat liver microsomal enzymes, and it also was found to occur in rat urine (163).Compound 79 is structurally related to TIQ 80, obtained on condensation of norepinephrine with formaldehyde (164),and to TIQ 81, detected in animal tissues after exposure to acetaldehyde (165). Acid-catalyzed dehydration of TIQ 82, the N-methyl analog of TIQ 79, should lead to the iminium species 83 (166),which on two-electron oxidation or by disproportionation should give the isoquinolinium salt 84. Such reactions, if occurring in uiuo, would parallel similar reactions seen with the neurotoxin MPTP in its conversions to MPDP+ and MPP+ (167) and could possibly explain the neurotoxic effects seen with 117 (168). A synthesis of 4-hydroxy-substituted TIQ has been developed in connection with plant isoquinolines (169,f70),and the glycine ester route used for a similar purpose (171) is shown in Fig. 23. Reaction of the benzyl bromide 85 with N-methylglycine ester gave 86; the ketoester 87 was obtained on Dieckmann condensation. Alkaline hydrolysis of ester 87 followed by treatment with mineral acid gave ketone 88; alcohol 89 was obtained on reduction of 88 with sodium borohydride. 2. Tetrahydroisoquinoline-1-carboxylic Acids

Another group of mammalian TIQs is represented by the amino acids 91-94 shown in Fig. 24. They are readily obtained by condensation of dopamine (90) with pyruvic acids under physiological conditions, requiring a slightly acid pH (pH 4-6). Amino acids 91 and 92 were prepared and

0

r

0 n

r

v

1

0

I

n

I

0

v

n

I

% I

0

0

% I

3

0

144

I

0 I

0 I

=

0

I

I

C

m 0

OH

OH

"HO

~

N

OH

H

"HO "*N-CH3

80

-

J

83 -

COOC2H5 -COOC2 CH3 0

89

CH2 Br

-

CH30

CHz /N\CH3

-

88 87 FIG.23. Synthesis and chemical transformations of 4-hydroxytetrahydroisoquinolines.

H5

146

ARNOLD BROSSI HO

0

HO

R ACOOH

90

HO

HO

1

HO

q X o H H3 c

H

O

T

OH

COOH H

/

/

RO

\

OH 91

92

93R= H 9 4 R = CH,

FIG.24. Synthesis and occurrence tetrahydroisoquinoline-I-carboxylicacids.

investigated by Hahn and Stiehl(172). The synthesis of amino acids 93 and 94 was similarly achieved by condensing dopamine with the appropriate phenylpyruvic acids (173,174). obtained from benzalhydantoins on hydrolysis with strong alkali (175). Oxidative decarboxylation of 6-hydroxy-substituted TIQ- 1-carboxylic acids to quinone methides under physiological conditions (pH 7 and above) was first reported by Bobbitt et ul. (176,177). This reaction was later investigated in detail with dideoxynorlaudanosoline- 1-carboxylic acid (92) (177). The yellow quinone methide 95, which was fully characterized, was converted by acid to the salt of DIQ 96 shown in Fig. 25. The spectral data collected for compounds 95 and 96 were in accord with this well-known interconversion of phenolic DIQs (147~1,157).DIQ 96 on chemical reduction gave TIQ 97, and on acetylation it gave triacetate 98 with an exocyclic double bond ( I 77). The synthesis of optically active TIQ- 1-carboxylic acids has recently been accomplished by the chemical reactions shown in Fig. 26. Carboxylic acids 99 and 100, after esterification and reaction of the methyl esters with optically active 1-phenylethyl isocyanates, gave ureas 101 and 102, respectively, which were separated from their diastereoisomers by chromatography (33,218). Ureas 101 and 102, on treatment with base or on

2.

H HO

O

T

\ /

O

H

T

H

op O

\

\ NH

6 95

92

98

I47

MAMMALIAN ALKALOIDS I I

HO

6 97

96

FIG. 25. Oxidative decarboxylation of 3’,4’-dideoxynorlaudanosolineI-carboxylic acid

heating, were converted to hydantoins 103 and 104, respectively, and underwent fragmentation in refluxing butanol to afford esters 105 and 106, respectively. Hydrolysis of 105 with 20% hydrochloric acid yielded ( S )salsoline-1-carboxylic acid (99a), whereas hydrolysis with 48% hydrobromic acid gave ( S )-salsolinol- 1-carboxylic acid (91a).Treatment of ester 106 with 48% hydrobromic acid similarly gave ( S )-dideoxynorlaudanosoline- I carboxylic acid (92a). The absolute configurations of the optically active compounds of this series were derived from X-ray analysis of 105.HBr and hydantoin 104 (218). An enantioselective synthesis of TIQ- 1-carboxylic acids 91a,b has recently been reported (219). Hydrolysis of the optically active methyl ether enantiomer of hydantoin 103 was accomplished by 20% sodium hydroxide in refluxing methyl cellosolve and led to the dimethyl ether analog of 91a, which was used to establish the absolute configuration of the products. Amino acids 91a,b have also been prepared by chemical resolution of the N,O-benzylated acid 108 with optically active I phenylethylamines. Catalytic debenzylation of enantiomer 109a gave (S)-salsolinol-l-carboxylicacid (91a) (220).

3. Tetrahydroisoquinoline-3-carboxylic Acids Tetrahydroisoquinoline-3-carboxylic acids, which can readily be obtained from L-dopa (110) with aldehydes, do occur in plants (148,149), but

R20

CH30

R'

H

I

COOH

R' 0

CH30

CH3

101 R' = CH,, R 2 = H 102 R ' = Bn, R 2 = CH,

99 R' = CH3, R 2 = H 100 R ' = Bn, R 2 = CH,

103 R ' = CH,, R 2 = H 104 R = Bn, R 2 = CH,

R 2O CH30

HO

105 R 1 = 106 R ' =

-

Bn, R 2 = CHJ

BnO

B BnOn o q N - B n

CH3 107 -

COOBn

BnO q

91a R = CH, 92a R = Bn -

CH,, R 2 = H

-

BnO

N

-

CH3

B

n

COOH

m

BnO

108 FIG.26. Synthesis of optically active tetrahydroisoquinoline-I-carboxylicacids.

N

-

CH 3 109 -

B

n

"'C 00 H

2.

149

MAMMALIAN ALKALOIDS 11

so far they have not been detected in mammals. These amino acids, however, deserve attention as intermediates in a chemical synthesis of optically active TIQs substituted at c-1 by alkyl or benzyl groups. This is illustrated in Fig. 27. Pictet-Spengler reaction of L-dopa (110) with formaldehyde gave amino acid 111 (178). Similar cyclization with methyl ether analogs yielded the methyl ethers of 111, but cyclization could not be accomplished in case of the 3-0-methyl ether under physiological conditions, and only after harsh reaction conditions were applied. Cyclization of 110 and its 0methyl ether analogs with acetaldehyde led to mixtures of diastereomers. With 110, the cis-acid 112a and the trans-acid 113b were obtained, with 112a being the major reaction product (178).Chemical correlation of these acids with optically active TIQs can be achieved with N-alkylated analogs

HO

HO

113 b

112a CH30 N-En

CH30

CH30

114 a

115 a -

68a -

116a -

FIG.27. Synthesis of optically active tetrahydroisoquinolines from L-dopa.

150

ARNOLD BROSSI

with phosphorus oxychloride, giving iminium compounds via decarbonylation (179). The iminium salts can either be reduced to optically active Nsubstituted TIQs or transformed into optically active polycyclic isoquinolines, such as berbines (180). A hypothetical synthesis of (S)-salsolidine (68a) by the Rapoport route is sketched in Fig. 27. The N-benzyl-protected TIQ 114a, on reaction with phosphorus oxychloride, affords the iminiurn salt 115a, which on reduction with sodium borohydride gives TIQ 116a and TIQ 68a following catalytic debenzylation of the latter compound. 4. Occurrence of Mammalian Isoquinolines

The mammalian isoquinolines so far detected and characterized are shown in Figs. 28 and 29. Tetrahydroisoquinoline (117) and its 1-methyl congener TIQ 118a were found in rat brain (181) and in food (182). TIQ 117 produces a Parkinson-like syndrome in rats, and the levels of dopamine were markedly decreased (168). GC-MS techniques and chiral reagents identified the major alkaloid present in animal tissue as the (S)-configured TIQ 118a (183,184). The 4-hydroxy-substituted TIQ 79, detected in rat liver and in rat urine ( 1 6 3 , is probably the most interesting of the simple mammalian TIQs, and its relevance to MPTP in parkinsonism was mentioned earlier. TIQ 119 was detected in alcoholic rats given amphetamine (185).It is believed that TIQ 119, which was not found in the absence of amphetamine, was responsible for the behavioral abnormalities seen in the animals. Salsolinol (64) is the best known of the mammalian isoquinolines (5a,6g,10a),and it is present in increased amounts in alcoholics (186a,b). The ( R ) enantiomer (TIQ 64b) predominates in the urine of healthy humans, whereas both enantiomers are found in alcoholics (187a-c). A metabolite of TIQ 64b, and of the amino acid 91, is DIQ 120, which was detected in rat kidneys (188) and in human urine ( 1 8 9 ~ - c ) . The methyl ethers salsoline (62a) and isosalsoline (l22b), shown in Fig. 28 as the ( R )and (S)enantiomers, respectively, were prepared by chemical synthesis (152). Both compounds were found in rat brain after intraventricular administration of salsolinol (64) (190). They are also excreted in the urine of normal human subjects, and their levels are markedly increased in alcoholics (191). Salsoline (62a) is a major metabolite of salsolinol (191-19.9, and only small amounts of isosalsoline (1226) were found in rat brain (194). The simplest of the catecholic TIQs detected in mammals is norsalsolinol (l23),which occurs in rats treated with L-dopa (195); its presence in rat brain was confirmed by GC-MS methods (196). The most important of the mammalian benzylisoquinolines is tetrahydropapaveroline ( 7 9 , often referred to as T H P and shown in Fig. 29 as the (S)enantiomer. T H P is the condensation product of dopamine with the aldehyde of its own oxidative deamination (160), and it is formed in

2.

I51

MAMMALIAN ALKALOIDS 11

WH H\\'

117 [181,182]

CH3 118a [181,182,183]

CH3 79 [163]

119 [185]

CH3

64b [5a,6g,186]

CH30

120 [188,189]

c HO H 3 0 w H\\\ N H CH3 62a [ 192,1931

CH3 122b [192,193]

123 [195]

FIG.28. Mammalian isoquinolines.

uiuo on incubation of dopamine with M A 0 preparations (197). It was reported by Holtz et al. that TIQ 75 was responsible for the antihypertensive effect noted in animals treated with M A 0 preparations, and its formation was blocked by M A 0 inhibitors (197). The finding that TIQ 75 was present in parkinsonian patients treated with L-dopa (198) initiated much research, the results of which have been summarized (6g,199).With the

I52

ARNOLD BROSSI

HO

+

OH

OH 75a [5a,6g,199]

Hom CH30

6

N

-

C H3

H E

OH

OCH3 121b [209]

FIG.29. Mammalian benzylisoquinolines.

availability of highly sensitive analytical methods (200),it was found that the levels of T H P were significantly increased when the animals were treated with L-dopa, and a further increase was seen when ethanol was administered in addition to L-dopa (201).T H P has proved to be a valuable probe to map THP-reactive sites in dopaminergic pathways (202). The sites involved in the biochemical process overlap with enkephalinic and dopaminergic systems (203).THP, when injected into rats by the intraventicular route, reversed the action of the decarboxylase inhibitor benserazide (204). Other important mammalian benzylisoquinolines which thus far have escaped detection in mammals are (S)-reticdine and its (R) enantiomer TIQ 121b, shown in Fig. 29. The latter compound is required for the biosynthesis of morphinandienones in the opium poppy, and its possible role in biosynthesis of mammalian morphine is discussed in Section IV. The I-carboxy-TIQs 91-94 shown in Fig. 24 have been detected only by GC-MS methods; it is not known whether they occur naturally as racemic mixtures or in the form of enantiomers. Salsolinol- 1-carboxylic acid (91) was detected in human urine (205) and in the caudate nucleus (206).Dideoxynorlaudanosoline- I-carboxylic acid (92) was found in small amounts in rat brain, but is present in enhanced amounts in phenylketonuric patients (207). It is believed that TIQ 92 may be responsible for the neurological disorders which develop during the disease. Carboxylic acids 93 and 94 were detected in the urine of parkinsonian patients undergoing L-dopa therapy, and the methyl ether 94 was the major metabolite (174). It should be pointed out that so far no aromatic mammalian isoquinolines have been found in mammalian tissues and fluids. Also, glucuronides and sulfates of catecholic or phenolic mammalian TIQs have not been found,

2.

MAMMALIAN ALKALOIDS I 1

I53

although incubation of rat liver with sulfatases resulted in the appearance of surprisingly large quantities of salsolinol (64) and its methyl ether analogs TIQs 62 and 122 (208).

5 . Metabolism of Mammalian Isoquinolines 0-Methylation of simple catecholic TIQs probably catalyzed by COMT, is a major metabolic pathway. Metabolites arising from N-methylation are rare, and products involving a chemical reaction of the activated 1methyl group in DIQs with carbonyl substrates have so far not been found. The benzylisoquinolines with a freely rotating benzyl group, however, are capable of undergoing a variety of chemical reactions prior to 0-methylation. By C-N coupling they afford dibenzopyrrocolines, by C-C coupling aporphines, and by insertion of a one-carbon unit tetrahydroprotoberberines (also called berbines). All these reactions take place in plant tissues and give rise to the many plant isoquinolines which are discussed in the classic texts (146,147). The biosynthesis of benzylisoquinolines has recently been investigated by Zenk and colleagues in great detail, and the results of their work have been reviewed (209a,b).Two major metabolites detected by incubation of THP with soluble rat liver and brain supernatants, which were found in the urine of rats given the alkaloid and in the urine of parkinsonian patients, were the berbines 124 and 125, shown in Fig. 30 (210).They were found to be identical with the compounds obtained in the condensation of THP with formaldehyde under slightly acidic conditions (211). The formation of 0-methylated metabolites of THP, and of berbine 124, with rat liver microsomal enzymes was investigated in some detail (212,213). It was shown by Davis and colleagues that intraventricular application of racemic and optically active THP and of berbine 124 led after 1 hr to the formation of metabolites which were identified and quantitated by GC-MS methods. The results are summarized in Fig. 30. It can be seen that 0-methylation of the hydroxy group at C-6 in both enantiomers of THP and the racemate necessary to connect with norreticuline (121)only was a minor pathway, and that 0-methylation of the hydroxy group at C7 was the preferred route. 0-Methylation of the hydroxy group at C-1 I in berbine 124 was preferred over that at C-10, as was the 0-methylation of the hydroxy group at C-2 over that at C-3. Condensation of the optical isomers of tetrahydropapaverine (74a,b) with acetaldehyde under nonphysiological conditions afforded, as shown in Fig. 3 1, the optical isomers of coralydine (126a,b) and O-methylcorytenchirine (127a,b) (214).0-Demethylation of these alkaloids was accomplished with 48% hydrobromic acid to give the C-8-methyl analogs of THP

HO

HO

3

HO

Ho%OH

'

HO?

OH

OH

\

OH

OH

OH 75

124

Compound

125

C-7

C-6

C-3

c-2

c-3

c-10

c-11

~~

(k)-THB ( 1 2 4 ) 69 (+I-(R I 73 (-)-(S) 59

6 24 0.4 26 13 21 FIG.30. 0-Methylation of racemic and optically active THP and derived berbines (% methylation). 2 0.3 7

CH30 CH,O

OCH,

OCH, OCH,

OCH,

RO R

o

y

113.31

74b

74a

A

A

$/

\

R RO

o

y

$/

\

OR

OR

126b 128b

R = CH, R = H

RO

H\\\

'

OR

R = CH, R = H

H \\'

/

\

OR

OR

OR

127b 129b

1%

/

127a 129a

R = CH, R= H

FIG.31. Acetaldehyde-derived berbines.

OR

126a 128a

R = CH, R = H

OR

156

ARNOLD BROSSI

(128a,b and 129a,b, respectively). Condensation of THP with acetaldehyde, when carried out in aqueous acetic acid, gave mixtures of berbines 128 and 129, two possible mammalian alkaloids. The biosynthesis of optically active TIQs, exemplified by the presence of (R)-salsolinol in human fluids (189b), requires some comment. This is particularly warranted since many investigators have concluded that the conversion of dopamine to TIQ 64b is the result of condensation of the amine with acetaldehyde originating directly from ethanol. As shown in Fig. 32, this reaction, when carried out in uirro, affords racemic salsolinol (64) (5a,10). Formation of optically active TIQ 64b from dopamine and acetaldehyde would require that the Pictet-Spengler reaction be enzymatically controlled, as observed in the condensation of dopamine with 4hydroxyphenylacetaldehyde in benzylisoquinoline-producingplants (209). The enzyme required to perform this reaction in mammalian systems has not yet been found. There are several observations which dispute such a reaction taking place in mammals: the finding of I-carboxy-TIQ 91 and DIQ 69 as major metabolites (189) and the very low levels of acetaldehyde detected in the brains of animals after alcohol consumption (215,216).This makes the acetaldehyde route to optically active I-methyl-substituted TIQ suspect. The reaction of optically active carbinolamines formed by an enzymatically controlled addition of acetaldehyde to amines, illustrated in Fig. 2, may be of theoretical interest, but lacks experimental verification; it also would require the presence of acetaldehyde. The more likely pyruvic acid route to optically active TIQs, however, also remains inconclusive. If it indeed proceeds through TIQ-l-carboxylic acids to DIQ intermediates by an oxidative decarboxylation (176,217,218), it requires that it be followed by an asymmetric enzymatic reduction. Although achieved in uitro ( 3 3 , this reaction has not been realized in uiuo. The formation of unequal amounts of the optical isomers of salsolinol and other TIQs in uiuo could arise from racemic I-carboxy-TIQ in an enzymatic decarboxylation, proceeding with ( S ) and (R) enantiomers at a different rate and thus affording different amounts of ( S ) - and (R)-TIQ. With the availability of optically active TIQ-l-carboxylic acids, this possibility can now be tested. There is at present no unifying concept which would explain the presence of optically active TIQs, TIQ- I-carboxylic acids, and noralkaloids in mammalian systems. The conclusion reached by several investigators that methylenetetrahydrofolate is responsible for the N-methylation of phenethylamines and indolylethylamines (221,222) makes it likely that the one-carbon unit present at C-1 in the noralkaloids is derived from formaldehyde, formed via nonenzymatic disassociation of methylenetetrahydrofolates (223,224).

2.

157

MAMMALIAN ALKALOIDS I 1

H* h HO O WH H: NH

HO O

CH3 Doparnine

64b

FIG.32. Optically active salsolinol from acetaldehyde

Do optically active 1-methyl-TIQs, as sketched in Fig. 32 for the synthesis of (R)-salsolinol, originate from a Pictet-Spengler reaction of dopamine with acetaldehyde derive from ethanol, or are they the result of a Pictet-Spengler reaction of biogenic amines with pyruvic acid, as sketched in Fig. 33? Based on the accumulated data it seems reasonable to propose that optically active TIQs are formed by the pyruvic acid pathway, and that the pyruvic acids may be derived from an impaired glucose metabolism or an impaired amino acid metabolism. Whether the intermediate TIQ- 1carboxylic acids 91a,b are enzymatically decarboxylated to afford 64a,b in a different enantiomeric ratio, or whether optically active TIQs are formed by oxidative decarboxylation of TIQ 91 to DIQ 120, followed by an asymmetric reduction, remains open to question.

91

HO

120=69

HO CH3 9lb

HO

HBC "'COOH 91a

64b

HO 64a

FIG. 33. Synthesis of optically active tetrahydroisoquinolines by the pyruvic acid pathway.

158

ARNOLD BROSSI

6 . Analytical Methods Gas chromatography with electron capture and GC-MS techniques using volatile derivatives are frequently used to characterize and quantitate mammalian isoquinolines (255-227a,b). Liquid chromatography and cation exchange not requiring derivatization have been reported, as was detection by reversed-phase chromatography, which readily separated biogenic amines and mammalian TIQs (226,228). Further improvement in the analysis of mammalian TIQs was achieved when reversed-phase chromatography was performed with 2-propanol in a linear gradient against a constant concentration of acetic acid (229). Several catecholic TIQs derived from dopamine were clearly separated, as shown in Fig. 34, by ion-exchange chromatography using 3,4-dihydroxybenzylamineas an internal standard with a citrate-ammonium phosphate buffer containing 8% methanol and a chemically bonded octadecylsilane as the stationary phase (230). Complete separation of salsolinol, salsolinol-1-carboxylic acid, dopamine, and 7-methylsalsolinol-1-carboxylic acid was achieved. Thin-layer chromatography has frequently been used to separate isoquinoline cactus alkaloids (231,232) and many of the phenolic isoquinolines (233). The oxidation of TIQs with an NH group to imines by mercuric 0.200 1 0.150

al 0

C

0

0.100

51

s

0.050

n

u

I

-h I

I

l

0

2

4

1

6

1

8

1

1

1

1

1

1

10 12 14 16 18 20

Time (min) FIG.34. Chromatographic separation of dopamine and derived salsolinoids. Salsolinol-lcarboxylic acid ( I ) , dopamine (2), salsolinol(3), and 7-methylsalsolinol-I-carboxylicacid (4) were separated by ion-exchange chromatography on a Nucleosil 10 SA column (4.6 X 250 mm, lO-pn particle size) with 8% methanol in a citrate-ammonium phosphate buffer at 1.0 ml/min as the mobile phase (inlet pressure 1100 psi). Detection was at 280 nm.

2.

+

HO

91

I59

MAMMALIAN ALKALOIDS I I

H o m N + C H 2 C O 0 HO

COOH

130

1 HO 131

FIG.35. Formation of fluorescent isoquinolines.

acetate (234) and photooxidation of secondary amines to isoquinolines, recognized by their fluorescence at 405 nm (235), were found to be useful methods to analyze and assay TIQ drugs and their metabolites. A highly fluorescent isoquinoline is formed on reaction of salsolinol-1-carboxylic acid (91) with glyoxylic acid; it was shown to be the betain 130, present above pH 7 as the quinone methide 131 (236). The formation of 131 and 91 is shown in Fig. 35. A radioimmunoassay method was developed that allows the measurement of picomole amounts of the alkaloid salsolidine (68)(237);it is useful in localizing the alkaloid in tissues and in cells. The agent was prepared by coupling 68 with bovine serum albumin by reductive N-alkylation.

7 . Biological Activities of Mammalian Isoquinolines The pharmacological properties of the mammalian alkaloids salsolinol (64) and THP (73, with a focus on alcoholism, were reviewed in depth by Melchior and Collins (6g) and by Hirst et al. (238). Additional data were reported later (153,186,239,240).The review by Melchior details the effects of mammalian isoquinolines on analgesia, narcosis, body temperature, endocrine function, motor activity, mutagenicity, toxicity, and their possible importance in diseases such as parkinsonism, phenylketonuria, schizophrenia, and alcoholism. Some of the conclusions reached, however, are seriously flawed, since the data were elaborated with racemic mixtures of compounds and not with optically pure enantiomers. It is well documented that (S)- and (R)-configured benzylisoquinolines show distinct differences in assays measuring inhibition of dopaminergic and adrenergic receptor

160

ARNOLD BROSSI

systems (241),and optical isomers of simple TlQs express clear differences in their inhibition of M A 0 (153).This criticism also puts into question the results of investigations on the inhibition of other enzymes by TIQs carried out in uitro (242,243). The hypothesis that mammalian isoquinolines may exacerbate ethanol and opiate dependences (244,245)was suspected by the finding that racemic salsolinol (64)and optically active 3-carboxysalsolinol (112a), when given to mice, generated a biphasic dose-response in the analgesic screening, which was blocked by naloxone, suggesting an interaction of TIQs with opiate receptors (246). Binding of morphine and chemically related isoquinolines to opiate receptors and the analgesic effects of these compounds are highly stereospecific, and they depend entirely on the presence of properly configured enantiomers (247),which again casts doubts on the data established for racemic TIQs in these assays. There is now substantial evidence that several TIQs and TBCs are endogenous substances which exist in increased amounts under certain pathological conditions. It remains to be seen, however, what role they play in biochemical processes and whether they are required for the expression of pathological symptoms. Biochemical insights, as they accumulate, continue to support the notion that mammalian alkaloids can act as false transmitters, with catecholic isoquinolines mimicking dopamine and 6-hydroxy-p-carbolines mimicking serotonin. The interactions of esters of 3-carboxy-BCs with the benzodiazepine receptor and the question whether the BC unit is part of an endogenous receptor ligand remain challenges for further research. Chronic rather than acute administration of these alkaloids is more likely to resemble pathological states. Whether the pathologies are related to the parent alkaloids or their metabolites is presently not known. As pointed out earlier, further studies have to be conducted with the individual optical isomers if meaningful data are to be obtained.

B. PYRIDOXAL-DERIVED ISOQUINOLINES Schiff bases resulting from condensation of pyridoxal with amino acids (248) and polyamines (249) occur in mammalian tissues and fluids. The Schiff base pyridoxylidenephenethylamine (132) obtained by synthesis equilibrates in aqueous solution with the carbinolamine 133 and the Schiff base 134 obtained from pyridoamine and phenylacetaldehyde (Fig. 36) (250). The material isolated from the urine of parkinsonian patients, on the basis of UV data and chromatographic comparison, is a mixture of 132-134 (25f,252).

N

I 0

($5 \ /

P P

6

P P

\ /

I

m

I

m

02

I

8 6 \a'

I

6

By P P

w

d

r.l

E

I62

ARNOLD BROSSI

TIQ 135 was reinvestigated by Kametani et al. (253). Pyridoamine, but not pyridoxal, prevented the accumulation of ketonic metabolites of aromatic amino acids by eliminating them as Schiff bases (254). Isoquinolines obtained from pyridoxal and dopamine, such as TIQ 135, did not inhibit the enzyme pyridoxal kinase (255). Bringmann has recently reinvestigated the formation and metabolic fate of the optically active TIQs 136-138 shown in Fig, 36. This was prompted by the belief that such products may be formed in homocystinuria, a metabolic disorder in which the lack of the enzyme cystathione synthase leads to an accumulation of homocysteine and its dimer homocystine (6h). It was found that little of the cis-TIQ 136 was formed when rats were given pyridoxal together with L-dopa, and that the phosphorylated congeners, TIQs 137 and 138, were formed instead, with the decarboxylated TIQ 138 being the major product. The analysis was performed with labeled reference compounds, subjecting the extracts prior to GC-MS analysis to a dephosphorylation with phosphatase. Whether phosphorylation occurs prior to or after the Pictet-Spengler reaction awaits further investigation (6h).

IV. Mammalian Morphine A. INTRODUCTION

Reports that mammals produce morphine and congener alkaloids identical to the alkaloids of the opium poppy (Papaver somnij-erirm) started a flurry of research activity, which not only confirmed this finding but suggested that they might be produced in mammals by a biosynthetic pathway similar to that established in the opium poppy. The chemistry of morphine and related alkaloids has repeatedly been reviewed (247,256~-d1, and the investigation of mammalian morphine greatly profits from the wealth of information garnered from the plant alkaloids. Concise information also exists on the biosynthesis of morphine from (R)-reticuline, the crucial isoquinoline required for the construction of morphinandienones and their further conversion to thebaine. codeine, and morphine (257). Details of the biosynthesis of the reticulines, almost exclusively elaborated by Zenk el al. (209a,b), are shown in Scheme I, summarizing the enzymatic steps involved in the biosynthesis of morphine from (S)-norcoclaurine, the first isoquinoline formed in benzylisoquinoline-producingplants. The occurrence of morphine-like materials in mammalian tissues was discovered in 1976, and their presence has since been proved to be real

2. MAMMALIAN

163

ALKALOIDS I 1

HO

OH

OH

(S)-N-MethylcoclaurineR'=H. R2=OH (S)-3-Hydroxy-N-meth lcoclaurineR'-Ff=OH' (S)-Reticuline RY=OH.R2=OCH,

DehydroreticuliniumSalt

"'""a

HO

H,CO

OCH3

R2

(S)-Norcoclaurine R'=OH. R2=H (S)-CoclaurineR'=OCH,. R2.H

HO

HO

-CH3

V

HO \u"' \

(-)-Salutaridinol-l

(-)-Oripavine

(-)-Morphinone

0

\

V

O

H

OCH3 (+)-Salutaridine'

(R)- Reticuline'

(-)-Thebaine

(-)- Neopinone

(-)-Codeine R-CH, (-)-Morphine R=H

(-). Codeinone

SCHEME 1. Biosynthesis of morphine in plants. Metabolic conversions marked by asterisks are highly stereoselective.

164

ARNOLD BROSSI

and beyond doubt (258-264). The biological consequences of morphine being present in mammalian tissues, besides opioid peptides, have been discussed (265). The discovery of thebaine in mammalian tissues and its enzymatic conversion to oripavine and morphine provided evidence that these alkaloids derived from (R)-reticuline are of endogenous origin (266a,b) and not from dietary sources, such as milk (267). The question whether the very small amounts of morphine present in mammals are high enough to exert an agonistic effect on p-opioid receptors, however, remains an open question. Morphine, when measured by an enzyme immunoassay, was found to be present in the urine of healthy human subjects in the 15.7 & 6.3 pmol/ml range, not accounting for conjugates and metabolites, and the total amount of “active” morphine might, therefore, be much higher and possibly of physiological significance (268). The analytical methods used to identify morphine and its congener alkaloids included ion-exchange HPLC and comparison with chemically pure standards (269), sensitive immunoassays, and GC-MS techniques.

PATHWAYS TO MAMMALIAN MORPHINE B. BIOSYNTHETIC First reports that mammalian liver can generate morphinandienones from reticulines were announced by Kametani et ul. ( 2 7 0 ~ - d ) .The reported conversion of racemic reticuline to racemic salutaridine (sino) racemic pallidine (270b,c), and of (R)-reticuline to acutine) ( 2 7 0 ~ and (-)-pallidine (270d), obtained together with the berbines coreximine and scoulerine with rat liver homogenates, are rendered suspect in the light of recent reports by Amann and Zenk (271). The nature of the morphinandienones detected by Kametani et al. strongly suggests that they do not result from an enzymatically controlled reaction, but rather from a chemical phenol-oxidative coupling. Conversion of reticuline to salutaridine by rat liver, the critical step that generates the morphine skeleton, was later realized (2631, and ambiguities clarified (271). This firmly established that mammalian liver is capable of converting (R)-reticuline to (+ )-salutaridine in a coupling reaction which occurs with the help of a microsomal cytochrome P-450 enzyme. The reaction was found to be NADPH dependent and enantiospecific since it did not proceed with (S)-reticuline. The pH optimum was found to be 7.8, and omission of oxygen or NADPH rendered the enzyme complex inactive. Typical inhibitors of P-450 enzymes, as well as exposure to carbon monoxide, halted the formation of (+)-salutaridine. These findings, together with a report that intravenous administration of (+)salutaridine,

2.

MAMMALIAN ALKALOIDS I 1

165

(-kthebaine, and (-)-codeine to rats resulted in significant increases of morphine levels in various tissues. They nicely connect mammalian isoquinolines with mammalian morphine by routes almost identical to that used by the opium poppy (266a), and shown in Scheme 1. In this scheme the configuration of salutaridinol is depicted as reported by Zenk’s group (2666). However, reticulines have not yet been found to occur in mammalian tissues. Is (R)-reticuline, which is required for the synthesis of morphine in plants (272,273) and formed from the (S)-enantiomer in benzylisoquinoline-producing plants (274) by an oxidation-reduction sequence (257), also the crucial intermediate in the biosynthesis of mammalian morphine? Confirmation that it is must await its detection. Conversion of (S)-reticuline to (R)-reticuline in Papuer sornniferum is achieved by enantiospecific oxidation of the (S)-enantiomer (273), and it is followed by an NADPH-dependent enzymatic reduction of the dehydroreticulinium ion, which also is highly substrate specific since no reduction of 1,2dehydronorreticuline was observed (275). Although dehydroreticulinium chloride has been identified as a natural alkaloid (276a),its chemical stability at pH 8.5, the optimum for enzymatic reduction (275),requires some comment. The amorphous base obtained on extraction of an aqueous solution of dehydroreticulinium iodide rendered alkaline with ammonium hydroxide suggests that it is an enamine with an exocyclic double bond (276b), which is readily converted to a quaternary iminium salt on addition of acid. Therefore, it cannot be excluded that the species reduced at pH 8.5 is the enamine. With the assumption that reticulines are also precursors in mammalian synthesis of morphine, it was challenging to investigate whether they could be produced by enzymatic reactions similar to those utilized in benzylisoquinoline-producingplants (274).This plan focused attention on reactions controlled by the enzyme catechol 0-methyltransferase (COMT), using S-adenosyl-L-methionine (SAM) for the methylation reaction. Mammalian COMT is present in mammalian tissues, particularly the liver, and an enzyme preparation from rat liver was used for the experiments. It was found that (S)-norcoclaurine, which is the first isoquinoline produced in benzylisoquinoline-producingplants, was similarly 0methylated in uitro by SAM in the presence of COMT, and a reverse proportion of methylated products was obtained with the (Rbenantiomer (277). Similar 0-methylation of (S)-4’-demethylreticuline (3’-hydroxy-Nmethylcoclaurine), prepared by total synthesis (162), however, afforded almost exclusively (S)-orientaline, with a methoxy group at C-3’ and not at C-4’ as in (S)-reticuline (Fig. 37) (162).

4

4

c HO H 3 0 q N \,#- C H 3

c HO H 3 0 q N\,/ - C H 3

+

+(R)-Reticuline,Morphine

Papaver somniterum

OH

OH

OH

OCH3

(SW-Demethylreticuiine

1

(S)-Reticuiine

Mammals

4

c HO H 3 0 q NN ,\ - C H 3

OCH,

?

c HO H 3 0 p H c H 3

+CH3O

/ \ (S)-isothebaine

OH

(S)-Orientaiine FIG. 37. Bioconversion of (S )-4’-demethylreticuline in plants and mammals.

2.

M A M M A L I A N ALKALOIDS I 1

I67

The O-methylation of the (R)-enamtiomer of 4’-demethylreticuline gave a mixture of (R)-orientaline and (R)-reticuline, with a slight excess of the former. O-Methylation of (S)-and (R)-4’-demethylnorreticulinesby SAM in the presence of mammalian COMT afforded a mixture of products, containing in each case, besides norreticulines and nororientalines, two unknown products (162).This result contrasts with that observed in benzylisoquinoline-producing plants, where the O-methyltransferase “4’OMT” directs the methylation of (S)-4’-demethylreticuline regiospecifically to the hydroxy group at C-4’ (278).This suggests that (S)-reticuline, should it serve as a precursor of (R)-reticuline and (+)-salutaridine in a mammalian biosynthesis of morphine, must originate by another route. (S)-Orientaline, the almost exclusive product in the methylation of (S)4’-demethylreticuline, is not capable of forming the morphinandienone structure on phenol-oxidative coupling, instead giving (-)-isothebaine (279). Two isoquinolines which represent potential intermediates of “mammalian recticuline” (Fig. 38) are (S)-norreticuline (78a) and (R)-tetrahydropapaveroline (75b). Norreticuline could be converted to the dehydroisoquinoline 139 by an oxidase to give, after reduction and N-methylation, (R)-reticuline (l21b).Oxidation of TIQ 78a with the oxidase STOX gave DIQ 139, which on chemical reduction yielded racemic TIQ 78 composed of 50% (R)-enantiomer, leading to a high-yield conversion of the (S) to the (R)-enantiomer if the former were recycled (280).It is known that the reaction of TIQ 78 with formaldehyde, when executed at slightly acidic pH, yields predominantly berbines (211) and at pH 7 (281). O-Methylation of the optical isomers of T H P gave only little of the desired norreticulines (Fig. 30) and mostly products which were methylated at the hydroxy groups at C-7 and C-3. Whether the proportion of methylated products would change in favor of those representing norreticulines if COMT of a different origin were to be used, or if THP would be chemically modified at the N H group (N-CH,, N-acyl) is not known. Further progress regarding the biosynthesis of mammalian morphine rests on the detection and isolation of mammalian TIQs which could convert to (R)-reticuline, and on the isolation of the enzymes responsible for these transformations. In attempts to enhance the production of mammalian morphine by administration of potential precursors in rats, some preliminary experiments have been initiated (282).Single and repeated administration of 30 mg/kg of the optical isomers of norreticuline (78u,b) and reticuline (121a , b )given subcutaneously did not result in a significant analgesic effect in the writhing test, which would have been seen with an increased production of morphine.

168

ARNOLD BROSSI

“““q! HO

+

0CH2

I

OH

@OH

OCH3

OCH3 78a

+ QOH OH 75b

OCH3 78b R=H 121b R=CH3

FIG.38. Possible biosynthetic pathways to mammalian (R)-reticuline.

V. Alkaloid Formation in Mammals as a Therapeutic Concept Bringmann and colleagues have recently extended their investigations of mammalian alkaloids to include the design of therapeutic approaches to metabolic disorders, such as hyperoxaluria, which is manifested by a massive formation of oxalic acid derived from glyoxylic acid (283).They demonstrated that glyoxylic acid could be trapped by D-penicillamine in uitro under physiological conditions, affording the 1,3-thiazolidines shown in Fig. 39. Reaction with dansyl chloride, which reacted only with the cis epimer (140), led to the conclusion that it produced an 80:20 mixture of

2.

MAMMALIAN ALKALOIDS I 1

..++ COOH

HC ,

SyNH

Fi'

169

COOH .++

sY

NH COOH

COOH

140 141 FIG.39. Formation of 1,3-thiazolidines with D-penicillamine.

the cis and trans isomers (140and 141). Pure 140 slowly epimerized on standing to an equilibrium mixture of 140 and 141 as a function of pH. It was found that the calcium salts of 140 and 141 were substantially more soluble than calcium oxalate and nontoxic when given to mice by intravenous injection. With the finding that thiazolidines 140 and 141 were formed and readily excreted when mice were treated with glyoxylic acid and Dpenicillamine, the concept of treating glyoxylate-induced oxaluriasis by forming mammalian alkaloids seems to be rational.

VI. Addendum A two-electron oxidation of N-acetyltyrosine ethyl ester with mushroom tyrosinase, or with periodate, afforded the N-acetyldopa ester 142, together with the (Z)-enamide 145 and the 6-acetoxydopa amide 146 (Fig. 40) (284). It is assumed that 145 originates from dopaquinone 143 via 144 by tautomerization. Michael addition of acetate to quinone 143 is believed to be the origin of 146. The formation of quinone methide 144 from dopa ester 142 by tyrosinase is reminiscent of the formation of iminochromes and quinone methides catalyzed by this enzyme in their formation from Qmethyl dopa ester (285),and such reactions may well occur in mammalian systems. Possible effects of TIQs on rat testicular endocrine function were measured in uitro (286). Gonadotropin-stimulated testosterone production by testicular Leydigs cells was inhibited by T H P (75)and by isosalsoline (l22),but much less by salsolinol (64), salsoline (62)or salsolidine (68). None of the TIQs interacted significantly with the testicular estrogen receptor. T H P (73,isosalsoline (122),and salsolinol (64)competitively inhibited substrate binding to steroidogenic cytochrome P-450with similar efficiency as estrogens. It was concluded that certain mammalian TIQs may amplify peripheral inhibitory effects of testicular endocrine function.

170

ARNOLD BROSSI

142

HomH::Et HO

OAc

146

FIG.40. Two-electron oxidation of N-acetyldopa ethyl ester.

It has been demonstrated that N-hydroxytryptophan can be converted to p-carbolines in two ways (Fig. 41). Pictet-Spengler reaction of 1 with acetals provided the N *-hydroxytetrahydro-p-carbolines ( 2 )(287).A modified Bischler-Napieralski reaction of 1 with trimethylorthoformate gave N 2-oxo-3,4-dihydro-p-carbolines (3), the nitrone function of which can undergo I ,3-dipolar cycloaddition with alkenes (288) and nitriles (289), providing isoxazolidine (4) and dehydro- I ,2,4-oxadiazoline(51, annulated TBCs, respectively. Nitrone 3 also was obtained by oxidation of the I ,4-benzoquinone N-hydroxy-p-carboline2 with 2,3-dichloro-5,6-dicyano(DDQ). N-Oxygenated TBCs showed no affinity for the benzodiazepine and tryptamine receptors (290). Unfortunately, no toxicity data were recorded for these substituted hydroxylamines. Electrochemical oxidation of racemic salsolinol (SAL) was investigated in aqueous solution at physiological pH (291) (Fig. 42). The initial step was a reversible two-electron oxidation of SAL to the short-lived orthoquinone E, isolated as the more stable quinone methide C.Further reaction of E, probably via quinone methide F and addition of water, yielded cis-alcohol A and trans-alcohol B, which are shown in an arbitrarily chosen configuration. Another product which was isolated is the fully aromatic isoquinoline D. Alcohols A and B are chemically related to the condensation product obtained from epinephrine and acetaldehyde (165).

.->m

172

ARNOLD BROSSI

HO

()$OH

H

CH3

CH3

SAL

0

H

O

~

N

H

+

H

C"3

E

CH3

F

\ o m \N

C

H O & N H +

CH3

A

H

O

~

CH3

B FIG.42. Electrochemical oxidation of racemic salsolinol (SAL).

CH3

D

VII. Conclusions Endogenous mammalian alkaloids occur in tissues and fluids at very low levels, and none has ever been isolated in amounts sufficient to allow determination of optical properties by direct measurements. Biological testing in uitro and in uiuo was performed almost entirely with racemic mixtures composed of optical isomers with different biological profiles. A recent report that optically active tetrahydroharmine racemized in the presence of acid suggests that TBCs may be subjected to racemization during workup and afford partially racemized material. For this reason it is difficult to judge whether the biological data reported for mammalian alkaloids are real or unreliable owing to optical inhomogenity. This field of research, although exciting and important in connection with problems associated with alcohol addiction, drug abuse, and disorders originating from interference with dopamine and serotonin formation and metabolism, in the opinion of the author, will only mature and become meaningful when appropriate standards and controls are implemented. A close collaboration of biochemists and clinicians with organic chemists is highly recommended so that research can result in medically meaningful advances.

Acknowledgments

The interest in the National Institutes of Health mammalian alkaloid program by Dr. Bernhard Witkop, NIH Institute Scholar, and his help in finalizing this review are gratefully acknowledged.

N

2.

MAMMALIAN ALKALOIDS I1

173

REFERENCES

I . D. W. Shoemaker, J. T . Cummins, and T. G. Bidder, Neuroscience 3, 233 (1978). 2. 0. Hesse, Ber. Dtsch. Chem. Ges. 11, 1542 (1878). 3. R. Reith, Liebigs Ann. Chem. 120, 247 (1861). 4. A. J. Blackman, D. J . Matthews, and C. K. Narcowicz. J . Nut. Prod. 50,494 (1987). 5a. M. A. Collins, in “The Alkaloids” (A. Brossi, ed.). Vol. 21, p. 329. Academic Press, New York, 1983. Sb. R. H. F. Manske, in “The Alkaloids” ( R . H . F. Manske and H. L. Holmes, cds.). Vol. 8, p. 47. Academic Press, New York, 1965. 5c. K. Stuart and R. Woo-Ming, Heterocycles 3, 223 (1975). 5d. B. Holmstedt, in “Beta-Carbolines and Tetrahydroisoquinolines,”p. 3. Alan R. Liss, New York, 1982. 5e. H. P. Husson, in “The Alkaloids” (A. Brossi, ed.), Vol. 26, p. I . Academic Press, New York, 1985. 5f. T. Hino and M. Nakagawa, in “The Alkaloids” (A. Brossi. ed.). Vol. 34, p. I . Academic Press, San Diego, 1988. 6a. A. Brossi, Heterocycles 7 , 521 (1980). 6b. D. Walterova, V. Preininger, and V. Simanek. Acta Univ. Palacki. Ulomuc. Fac. Med. 97, 121 (1981). 6c. A. Brossi, in “Beta-Carbolines and Tetrahydroisoquinolines,” p. 125. Alan R. Liss, New York, 1982. 6d. G. Bringmann, Nachr. Chem. Tech. Lab. 34, 222 (1986). 6e. M. M. Airaskinen and I. Kari, Med. B i d . 59, 21 (1981). 6f. H. Rommelsbacher, T. May, and R. Susilo, Planta Med. I , 57 (Suppl. I). S.85 (1991). 6g. C. Melchior and M. A. Collins, Crit. Rev. Toxicol. 9, 313 (1982). 6h. G. Bringmann, D. Feineis, H. Friedrich, and A. Hille, Planra Med. 57 (Suppl. I), s.73 (1991). 7. 1. W. Southon and J. Buckingham, in “Dictionary of Alkaloids.” p. 76. Chapman & Hall, London, 1989. 8. V. Erspamer, T . Vitali, M. Roseghini, and J. M. Cei. Experientirr 19, 346 (1963). 9. G. G. Habermehl and W. Ecsy, Heterocycles 5, 127 (1976). IOa. W. M. Whaley and T. R. Govindachari, Urg. React. 6, 151 (1951). lob. A. Brossi, Planta Med. 57 (Suppl. I ) , S.85 (1991). 11. R. A. Abramowitch and I. D. Spenser, in “Advances in Heterocyclic Chemistry” (A. R. Katritzky. A. J. Boulton, and J. M. Lagowski, eds.), p. 79. Academic Press. New York, 1964. 12. “The Merck Index,” 10th Ed., No. 4502 (harman). Merck & Co., Rahway. New Jersey, 1983. 13. E. Spath and E. Lederer, Ber. Dtsch. Chern. Ges. 63, 2102 (1930). 14. G. Hahn and K. Ludwig, Ber. Dtsch. Chem. Ges. 67, 2031 (1934). 15. B. T. Ho, W. Maclsaac. K. E. Walker, and V. Estevez. J. Pharm. Sci. 57,269 (1968). 16. G. Blasko, J. Kardos, M. Simonyi, and C. Szantay, Planta Med., 41 (1986). 17. H. Kulinska, P. Taufmann, H. Frycova. and M. Protiva, Collect Czech. Chem. Commun. 53, 373 (1988). 18. W. P. Gessner, A. Brossi, M. E. Bembenek. and C. W. Abell, Arch. Pharm. ( Weinheim. Ger.) 321, 95 (1988). 19. J. Gynther, P. Peura, and S. Salmi, Acta Chem. Scand. B39, 849 (1985). 20. B. T. Ho, W. M. McIsaac, and L. W. Tansey, J. Pharm. Sci. 58, 999 (1969).

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AMPHIBIAN ALKALOIDS JOHN W. DALY,H . MARTINGARRAFFO, A N D THOMAS F. SPANDE Laboratory of Bioorganic Chemistry National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda. Maryland 20892

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187

111. Bicyclic Alkaloids ............................................................................. A . Histrionicotoxins (Azaspiro[5.5]undecanols)...................................... B. Decahydroquinolines ...............................

199 200

B. Samandarines .........................................

D. Pyrrolizidines .........

....................................

225

F. Quinolizidines ..................................................................... IV. Tricyclic Alkaloids ....... A. Gephyrotoxins ............................................................................. B. Coccinellines .................................................................... C. Cyclopenta[h idines ..................... D. Pyrrolizidine .................................................................... V. Monocyclic Alkaloids ........................................................................

249 251

....................................

255

.......................................

251 261

V1. Pyridine Alkaloids A. Epibatidine ............................................ V11. lndole Alkaloids ..... A. Pseudophrynamines B. lndole Amines C. Dehydrobufote

....................................

238 242 245

...........................

VIII. Imidazole Alkaloids .................................... 263 IX. Morphine .................................................... X. Guanidinium Alkaloids ....................................................................... 264 A . Tetrodotoxin .......... .................. 264

C. Zetekitoxin .............. XI. Other Alkaloids ........................................... I85

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THE ALKALOIDS. VOL.. 43

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XII. Summary .........................................................................................

275 Appendix ...... 277 References ....................................................................................... 281

I. Introduction

Amphibians have developed a wide range of biologically active compounds that are present in skin, often in relatively large amounts. It would appear that many such compounds serve as chemical defense, being released onto the skin surface from cutaneous granular (poison) glands. These compounds include biogenic amines, peptides, proteins, the steroidal bufadienolides and cardenolides, and more than two dozen classes of alkaloids. A general taxonomic survey of the occurrence of such noxious/ toxic substances in skin of amphibians was presented in 1987 (I). Many of these compounds, because of noxious or toxic effects on nerves and muscles of buccal tissue, clearly could serve the host amphibian in defense against predators. Certainly among the amphibian alkaloids, very potent neurotoxins, such as batrachotoxins, samandarines, and tetrodotoxin, are admirably suited as chemical defenses. However, among the peptides there are certain examples, such as the magainins, that in view of potent antimicrobial activity probably serve as a defense against infections from protozoans, fungi, and bacteria (2). Consonant with the hypothesis (see discussion in Ref. 1 ) that the unusual secondary metabolites present in amphibian skin serve in chemical defense against predators and/or microorganisms, most such compounds, including the many classes of alkaloids, exhibit marked biological activity. The uniqueness of the structures of the different alkaloids, which often occur only in a single genus, has focused attention on their taxonomic significance and biosynthetic source. Such amphibian alkaloids also have afforded a challenge for chemical synthesis. A number of reviews in the last decade (3-5)have focused on synthesis of amphibian alkaloids. The present chapter will not treat synthesis but will document only the structural diversity, spectral and chemical properties, biological activity, and distribution in Nature of the nearly 300 known amphibian alkaloids.

3. AMPHIBIAN ALKALOIDS

187

11. Steroidal Alkaloids

Two classes of steroidal alkaloids have been discovered in amphibians, the batrachotoxins and the samandarines. Both discoveries had as their starting point the folk knowledge that a brightly colored amphibian was poisonous. In the case of batrachotoxins, it was the knowledge of Indians that skin secretions of certain brightly colored frogs native to rain forests west of the Andes in Colombia were sufficiently toxic to be used in poison blow darts (see Refs. 5 and 6 for reviews of early literature). Studies initiated at NIH in 1962 (6) led ultimately to the isolation and structure elucidation of the unique steroidal alkaloid batrachotoxin and several congeners. The existence of amphibian alkaloids, indeed of alkaloids from an animal rather than a plant source, had been established in 1866 (71) during investigations of the European fire salamander, a brilliant black and yellow amphibian known since ancient times to be poisonous. The characterization of the steroidal alkaloid samandarine and its congeners from this salamander was initiated in the 1930s, leading, over the following 30 years, to the structural elucidation of all the major alkaloids of the samandarine class (8-10). The batrachotoxins and samandarines were major challenges from the standpoint of structure elucidation, and X-ray crystallographic analysis played a major role.

A. BATRACHOTOXINS I . Structures

The isolation and characterization of batrachotoxin and its congeners from extracts of the skin of the poison-dart frog Phyllobates aurotaenia (6,11-13), and later from the poison-dart frog Phyllobates terribilis ( 1 4 , have been reviewed in detail (5). The key events were the preparation of a crystalline 4-bromobenzoate of a less toxic but stable congener, batrachotoxinin A, and X-ray analysis of this derivative (12), followed by a reevaluation of the spectral properties of the much more toxic batrachotoxin and its major congener, homobatrachotoxin (13). It should be noted that, because of early incorrect interpretations of mass spectra, homobatrachotoxin was initially referred to as “isobatrachotoxin” (12). X-Ray analysis of the 4-bromobenzoate demonstrated that the structure of batrachotoxinin A is 3a,9a-epoxy-l4P, 18-(2’-oxyethyl-N-methylamino)-5Ppregna-7, 16-diene-3/?,1la,20a-triol, as shown in I.

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JOHN W . DALY E T A L .

OH

HO

Batrachotoxinin A (1)

The absolute configuration at C-14 differs from cholesterol, and instead is reminiscent of the bufadienolides and cardenolides. The conformation is markedly constrained by the 3,9-oxygen bridge and the homomorpholine bridge at the C,D ringjuncture. The reevaluation of the spectral properties and comparison to model ethyl pyrrole 2- and 3-carboxylates led to the conclusion that batrachotoxin was the 20a-2,4-dimethylpyrrole-3carboxylate, and that homobatrachotoxin was the 20a-2-ethyl-4-methyIpyrrole-3-carboxylate. The structure of batrachotoxin was confirmed by acylation of the 20a-hydroxyl group of batrachotoxinin A with a mixed anhydride from 2,4-dimethylpyrrole-3-carboxylicacid (13). The structures of batrachotoxinin A, batrachotoxin, homobatrachotoxin, and the 4phydroxy congeners of batrachotoxin and homobatrachotoxin are shown in Fig. 1. The structure of pseudobatrachotoxin, a labile congener that yields batrachotoxinin A during storage, remains unknown. Chemical properties of the batrachotoxins were assessed initially on a microscale (see review in Ref. 5 ) . The Ehrlich reaction proved to be a sensitive indicator of the presence of a pyrrole moiety. Physical and spectral properties of batrachotoxins are presented in Table I. Mass spectra have been presented and interpreted (3,13,14). The parent ion of batrachotoxin is virtually nondetectable by direct probe methods, and instead an apparent molecular ion of m l z 399 is seen, probably because of pyrolytic elimination of the pyrrole carboxylate moiety. Batrachotoxin alkaloids do not chromatograph on capillary gas chromatographic columns, but a pyrolysis product has been detected at 280°C on the temperature-programmed, packed OV-1 columns used for analysis of other dendrobatid alkaloids (see Appendix). The pyrrole carboxylate moiety is responsible for major ions of C,H9N02+ ( m l z 139), C,H9N+

3. AMPHIBIAN

I89

ALKALOIDS

1

R=

H

H

OH H

FIG.1 . Structures of batrachotoxinin A (A, R

H), batrachotoxin (A, R = 2), homobatrachotoxin (A, R = 3), 4P-hydroxybatrachotoxin (B, R = 2). and 4P-hydroxyhomobatrachotoxin (B, R = 3). =

(m/z95), and C,H,N+ (mlz94) in batrachotoxin and C,H,,NO,+ (mlz 153), C7H,N02+ (mlz 138), C 7 H I , N +(mlz 109), and C6H8N+(mlz 94) in homo-

batrachotoxin. The homomorpholine ring yields a major fragment ion of C,H,,NO+ (mlz 88). Proton magnetic resonance spectra have been presented and discussed (3,5,13-15). Carbon- 13 magnetic resonance assignments have also been reported (14).

2 . Biological Activity Batrachotoxin, not unexpectedly in view of its use as a dart poison by South American Indians, is an extremely toxic substance. The LD,, on subcutaneous injection in mice is about 40 ng. Homobatrachotoxin is only slightly less toxic, while batrachotoxinin A is 500-fold less toxic. The nature of the ester function at the 20a position is of critical importance to toxicity. Thus, the 20a-benzoate of batrachotoxinin A is fully as toxic as batrachotoxin, whereas the 20a-4-bromobenzoate has very low toxicity. For a summary of the toxicities of natural and synthetic batrachotoxins, see Ref. 5 . The pharmacology of batrachotoxins that underlies their high toxicity is the result of a specific high-affinity interaction of batrachotoxin with a

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JOHN W . DALY E T A L .

TABLE I PHYSICALA N D SPECTRAL PROPERTIES OF BATRACHOTOXINS ( 5 , lI, 13-16) Batrachotoxin, C31H42N206 Mass spectrum: mlz 538 (el),399(3), 312(13), 294(10), 286(10), 184(30), 139(65), 138(24), 122(20), 121(13), 120(10), 109(15), 95(100), 94(100), 88(34), 71(26) Ultraviolet: A, 234 nm, E 9800: 262 nm, E 5000 Infrared: 1690 cm - I Optical rotation: [a]:& - 5 to - lo", [a]&-260" (0.23, CH,OH) Rfvalue: 0.45' Homobatrachotoxin, C32HuN206 Mass spectrum: rnlz 552(41), 399(6), 312(25), 294(20), 286(22), 184(60), 153(90), 139(22), 138(100), 122(12), 121(15). 120(26), 109(60), 95(23), 94(94), 88(72), 72(28) Ultraviolet: A,,233 nm. E 8900, 264 nm, E 5000 Infrared: 1690 cm - I Rfvalue: 0.50 4P-Hydroxybatrachotoxin, C3,H42N207 Mass spectrum: rnlz 554 (el),415(6), 386(5), 328(30), 310(18), 184(38), 139(68), 83100) Rfvalue: 0.10

4~-Hydroxyhomobatrachotoxin,CI2HMN2O7 Massspectrum: mlz568(~1),415(5), 386(6). 328(7),310(17), 184(14),153(60),139(100),8466) Rr value: 0.10 Batrachotoxinin A, C24H35N05 Mass spectrum: mlz 417(2), 399(11), 330(100), 312(30), 202(15), 184(11), 158(14), 88(60) mp: 160-162°C (synthetic) Ultraviolet: end absorption Optical rotation (synthetic): [a]! - 42" (0.45, CHIOH) Rfvalue: 0.28 pK,: 8.2 Pseudobatrachotoxin' Mass spectrum: mlz 399(6), 312(17), 294(15), 286(6), 202(3), 184(48),88(100),71(45); ions at mlz 342 (C22H32N02. 6) and 166 (CIOHl6NO, 12) were detected, but are probably due to an impurity Ultraviolet: end absorption Rfvalue: 0.25 a Ultraviolet spectra are shown in Ref. 13. 'Thin-layer chromatography: silica gel, HCCI,-MeOH (9: I , vlv). Pseudobatrachotoxin converts to batrachotoxinin A at room temperature.

3. AMPHIBIAN ALKALOIDS

191

site on voltage-dependent sodium channels of nerve and muscle. Interaction of batrachotoxin with this site stabilizes the sodium channel in an open formation, leading to a massive influx of sodium ions and depolarization of nerve and muscle. Tetrodotoxin, through a specific blockade of sodium channels, can prevent batrachotoxin-elicited depolarization. The activation of sodium channels by batrachotoxin is time- and stimulus-dependent, suggesting that batrachotoxin acts preferentially on an open channel. Certain other alkaloids, namely, veratridine and aconitine, and the diterpene grayanotoxin, appear to interact at the same site as batrachotoxin, but they are much less potent and efficacious in maintaining the channel in an open, conducting form. The action of batrachotoxin on sodium channels can be enhanced allosterically by certain polypeptide neurotoxins, namely, a-scorpion toxin and anemone toxins, and by the terpenoid brevetoxins from marine organisms. Conversely, many local anesthetics appear to reduce allosterically the action of batrachotoxin. The development of a batrachotoxinin A 20a-[3H]benzoate as a radioligand for batrachotoxin-binding sites on sodium channels has proved invaluable in studying the allosteric regulation of such interactions. Batrachotoxin at present remains an important, indeed often essential, tool for mechanistic studies of the function of voltage-dependent sodium channels and for the investigation of the role of depolarization and/or influx of sodium ions on physiological functions. Batrachotoxin has been particularly useful in the study of the function of sodium channels, purified and reconstituted into artificial lipid bilayers. A summary and overview of the extensive studies with batrachotoxin appeared in 1986 (5). Since that time more than 100 articles dealing with the activity of batrachotoxin and/ or the radioligand batrachotoxinin A 20a-[3H]benzoate have appeared, and it is beyond the scope of the present review to summarize this extensive recent literature. A few selected developments are as follows: allosteric enhancement of the action of batrachotoxins by pyrethroid insecticides (17,18) and inhibition by polyunsaturated alkanoic N-alkylamide insecticides (e.g., pelliterine) (19);allosteric enhancement of binding of batrachotoxinin A 20a-[3H]benzoateby the polypeptide striatoxin isolated from a marine snail (20); stimulation of phosphoinositide breakdown by batrachotoxin and dependence on influx of sodium ions (21); structure-activity relationships for derivatives of 7,8-dihydrobatrachotoxininA (22); an apparent interaction of muscarinic receptors and sodium channels that can affect the binding and action of batrachotoxin (23);development of irreversible blockers of binding batrachotoxinin A 20a-[3H]benzoate(24,25); demonstration of cyclic AMP-dependent regulation of binding sites for batrachotoxinin A 20a-[3H]benzoate(26).

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JOHN W . DALY E T A L .

3. Occurrence

The batrachotoxins represent a unique class of steroidal alkaloids whose structures contain several unprecedented elements, in particular the homomorpholine ring sharing the steroidal C,D ring juncture, the 2,4-dialkyl pyrrole-3-carboxylate moieties, and the 3,9a-hemiketal oxygen bridge. There is, to our knowledge, no other natural compound closely related in structure to the batrachotoxins, and hence the biosynthetic origin is of some interest. A preliminary study revealed no detectable incorporation of radiolabeled acetate, mevalonate, cholesterol, or serine into batrachotoxins of the poison-dart frog Phyllobates aurotaenia or into the bicyclic alkaloids ofDendrobatespumilio after 6 weeks of intraperitoneal injections (27). Batrachotoxins have been detected from amphibians only in the five species of Phyllobates, the true poison-dart frogs, which were defined as a monophyletic group, partly in recognition of the occurrence of batrachotoxins in their skin (28). Batrachotoxins are virtually absent in other tissues of these frogs. Only the three Colombian species of Phyllobates have high enough levels of batrachotoxins in their skin to make them useful for poisoning blow darts. The highest levels occur in Phyllobates terribilis, which has roughly 500 pg batrachotoxin, 300 p g homobatrachotoxin, and 200 pg batrachotoxinin A per frog skin ( 1 ) . This frog is so toxic that Indians merely scrape the grooved tips of blow darts across the frog’s back (28). With the less toxic Phyllobates bicolor and Phyllobates aurotaenia, both of which contain roughly 20 pg batrachotoxin, 10 p g homobatrachotoxin, and 50 p g batrachotoxinin A per frog skin (I), the frog is impaled on a stick in order to provoke a profuse skin secretion containing the batrachotoxins that is used to poison the blow darts. The remaining two species of Phyllobates, both Central American species, have very low levels of batrachotoxins. The Costa Rican species, Phylfobates vittatus, has only 0.2 p g batrachotoxin, 0.2 pg homobatrachotoxin, and 2 pg batrachotoxinin A per frog skin ( 1 ) . Several populations of the Panamanian-Costa Rican species Phyllobates lugubris do not have levels of batrachotoxins sufficient to be detectable either by toxicity or by the sensitive Ehrlich color reaction (Ref. 1 and J. W. Daly, unpublished data). One Panamanian population does contain very low levels (estimated) of 0.2 p g batrachotoxin, 0.1 pg homobatrachotoxin, and 0.5 p g batrachotoxinin A per frog skin (1). Frogs of the genus Phylfobates contain other classes of alkaloids. Usually these are present only in trace amounts, although the montane Colombian species Phyllobates bicolor contains significant amounts (100 p g or more per frog skin) of decahydroquinolines and histrionicotoxins (I). In view of the apparently extremely limited occurrence of batrachotoxins in Nature, namely, in the five monophyletic species of the dendrobatid

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genus Phyllobates, and their absence in other dendrobatid species and in many other species of amphibians examined for lipophilic alkaloids (see Ref. I ) , it seems likely that a complete set of biosynthetic enzymes responsible for formation of batrachotoxins has evolved in this monophyletic group of dendrobatid frogs. A dietary source seems unlikely. However, levels of batrachotoxin/homobatrachotoxinin Phyllobates terribilis did decline slowly when wild-caught frogs were maintained in terraria on fruit flies and crickets (29). After 3 years, levels were 320 and 480 pg in two frogs. After 6 years, levels were 250 p g in one frog, but were 1150 p g in another frog sacrificed because of bloating. Wild-caught frogs had levels of 1140 ? 140 p g ( n = 10). Second generation terrarium-reared Phyllobates terribilis did not have detectable levels of batrachotoxins; that is, if present at all, levels of batrachotoxins were more than 10,000-fold lower than in the wild-caught parents (29). Thus, unresolved questions concerning the synthesis of batrachotoxins by the poison-dart frogs are raised. Are they present in the food chain? If so, how are they concentrated selectively by Phyllobates terribilis > P . aurotaenia and P . bicolor % P . vittatus > P . lugubris, and not by other dendrobatid frogs? The diet of Phyllobates and other dendrobatid frogs consists entirely of small insects, such as ants, fruit flies, mosquitoes, crickets, and termites. Is there an essential cofactor or precursor present in the diet of wild Phyllobates, but not in the fruit fly and cricket diet of frogs maintained in terraria? If so, it seems likely not to be from the wild insects per s e , but more likely to be from the gut content of the insects. Or is there a symbiotic organism present in the wild frogs that is needed for elaboration of batrachotoxins? Or is there an environmental trigger that turns on and/or maintains expression of frog enzymes responsible for synthesis of batrachotoxins? It is noteworthy that P . aurotaenia and P . terribilis, both wild-caught and captive-raised, have voltage-dependent sodium channels that are insensitive to batrachotoxin (29,30).Thus, such frogs have evolved a batrachotoxin-resistant sodium channel. Other dendrobatid frogs, for example, Dendrobates histrionicus, have sodium channels sensitive to batrachotoxin (cited in Ref. 29). The same questions concerning the origin of batrachotoxins in true poison-dart frogs pertain to other dendrobatid alkaloids, since dendrobatid poison frogs of the genera Dendrobates and Epipedobates also do not contain alkaloids when reared in captivity (31). One of the batrachotoxin alkaloids has now been discovered in a nonamphibian source. Remarkably, the source is the skin and feathers of New Guinea birds of the genus Pitohui (family Pachycephallinae) (32). Of the three species examined, highest levels occurred in the hooded pitohui (Pitohui dichrous), where it is estimated that the skin and feathers of one bird contained about 20 p g of homobatrachotoxin. Batrachotoxin was not

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detected. Striated muscles contained much lower levels of homobatrachotoxin. The bird is recognized by natives of New Guinea as being toxic, and they advise that it should be skinned prior to cooking if it is eaten. This discovery of homobatrachotoxin in a bird suggests the independent development of biosynthetic pathways leading to batrachotoxins in birds (Pitohui) and frogs (Phylfobates).Birds of the genus Pitohui subsist on insects and seeds. None of the frogs of New Guinea are from a genus that produces alkaloids. The levels of batrachotoxins in the skin of Phyllobates terribilis (-2000 pg/g skin) are orders of magnitude higher than the levels of hombatrachotoxin in the skin of the hooded pitohui (-5 pg/g skin).

B. SAMANDARINES

I . Structures The isolation and characterization of samandarine and its congeners from the parotoid skin glands of the European fire salamander (Salamandra salamandra) and alpine salamander (Salamandra atra) have been reviewed in detail (8-lo), most recently in 1986 (5).The majority of isolations have been from two subspecies of Salamandra salamandra. The 1986 review also covers synthetic approaches to the samandarines. Samandarine (11) proved to be a steroidal alkaloid containing an oxazolidine in an altered A ring. The ring junctions are as in cholestane. Since these studies were essentially completed prior to the emergence of mass spectrometry and nuclear magnetic resonance spectroscopy, structure elucidation relied mainly on chemical conversions, infrared and ultraviolet spectroscopy, and X-ray crystallography. The majority of the samandarine alkaloids contained the oxazolidine ring (samandarine, 0-acetylsamandarine, sa-

19

OR

Samandarine (11)

3. AMPHIBIAN

195

ALKALOIDS

mandarone, samandaridine, samandenone, samandinine), which affords a diagnostic pair of infrared absorbances in the region from 830 to 875 cm-' (33). The salts of the samandarines show only a weak absorbance at 870 cm-' . Structures of the nine naturally occurring samandarine alkaloids are shown in Fig. 2. Cycloneosamandione did not contain the oxazolidine ring, but rather a tautomeric aminocarbonyl carbinolamine system (34). Two structures were proposed, one a carbinolamine formed with a C-19 aldehyde, the other a carbinolamineformed with a 6-keto group. Ultimately, the cyclone-

*

dO H0 Samandarine 0-Acety lsamandarine

Samandarone

R=H R = COCH3

HN O,

HN ,.@

i

yp \

Samandaridine

Samandinine

HN/A O, :

@o N

fl0 Samandenone

HN&OH

-manine

Cycloneosamandione N "1'.

I......,

... 6

k

OH

lsocycloneosamandaridine FIG.2. Structures of samandarine alkaloids isolated from salamanders of the genus Salamandra.

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JOHN W. DALY E T A L .

osamandione was demonstrated to have the C-19 carbinolamine structure shown in Fig. 2 (35;see also Ref. 5 ) . One other samandarine alkaloid, namely cycloneosamandaridine, contained a tautomeric aminocarbonyl e carbinolamine system, and it was initially thought to be related to samandaridine just as cycloneosamandione was related to samandarone (34). However, synthetic cycloneosamandaridine with the carbinolamine formed with a C- 19 aldehyde was not identical with the natural compound (36), which thus appears to have a carbinolamine formed with a 6-keto group. Retention of the original name, cycloneosamandaridine, for the natural compound would be unfortunate, since it implies the presence of the cycloneosamandione A,B ring system. The name isocycloneosamandaridine, proposed in Ref. 5 , is used in this chapter (see Fig. 2). The name cycloneosamandaridine was retained for the synthetic material, which is shown in Fig. 3; it has not been reported in Nature. There is one other natural samandarine alkaloid that does not contain the oxazolidine ring, but instead a seven-membered nitrogen-containing A ring. The structure of this alkaloid, samanine, is shown in Fig. 2. A synthetic alkaloid, isomeric with samandarine, has been erroneously reported to be isolated from a natural source, namely, the salamander Cryprobrunchus muximus (37). However, S. Hara has informed us that this alkaloid was never isolated from Nature (see discussion in Ref. 5). It is referred to in Fig. 3 as the Hara-Oka alkaloid. Physical and spectral properties of samandarine alkaloids are presented in Table 11. Mass spectra of various samandarine alkaloids and derivatives have been presented (38-43). Fragments of C,H,NO+ (mlz 86) and C,H,NO ( m / z 85) are typical of oxazolidine-containing samandarine alkaloids. Infrared spectra of various samandarine alkaloids have been published (33,34,43-46 and references therein). Proton magnetic resonance spectra for samandarone, samandenone, and cycloneosamandione have been presented (38,40,41). Samandarine, samandarone, and +

Cycloneosamandaridine "Hara-Oka alkaloid" FIG. 3. Synthetic sarnandarine alkaloids. These compounds have not been detected in Nature.

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I97

TABLE I1 PHYSICAL A N D SPECTRAL PROPERTIES OF SAMANDARINE ALKALOIDS (33-36.38-47) Samandarine, C IN02 Mass spectrum: rnlz 305(35), 277(14), 86(94), 85(100). 57(36), 56(50) Infrared: 853,832 cm-' Optical rotation: [a];+ 29.5" mp (free base): 188°C Rr value: 0.34" 0-Acetylsamandarine, C21H31N0, Mass spectrum: unpublished Infrared: 1730, 1240,840, 830 cm-' mp (free base): 158- 159°C Rr value; 0.49 Samandarone, C 19H29N02 Mass spectrum: rnlz 303(27), 275(12), 86(24), 85(lOO), 57(32), 56(66) Infrared: 1740,845, 831 cm-' Optical rotation: [a];- 115.7" mp (free base): 190°C Samandaridine, CZlHI I NO, Mass spectrum: rnlz 34326). 317(13), 86(17),85(100), 57(33), 56(44) Infrared: 1760, 840, 830 cm-' Optical rotation: [a]:;,+ , 14.1" mp (free base): 287-288°C Rr value: 0.40 Samandenone, C22H,lN02 Mass spectrum: mlz 343(100),328(18),31320). 300(8), 283(1 I ) , 259(13), 245(10), 148(17), 86(85), 85(93), 57(67),56(70) Infrared: 1678, 1611, 845, 828 cm-1 mp (free base): 189-191°C Samandinine, C24H19N01 Mass spectrum: rnlz 389, 85,57, 56 Infrared: 1725, 1240, 845. 820 cm-' mp: 170°C Cycloneosamandione, C19H?9N02 Mass spectrum: rnlz 303(l8), 275(10), 274(26), 57(48), 56(35) Infrared: 1750 cm-' mp (free base): 118-1 19°C Rr value: 0.43 (conrinued)

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w. DALY

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TABLE I1 (Continued) Isocycloneosamandaridine,bC21H,IN03 Mass spectrum: mlz 345(3), 344(14), 330(53), 97(26), 9315). 85(32), 83(30), 71(41), 69(55), 57(55), 56(100) Infrared: 1780 cm-I mp (free base): 28 1-282°C Samanine, C19H3,N0 Mass spectrum: mlz 291(100), 276(71), 96(38), 82(22), 70(28), 57(70), 56(90) Infrared (N.0-diacetyl): 1735, 1640 cm-I mp (free base): 193-195°C "Thin-layer chromatography: silica gel, CHC1,-MeOH (9:I , vlv). Formerly referred to as cycloneosamandaridine (43).

0-acetylsamandarine elute at 220, 223, and 243"C, respectively, on the temperature-programmed, packed OV- 1 columns used for the analysis of dendrobatid alkaloids (see Appendix).

2 . Biological Activity Samandarine is a relatively potent, centrally active neurotoxin with an injected lethal dose for a mouse of about 70 pg (see Ref. 5 , and references therein). Samandarone is somewhat less toxic. Convulsions, respiratory failure, cardiac arrhythmias, and partial paralysis precede death. The fire salamander is sensitive to its own toxin. There have been no recent studies on the pharmacology of samandarine alkaloids. Samandarine is a potent local anesthetic (48). Cardiac depressant effects (48,49)and inhibition of binding of a radiolabeled batrachotoxin analog to sodium channels (50) are consonant with the potent local anesthetic activity of samandarine. Samandarine alkaloids show antimicrobial activity (51 and references therein). 3. Occurrence

The European fire salamander (Salamandra salamandra) and the alpine salamander (Salamandra atra) are the only amphibians known to contain samandarine alkaloids. These are the only two species in this genus. The proposal that extracts of the brilliant black and yellow Australian myobatrachid frog Pseudophryne corroboree contained samandarine alkaloids (52) has proved to be incorrect, and this and other frogs of the genus Pseudophryne instead contain pumiliotoxins and pseudophrynamines (see Sections III,C and VI1,A). The major alkaloids of Salamandra salamandra are samandarine, samandarone, and 0-acetylsamandarine. There do not

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199

appear to be any alkaloids related in structure to samandarines found elsewhere in Nature. In contrast to dendrobatid alkaloids, which are not present in captiveraised dendrobatid frogs, fire salamanders produce samandarine alkaloids when reared in captivity (G. Habermehl, personal communication, 1989). No apparent differences in alkaloid content occurred for at least three generations. Incubation of secretions from salamander parotoid glands with radiolabeled cholesterol in buffer for 3 days at room temperature led to some apparent incorporation of radioactivity into samandarine alkaloids

(53). 111. Bicyclic Alkaloids Most amphibian alkaloids are not as complex in structure as the steroidal batrachotoxins and samandarines. Of the 300 known amphibian alkaloids, most have been characterized from the skin extracts of frogs of the family Dendrobatidae and, hence, have been referred to as dendrobatid alkaloids. The major bicyclic classes of dendrobatid alkaloids are the histrionicotoxins, decahydroquinolines, and pumiliotoxin-A class. Because of the presence of a piperidine ring in most dendrobatid alkaloids, they also have been referred to as piperidine-based alkaloids. It was apparent by the late 1970s that hundreds of different alkaloids would be detected in skin extracts of dendrobatid frogs, and, therefore, a code system was developed using the nominal molecular weight of the alkaloid with an added letter to distinguish alkaloids with the same nominal molecular weight. Thus, the first alkaloid reported of a particular molecular weight had no attached letter, but later addition of an A was required for this alkaloid when another alkaloid with the same nominal molecular weight was discovered (e.g., 219 became 219A, 252 became 252A). In some cases it appeared initially that two alkaloids were present as an inseparable mixture, for example, a mixture of 223A and 223B; however, several were later shown to be a single compound, and a combination of the original codes was retained, as in 223AB, 239AB, 239CD, and 269AB. To characterize isomers, prefixes (e.g., cis, trans, epi, iso) and primes (Ar,A”) have been introduced. In some cases, for example, 307A, two diastereomers 307A’ and 307A” have been isolated, and the name 307A is now used to indicate that the diastereomeric composition of the alkaloid was, or is, unknown. Certain of the dendrobatid alkaloids also have been given trivial names, as in the various histrionicotoxins, pumiliotoxin C (now referred to as the decahydroquinoline cis-195A), pumiliotoxins A and B, gephyrotoxin, and epibatidine. Structure elucidation of major

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200

alkaloids has been mainly by X-ray crystallography and nuclear magnetic resonance spectroscopy, while minor and trace components have been characterized by gas chromatography-mass spectrometry, gas chromatography-Fourier transform infrared (FTIR) spectroscopy, and microchemical manipulations (see Appendix). A. HISTRIONICOTOXINS (AZASPIRO[5.5]UNDECANOLS)

I . Structures The structures of histrionicotoxin (283A)and dihydroisohistrionicotoxin (285A)were determined by X-ray crystallography (54) to be as shown in 111 and IV. Structures of most of the other histrionicotoxins have been

Histrionicotoxin 283A (111)

Dihydroisohistrionicotoxin 285A

(1V)

3.

20 1

AMPHIBIAN ALKALOIDS

based on nuclear magnetic resonance spectral analysis. At present, sixteen histrionicotoxins have been detected (I ,55). Nine have unsaturated pentyl (2 position) and butyl (7 position) side chains, while another seven have instead a three-carbon side chain at the 2 position. Three of the latter group have a two-carbon side chain at the 7 position. Structures of the known histrionicotoxins are shown in Fig. 4. Mass spectra of histrionicotoxins show a characteristic pathway for loss of the 2-substituent and a characteristic fragment ion at mlz 96 (C,HIoN+).Mass spectral fragmentation pathways for histrionicotoxins have been discussed (55,57; see also tabulations in Ref. 3 and data in Refs. 54,58,59). The properties of the natural histrionicotoxins are presented below in a format introduced in 1978 (60) for the dendrobatid alkaloids. The entries are as follows: (1) the code designation based on molecular weight and identifying letter(s) in boldface; (2) the trivial name, if any; (3) an empirical formula based on high-resolution mass spectrometry (tentative formulas

OH

R'

15-Carbon H i s t r i o n i c o t o x i n s 235A 237F 239H

-CH,CH=CH, -CH,CH,CH, -CH,CH,CH,

-CH=CH, -CH=CH, -CH,CH,

17-Carbon H i s t r i o n i c o t o x i n s 259A 261 263C 265E

-CH,CH=CH, -CH2CH=CH2 -CH,CH=CH, -CH,CH,CH,

cis-CH=CHC-CH cis-CH=CHCH=CH, -CH,CH,CH=CH, -CH2CH2CH=CH2

19-Carbon H i s t r i o n i c o t o x i n s 283A 285A 2858 285C 285E 287A 2878 287D 291A

c i s-CH,CH=CHC=CH -CH,CH CH-C-CH, c i~ - C & C H ~ ~ C = C H -CH2CH CH C=CH c i s-C&Ctf=cHCH=CH, -CH,CH CH=C=CH c i s-C&CH=CHCtf=CH, -CH,CH,CH,C=CH -CH2CH2CH2CH=CH2

cis-CH=CHC=CH cis-CH=CHC-CH cis-CH=CHCH=CH, cis-CH=CHCsCH cis-CH=CHC-CH cis-CH=CHCH=CH, cis-CH=CHCH=CH, cis-CH=CHCH=CH, -CH,CH,CH=CH,

FIG.4. S t r u c t u r e s of h i s t r i o n i c o t o x i n s from dendrobatid frogs.

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JOHN W. DALY E T A L .

are indicated by single quotes); (4) an &value (silica gel, CHC1,-CH,OH, 9:l,v/v); (5) the emergent temperature on a 1.5% 6-ft OV-I-packed gas chromatographic column programmed from 150 to 280°C at 10°C per minute; (6) the electron impact-mass spectral ions followed in parentheses with intensities relative to the base peak set equal to 100, although not all peaks, in particular low-mass (rnlz < 60) and low-intensity peaks, are reported (in some cases pseudo-electron impact spectra obtained with an ion trap instrument are reported); (7) the number of hydrogens exchangeable with deuteroammonia (OD, ID, etc., meaning no exchangeable, one exchangeable, etc.); (8) citations to vapor phase-FTIR spectra or data (infrared data for solutions are so indicated); (9) perhydrogenation derivative (H,, no addition of hydrogen; H2, addition of two hydrogens, etc.); and (10) pertinent comments. Omissions indicate that no or only ambiguous data are available (for further details, see Appendix). Histrionicotoxins

235A. CI5H2,NO, 0.36, 176"C, rnlz 235(5), 234(2), 218( 1 9 , 194(76), 176(25), 150(8), 96(100). 2D. Infrared spectrum (55). H, derivative, rnlz 239, 196, 178, 96. 237F.CI5H2,NO,-, 180"C, rnlz 237(5), 220(3), 194(54),96( 100). 2D. H, derivative. 2398.C15H2,N0,-, 182"C, rnlz 239(7), 238(4), 221(6), 196(35), 178(lo), 96( 100). 2D. H, derivative. 259A. CI7Hz5NO,0.36, 190"C, rnlz 259(4), 242(2), 218(18), 200(6), 96( 100). 2D. Infrared spectrum (55). H, derivative, rnlz 267(20), 250( 13), 224(39), 196(15), 168(19), 152(100), 96(68). 261. C17H2,N0,-, 19OoC, rnlz 261(8), 220(100), 204(10), 96(68). 2D. Infrared spectrum (55).H, derivative. 263C.C17H,,N0, -, 192"C, mlz 263(1), 222(100), 204(10), 96(48). 2D, H, derivative. 2653.C,,H,,NO, -, 194"C, rnlz 265(5),264(3),248( lo), 224(48), 222(20), 152(loo), 139(63),96(95). 2D. Infrared spectrum (55). H, derivative. 283A. Histrionicotoxin, CI9Hz5NO,0.50, 210"C, rnlz 283(9), 282(2), 266(5), 218(48), 200(27), 160(22), 96( 100). 2D. Infrared spectrum (55). H,, derivative, 0.36, 214"C, m l z 2 9 3 12), 294(2), 278( 131, 252( 18), 224(73), 196(27), 180(IOO), 168(39), 96(68). A A-17-trans-histrionicotoxin (283A') can occur in trace amounts with 283A but may be an artifact (55). 285A.Isodihydrohistrionicotoxin, C,,H,,NO, 0.39, 215"C, rnlz 285(7), 284(2), 268(8), 252(12), 238(3), 218(6), 200(9), 176(24), 162(18),96(100).2D. Infrared spectrum (55). HI, derivative.

3. AMPHIBIAN

ALKALOIDS

203

285B.Neodihydrohistrionicotoxin, C,,H,,NO, 0.46, 21 l"C, mlz 285(4), 284( l), 268(3), 220(37), 202(9), 160(20), 96( 100). 2D. Infrared spectrum (55). HI, derivative. 285C. Allodihydrohistrionicotoxin, C,,H,,NO, 0.40, 21 1"C, mlz 285(4), 284(1), 268(2), 218(5), 176(15), 162(17), 96(100). 2D. Infrared spectrum (55). HI, derivative. 285E. Dihydrohistrionicotoxin, CI9H2,NO, 0.50,212"C, mlz 285( 13), 268( lo), 218( loo), 200(84), 176(13), 96(78). 2D. Infrared spectrum (55). HI, derivative. 287A. Isotetrahydrohistrionicotoxin, C,,H,,NO, 0.42, 216"C, mlz 287( 12), 286(4), 270(3), 220(30), 202(34), 176(45), 162(60), 148(24),96( 100). 2D. Infrared spectrum (55) . H, derivative. 287B.Tetrahydrohistrionicotoxin, C,,H,,NO, 0.43,213"C, mlz 287( 13), 286(2), 270(2), 220(43), 202(18), 96( 100). 2D. Infrared spectrum (55). H, derivative. 287D. Allotetrahydrohistrionicotoxin, CI9H,,NO, 0.35. 215"C, mlz 287( 14), 270(8), 220(24), 202(36), 176(38), 162(49), 96( 100). 2D. H, derivative. 291A.Octahydrohistrionicotoxin, C,,H,,NO, 0.35, 212"C, mlz 291( 12), 290(2), 274(14), 250(54), 222(24), 194(18), 192(12), 178(loo), 96(52). 2D. Infrared spectrum (55). H, derivative. Other physical and spectral properties of the histrionicotoxins are presented in Table 111. Proton and carbon-13 magnetic resonance assignments have been presented (57-59) and reviewed (3). For proton spectra, see Refs. 3,57-59; for carbon-13 assignments, see Ref. 55. The diagnostic infrared peaks for various histrionicotoxins in solution have been tabulated ( 3 3 . Vapor-phase FTIR spectra of histrionicotoxins and derivatives have been presented and discussed (55). The pK, values for histrionicotoxin and its reduction product, perhydrohistrionicotoxin,were in the range 9.0-9.3 as determined by titration. Butylboronic acid derivatives of histrionicotoxins can be easily prepared and provide several advantages for gas chromatographic-mass spectral and FTIR analysis (55). The cis-diene-containing histrionicotoxins (e.g., 285B, 2853, 287A, 287B) are slowly isomerized to a photostationary state containing 55% truns-dienes by ambient light (55). The cis-enyne moiety, although not so easily photoisomerized, also undergoes isomerization to truns-enynes, making it likely that 283A', a previously isolated 7-(truns-buten-3-yne) isomer of histrionicotoxin (59), is an artifact. Other possible artifacts are two formaldehyde condensation products (oxazolidines) of molecular weights 297 and 299 from 285A (or 285C) and 287A,respectively, detected in some extracts and evidently arising from traces of formaldehyde in the methanol used for extraction (55).

204

JOHN W . DALY E T A L .

TABLE 111 PHYSICAL A N D SPECTRAL PROPERTIES OF HISTRIONICOTOXINS (535) 235A Optical rotation: [ale - 38.6"' (1.75, CHCI,) Histrionicotoxin (283A) mp (free base): 79-80°C Ultraviolet: A,, 224 nm, E 22,300 (C2H50H) Optical rotation: [a];5- 96.30b (HCI, I .O, C2H50H)

Isodihydrohistrionicotoxin (USA) Ultraviolet: A,,, 225 nm, E 8100, 235 nm, E 7200 (C2H50H) Optical rotation: [a]$-35.3" (HCI, 0.5, C2H50H) Neodihydrohistrionicotoxin (285B) Ultraviolet: A,,, 225 nm, E 17,300 (C2H50H) Optical rotation: [a];' - 125.9" (HCI, 1.1, C2H50H)

Allodihydrohistrionicotoxin (285C) Optical rotation: [a];' -43.4" (HCI, 1.2, C2H50H) Dihydrohistrionicotoxin (285E) Ultraviolet: A,,, 226 nm, E 24,700 (C2H50H) Optical rotation: [aid5- 122" (HCI, 1.0. C2H50H)

Isotetrahydrohistrionicotoxin (287A) E 19,200 (C2H50H)

Ultraviolet; A,,

228 nm,

Ultraviolet: A,,,

228 nm,

Tetrahydrohistrionicotoxin (287B) E 3900 (C2H50H)

Perhydrohistrionicotoxin (synthetic reduction product of 19-carbon histrionicotoxins) Optical rotation: [a]$- 34.6", -36.2" (HCI, 1.0, C2H50H,CHCI,) "Synthetic 235A had an [a]%of bSynthelic 283A had an [a]?of

-

102" (1.82. C2HcOH)(56). 114" (1.06, C,H,OH) (56).

2. Biological Activity

Histrionicotoxins have relatively low toxicities, and thus the toxin designation is a misnomer. A subcutaneous dose of 1000 p g of either histrionicotoxin o r isodihydrohistrionicotoxin in a mouse causes locomotor difficulties and prostration (54,60). Pharmacologically, the histrionicotoxins affect at least three classes of channels in nerve and muscle. The first class of channels are the receptorregulated channels, in particular the nicotinic acetylcholine receptorchannel, where histrionicotoxins, in a time- and stimulus-dependent man-

3.

AMPHIBIAN ALKALOIDS

205

ner, block the channel conductance and accelerate the desensitization or inactivation of the channel. The effects are those of a so-called noncompetitive blocker, and indeed the histrionicotoxins now represent classic noncompetitive blockers for nicotinic receptor-channel complexes (61-64). The development of a [3H]perhydro derivative of histrionicotoxin has afforded a radioligand for investigation of noncompetitive binding sites on the nicotinic receptor-channel complex. Histrionicotoxins also block conductance of a receptor-regulated channel that is activated by glutamate or N-methylaspartate (65,66). The second class of channels are the voltage-dependent sodium channels, where histrionicotoxins reduce conductances in a manner reminiscent of local anesthetics (61). The third class are the voltage-dependent potassium channels, where histrionicotoxins reduce conductances in a time- and stimulus-dependent manner. Structure-activity relationships for histrionicotoxins differ at the three classes of channels (61). A summary and overview of the extensive studies of the biological effects of histrionicotoxins appeared in 1986 (5). Most of the studies have focused on cholinergic systems. Since that time, many articles have appeared. It is beyond the scope of the present review to do more than continues highlight selected developments. [3H]Perhydr~histrionicotoxin to be used as a probe for noncompetitive blocker sites on the muscle-type nicotinic receptor-channels of the electric ray electroplax (64). Recent studies on ganglion (67), central neuronal (63), pheochromocytoma ( 6 4 , and adrenal chromaffin cells (68) demonstrate that histrionicotoxins are not only potent noncompetitive blockers of muscle-type nicotinic receptor-channels, but also noncompetitive blockers of ganglionic-type and central neuronal-type nicotinic receptor-channels. Histrionicotoxin blocks neuromuscular transmission in preparations of Dendrobates histrionicus, but higher concentrations are required to cause blockade than in the ranid frog Rana pipiens (69). 3. Occurrence

Histrionicotoxins represent a unique structural class of alkaloids found only in dendrobatid frogs (see below). Somewhat similar hydroxyazaspiro-undecanes, namely, sibirine, nitramine, and isonitramine, occur in certain plants of the genus Nitraria (cf. Ref. 70). Histrionicotoxins were first discovered in the extremely variable dendrobatid species Dendrobates histrionicus of western Colombia and northwestern Ecuador (54). Histrionicotoxin (238A),allodihydrohistrionicotoxin (285C), and isodihydrohistrionicotoxin (285A) represent major alkaloids in nearly all populations of this species (1,71). In one population from northwestern Ecuador, octahydrohistrionicotoxin (291A)is a major alkaloid (71), whereas in one population from the lower Rio San Juan

206

JOHN W . D A L Y E T A L .

drainage of western Colombia the lower molecular weight histrionicotoxins 235A and 259A have replaced the 19-carbon 283A, 285C, and 285A as major alkaloids ( J. W. Daly, unpublished). No histrionicotoxins are present in the “sibling” Colombian species Dendrobutes lehmanni. Histrionicotoxins are present in skin extracts from many dendrobatid frogs, representing major alkaloids in Dendrobates auratus, D . azureus, D . granuliferus, D . histrionicus, D . leucomelus, D . occultator, D . pumilio (some, but not all populations), D . quinqueuittatus, D . reticulatus, D . tinctorius, D . truncatus, and D . uentrimaculatirs (I). Histrionicotoxins are absent or trace alkaloids in D . arboreus, D . new species (Panama), D. lehmanni, D . pumilio (some populations), and D . speciosirs. In the dendrobatid genus Epipedobates, histrionicotoxins are major alkaloids in E. espinosai, E . paruulus, E . petersi, E . pictus, and E . triuittatirs, but are absent or trace alkaloids in the six other species examined (I). In the dendrobatid genus Minyobates, histrionicotoxins are absent in the nine species that have been examined (I). In the dendrobatid genus Phyllobates, a genus that is typified by the presence of batrachotoxins, histrionicotoxins occur as major alkaloids in P . bicolor, but are trace alkaloids or absent in the remaining four species (I). The distribution of histrionicotoxins within species and genera of dendrobatid frogs argues strongly for genetic determinants for their occurrence. Histrionicotoxins are often accompanied by highly unsaturated decahydroquinolines in dendrobatid frogs. N o deoxyhistrionicotoxins have yet been detected. In 1984, dendrobatid alkaloids were reported from one genus of each of three other amphibian families, namely, Bufonidae, Myobatrachidae, and Ranidae (72). Histrionicotoxin (283A), allodihydrohistrionicotoxin (285C),and isodihydrohistrionicotoxin (285A) were present as major alkaloids in a single skin of the Madagascan ranid frog Mantella madagascariensis obtained from a commercial dealer (72). Subsequent fieldcollected specimens of Mantella mudagascariensis have contained a variety of alkaloids, but no histrionicotoxins (73). Thus, histrionicotoxins may prove to be unique to certain dendrobatid species of the genera Dendrobates, Epipedobates, and Phyllobates.

B. DECAHYDROQUINOLINES I . Structures The first decahydroquinoline found in amphibians was isolated, along with two other alkaloids, from skin extracts of a Panamanian dendrobatid frog, Dendrobates pumilio. The three alkaloids were designated pumiliotoxins A, B, and C (74,75). Pumiliotoxins A and B were quite toxic, and

3. AMPHIBIAN

ALKALOIDS

207

are the parent members of the pumiliotoxin-A class of alkaloids (Section 111,C). Pumiliotoxin C proved to be a decahydroquinoline, namely, (2S,4aS,5R,8aR)-5-methyl-2-n-propyl-cis-decahydroquinoline, as shown in V. It was relatively nontoxic, and hence the toxin designation is inappropriate; the nomenclature decahydroquinoline cis-l95A, used in recent publications and in this chapter, is much preferable, particularly to avoid confusion with the structurally dissimilar pumiliotoxin-A class. A range of decahydroquinolines have been isolated from skin extracts of dendrobatid frogs. Structures of several have been determined (76-78) through the analysis of mass spectra, nuclear magnetic resonance spectra, and FTIR spectra. Both cis- and trans-fused decahydroquinolines have been isolated. The decahydroquinolines show a major mass spectral fragment ion corresponding to loss of the 2-substituent and a minor fragment ion from loss of the 5-substituent. Although the mass spectra of cis- and trans-isomers are virtually identical, the FTIR spectra are not and appear to be diagnostic for the ring-fusion stereochemistry (78). Thus, transdecahydroquinolines, such as trans-219A, have sharp, single peaks at approximately 1 100 and 1300 cm-', whereas cis-fused decahydroquinolines, such as cis-195A and cis-219A, show doublets at around 1120 and 1340 cm-' (78). The doublets were proposed to arise from contributions from two cis-fused conformations (78). Several diastereomers of cis-195A have been synthesized, namely, the 2-epi (78a),the 5-epi and 8a-epi (79), and the 2-epi-8a-epi analogs (80). Structures of 15 of the relatively wellcharacterized decahydroquinolines are shown in Fig. 5 . A number of other dendrobatid alkaloids appear to be decahydroquinolines based on spectral and chemical properties. All of these alkaloids are secondary amines; no simple N-alkyl derivatives have been found. FTIR spectra are not yet available for many of these alkaloids. Many decahydro-

Decahydroquinoline cis-195A Purniliotoxin C

(V)

208

JOHN W . DALY E T A L .

br, /J 8a N

.b 2

H o ~ . . \ " ,

H

H

I H

H

cis-195A

cis-21 1 A

cis-219A

cis-223F

I

H

H

H

cis-249D

cis-243A

cis-275B

A-

r

&l OJ J0 J T C ) J l- i N l

H I

H

trans-1 95A

H I

H

trans-219A

trans-223F

H I

H

trans-243A

lh/b&elH0& H I NI

5-epi-trans-243A

H HI

trans-249D

l iH l

trans-249E

l iH l

trans-253D

FIG.5 . Structures of decahydroquinolines from amphibians. Absolute configurations of natural cis-195A and rrans-219A are known. Only the relative configurations are known for the others.

quinolines show a major fragment ion corresponding to Cl,H18N+ (mlz 152), as in the case of cis-195A. These would have a methyl group as a 5-substituent. One alkaloid (189)that appears to be a tetrahydroquinoline and one (193D) that appears to be an octahydroquinoline have been reported from ranid frogs of the genus Manteffa(73). The decahydroquinoline alkaloids are tabulated below, along with the tetrahydroquinoline and the octahydroquinoline. Tentative structures are suggested for several alkaloids that are not shown in Fig. 5. Decahydroquinolines 153A. 'C,oH,9N,'--,1540C, mlz 153( loo), 152(60). ID. H, derivative. Tentative structure: a 5-methyldecahydroquinoline.

3. AMPHIBIAN ALKALOIDS

209

167D. ‘Cl,H2,N,’-, 154”C, mlz 167(100), 166(53). ID. H, derivative. Tentative structure: a 5-ethyldecahydroquinoline. 181D. ‘C,2H23N,’--, 156”C, mlz 181(3), 152(100). ID. Tentative structure: a 2-ethyl-5-methyldecahydroquinoline. 181E. ‘CI2H,,N,’--, 156”C, mlz l8l( loo), 180(46). ID. H, derivative. Tentative structure: a 5-propyldecahydroquinoline. 189. ‘C13H19N,’-,-, ion trap, mlz 190(10), 174(26), l6l( IOO), 146(94), 91( 10). OD. Infrared data (73). Tentative structure: a 2-propyl-5-methyl-

5,6,7,8-tetrahydroquinoline. 193D. ‘C13H23N,’-,-, ion trap, mlz 193(5), l50( IOO), 122(12), 96( 12). ID. Infrared data (73).Tentative structure: a 2-propyl-5-methyl octahydroquinoline. 195A. Pumiliotoxin C, C,,H,,N, 0.20, 157”C,mlz 195(3), 194(5), 180(1), 152(100), 109(8). ID. Infrared spectrum (78), infrared data (73). H, derivative. N-Acetyl derivative. Both cis and trans isomers occur, with pumiliotoxin C being the cis isomer. 211A. C,,H,,NO,--, 166”C, mlz 21 l(3), 210(2), 168(100), 152(32), 150(13). 2D. H, derivative. Only a cis isomer has been detected (77). 219A. C,,H,,N, 0.32, 165°C mlz 219( I), 218(2), 178(100). ID. H,derivative, mlz 223, 180. Infrared spectra (78). N-Acetyl derivative. Two naturally occurring isomers, cis-219A and truns-219A, do not separate on packed columns. Another isomer 219A‘ emerging at 164°C may represent a 2-epi-cis-219A. 219C. C,,H,,N,-, 170”C, mlz 219(<1), 152(100). ID. H4 derivative, mlz 223, 152. Tentative structure: a 2-pentadienyl-S-methyldecahydroquinoline. 219D. CI5Hz5N,-, 175”C, mlz 219(3), 180(100). 1D H, derivative, mlz 223, 180. Tentative structure: a 2-propargyl-5-propyldecahydroquinoline. 221C. C,,H,,N,-, 166”C,rnlz 221(2), 152(100). ID. H2 derivative mlz 223, 152. Tentative structure: a 2-pentenyl-5-methyldecahydroquinoline. 221D. C,,H,,N,-, 168”C,rnlz 221(3), 180(100). ID. H2derivative. Tentative structure: a 2-allyl-5-propyldecahydroquinoline. 223F. T15H29N,’-, 163”C, mlz 223(3), 222(3), 180( 100). 1D. Infrared spectra (81). H, derivative. Both cis and trans isomers occur. 231E. ‘CI6H2,N,’-, 160”C,mlz231(<1), 230(3), 152(100). ID. H,derivative, mlz 237, 152. Tentative structure: a 2-hexenynyl-5-methyldecahydroquinoline. 243A. C,,H2,N, 0.36, 182”C,mlz 243(2), 242(1), 202(CI4H2,N,100). ID. Infrared spectra (78). H, derivative, mlz 251, 208. N-Acetyl derivative. Two naturally occurring isomers, cis-243A and truns-243A, do not separate on packed columns. A 5-epi-truns-243A also occurs (78). Another isomer formerly designated as 243A‘ emerges at 179°C and appears to correspond to a 2-epi-cis-243A.

210

JOHN W . D A L Y E T A L .

2498. 'CI7H3,N,'-,-, mlz 249(2), 248(3), 206(100), 180(15). ID. Infrared data (81). H, derivative. Both cis and trans isomers occur. 2493. 'C17H31N,'-,-, ion trap mlz 250(15), 206(29), 180(100). ID. Infrared data (81). H, derivative. Only a trans isomer has been detected. 251A. 'C,7H33N,'-, 170"C, mlz 251(2), 208(6), 152(100). ID. H, derivative. Tentative structure: a 2-heptyl-5-methyldecahydroquinoline. 253D. C15H29N02,-,-, rnlz 253(<1), 222(6), 213(16), 212(100). 3D. Only a trans isomer has been detected. 269AB. C,,H,,N, 0.35, 207"C, mlz 269(4), 268(12), 204( IOO), 202(50). ID. HI, derivative, mlz 279,208. N-Acetyl derivative. Tentative structure: a 2-(3,4-pentadienyl)-5-(2-penten-4-ynyl)decahydroquinoline(see Ref. 5 ) . However, the occurrence of two major fragment ions appears remarkable for such a structure. A vapor-phase FTIR spectrum indicates a trans ring fusion for 269AB. Although 269AB appears to be a single compound, there also is evidence for a 269A and a 269B. 269A. 'C19Hz7N,'-, 207"C, mlz 269(4), 204( 100). ID. HI, derivative, mlz 279, 208. Related in structure to 269AB. 269B. 'C19Hz7N,'-, 202"C, mlz 269(4), 202( 100). ID. HI, derivative, mlz 279, 208. Related in structure to 269AB. 275B. 'C19H33N,'-r195"C, ion trap, mlz 276(1 I ) , 232(12), 206(100). ID. Infrared data (81).H, derivative, mlz 279,206. Only a cis isomer has been detected. A significant loss of propyl to yield mlz 232 is unexpected for the proposed structure (81). 293A. 'CZ0H3,N,'-, 192"C, mlz 293(2), l52( 100). ID. H, derivative. Tentative structure: a 2-decyl-5-methyldecahydroquinoline. Optical rotations of decahydroquinolines are presented in Table IV. Proton magnetic resonance spectra have been presented (75-78),as have TABLE IV OPTICALROTATIONS OF DECAHYDROQUINOLINES (76-78) Decah ydroquinoline cis-19SA.HCI cis-211A cis-219A trans-219A cis-243A.HCI trans-243A 5-epi-trans-243A

- 13.1" (0.3, CHIOH, 20°C)" - I 1.7" ( I .O, CHCIJ +5.8" (0.3, CHCII) + 9.7" (2.0, CH,OH, 24°C) + 10.1"(2.4, CHCI,) - 18.6", -0.96" (0.73, CHCI3, CHIOH, 16°C) -30.7". - 15.2" (1.57, CHCI,, CHJOH. 16°C)

'Synthetic (2s.4aS, 5R. 8aR)-cis-195~has an identical rotation (80;see also Ref. 5 and references therein).

3.

AMPHIBIAN ALKALOIDS

21 1

carbon-13 assignments (76-78)(see Ref. 78 for recent assignments and for clarification of a transposition of data for two isomers of trans-243A that occurred in Ref. 76).FTIR spectra for several decahydroquinolines have been presented and discussed (78). The Bohlmann band (v,,for N-CH, 2850-2600 cm-I) patterns in the infrared spectra of cis- and trans-2,6dialkyl piperidines permit the facile differentiation of isomers: the cis isomer shows clearly a more pronounced pattern (H. M. Garraffo, T. H. Jones, T. F. Spande, and J. W. Daly, unpublished). In the same way FTIR spectra permit assignment of the relative configuration of the 2-alkyl substituent in the cis- and transfused decahydroquinolines. Thus, both cis-195A from Dendrobates pumilio (78)and a trans-195A isomer from Epipedobates bassleri have a Bohlmann band pattern typical of a cis-2,6disubstituted piperidine. In this context, it should be noted that the absolute configurations of C-2 and C-8a as determined by X-ray diffraction in cis-195A and trans-219A are opposite to one another (75,76), even though both could be considered to be derived from cis-2,6-disubstituted piperidines.

2. Biological Activity Decahydroquinolines have relatively low toxicities, and for this reason and, more importantly, to avoid confusion with the pumiliotoxin-A class of alkaloids, the name pumiliotoxin C has been replaced with the name decahydroquinoline cis-195A. Decahydroquinoline cis-195A at a subcutaneous dose of 100 pg in a mouse causes difficulties in locomotor activity (60,71). The minimum lethal dose was estimated to be 400 pg. Decahydroquinoline 219A (presumably trans) at 80 pg caused paralysis of hind limbs, salivation, and piloerection in a mouse (71). The pharmacological activity of decahydroquinoline cis-195A and analogs in neuromuscular preparations appears to involve noncompetitive blockade both of nicotinic receptor-channels and of voltage-dependent sodium and potassium channels (see reviews in Refs. 3 and 5 ) . Recently, both cis- and trans-decahydroquinolines were shown to block ion flux through nicotinic receptor channels in pheochromocytoma cells and to enhance the rate of desensitization of such nicotinic channels (64,82).

3. Occurrence Simple 2,S-disubstituted decahydroquinolines do not appear to have been reported to occur in Nature except in certain amphibians. Decahydroquinolines, like histrionicotoxins, occur in a range of dendrobatid frogs. These two classes of dendrobatid alkaloids often occur together in the same species or population. Indeed, the 19-carbon decahydroquinoline 269AB (or 269A and 269B) always appears to accompany the 19-carbon

212

J O H N W . DALY E T A L .

histrionicotoxins. First discovered as a major alkaloid in a population of the Panamanian poison frog Dendrobates pumilio ( 7 3 , decahydroquinoline cis-195A (pumiliotoxin C) or other decahydroquinolines are major alkaloids in many populations of this extremely variable frog but are completely lacking in some populations ( I ). Decahydroquinolines also occur as major alkaloids in Dendrobates auratus, D . azureus, D . granuliferus, D . histrionicus, D . quinquevittatus, D . speciosus, D . tinctorius, D . trivittatus, D . truncatus, and D . ventrimaculatus, while being absent or trace alkaloids in D . arboreus, D . new species (Panama), D . lehmanni, D. leucomelas, D . occultator, and D . reticulatus. In the dendrobatid genus Epipedobates, decahydroquinolines are major alkaloids in only four species, E. parvulus, E. petersi, E. pictus, and E. triuittatus, while being trace alkaloids or absent in the seven other species examined ( I ) . In the dendrobatid genus Minyobates, decahydroquinolines, like histrionicotoxins, appear to be absent, although one presumed decahydroquinoline, 219D, was reported in M . new species (Panama) ( I ) . In the dendrobatid genus Phyllobates, decahydroquinolines, like histrionicotoxins, are major alkaloids only in the Colombian montane species, P . bicolor, and have been detected in only two ( P . aurotaenia and P . lugubris) of the remaining four species. In 1984, the decahydroquinoline 195A was reported as a trace constituent in extracts of a single skin of the Madagascan ranid frog Mantella madagascariensis (72). This frog, obtained from a commercial dealer, also contained histrionicotoxins and pumiliotoxin A class alkaloids. The occurrence of cis-195A as a trace constituent in Mantella has been confirmed with field-collected specimens (73). A variety of cis- and transdecahydroquinolines have been detected in skin extracts of bufonid toads of the genus Melanophryniscus (81). The apparent occurrence of decahydroquinolines as major alkaloids in dendrobatid frogs of the genera Dendrobates, Epipedobates, and Phyllobates, and in bufonid toads of the genus Melanophtyniscus, strongly suggests the independent evolutionary development of biosynthetic pathways to decahydroquinolines in two separate amphibian lineages. C. PUMILIOTOXIN-A CLASS 1 . Structures The structures of pumiliotoxin A (307A) and pumiliotoxin B (323A), first isolated in the late 1960s ( 7 4 , proved elusive until the structure of a simpler congener, pumiliotoxin 251D, was revealed by X-ray analysis a full decade later (83).The structure of pumiliotoxin 251D is as shown in VI. The structures of the more complex pumiliotoxins A and B then

3. AMPHIBIAN

ALKALOIDS

213

became evident on analysis of their nuclear magnetic resonance spectra in comparison with the spectra of pumiliotoxin 251D (83). During the decade between the isolation of pumiliotoxin A and B and their structural elucidation, more than 20 dendrobatid alkaloids were determined to be closely related in structure to pumiliotoxins A and B. This conclusion was based primarily on mass spectral similarities, namely, a prominent ion of C,H,N+ (mlz 70) and another ion of either C,,H,,NO+ (mlz 166), as in (mlz 182), as in the pumiliotoxins A and B and 251D, o r CIoHl6NO2+ allopumiliotoxin subclass. The allopumiliotoxins proved to be pumiliotoxins with a hydroxyl group in the 7 position of the indolizidine ring (83,84). At present 23 pumiliotoxins and 15 allopumiliotoxins can be included in the pumiliotoxin-A class of dendrobatid alkaloids (I ,77,95; see below). The structures of 26 relatively well-characterized pumiliotoxins and allopumiliotoxins are shown in Figs. 6 and 7. Certain alkaloids of the pumiliotoxin A class isolated from dendrobatid skin extracts appear to be artifacts formed by rearrangement of the side chain allylic C-15 alcohol, or by reaction with methanol at the same allylic center (95). These are shown in Fig. 8. A dendrobatid alkaloid exhibiting prominent mass spectral fragment ions of C5HloN+(mlz 841, instead of C,H,N+ (mlz 701, and C,,H,,NO+ (mlz 180), instead of C,,H,,NO+ (mlz 1661, was included in the pumiliotoxin A class, even though it has a quinolizidine ring instead of the indolizidine ring of the pumiliotoxins and allopumiliotoxins. This is homopumiliotoxin 2236 (77). Several other homopumiliotoxins have now been characterized in skin extracts of bufonid toads of the genus Melanophryniscus (81). Structures of four homopumiliotoxins are shown in Fig. 9. The alkaloids that appear to belong to the pumiliotoxin-A class are listed below as pumiliotoxins, allopumiliotoxins, or homopumiliotoxins. For many, the data do not yet allow formulation of a tentative structure. Also, it should be noted that some of the structures in Figs. 6, 7, and 9 deserve further study, particularly with regard to substituent configurations in side

214

209F

JOHN W . DALY ET A L .

R=

H&.

307A'

R=

(PTX-A)

225F

HO-

R=

3078

& 7 1

OH

R= OH

237A

v

R=

307F'

R=

0

251 D

R=

267C

R=

2778

R=

281A

R=

w

307F" and 307F"'

R=

307G' and 307G"

R=

4 0

YOH

3058

R=

3258

R=

I

OH

FIG.6. Structures of pumiliotoxins. Absolute configurations are known for Z l D , WA', and 323A and are assumed to be the same for the others. All occur in dendrobatid frogs except for 305B, which has been detected only in ranid frogs (Manrella)(73). and eryfhro323A, which has been detected only in ranid (Manrella) and myobatrachid frogs (Pseudophryne) (73,85,86).

3.

225E

R=

215

AMPHIBIAN ALKALOIDS

H$.

309D

R= 13

253A

R=

w

267A

R=

w

3238'

325A' and 325A"

R= OH

297A

3396 epimer of 339A at C-7

FIG. 7. Structures of allopumiliotoxins. Absolute configurations are known for 267A, 339A, and 339B and are assumed to be the same for the others. All occur in dendrobatid frogs. Some also occur in ranid (Manrella), bufonid (Melanophryniscus), and myobatrachid (Pseudophryne) amphibians (73,73,8/).

chains, when sufficient material becomes available for nuclear magnetic resonance spectral analysis. Pumiliotoxins

207B. CI3H2,NO,0.47, 161"C, mlz 207( lo), 190( 1 3 , 166( loo), 70(80). ID. H, derivative, m/z 209, 70. A reasonable structure fitting these data is not obvious. 2WF. C13H23N0,--,-, mlz 209(22), 166(70),70(100). Infrared spectrum (95). 1D.

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JOHN W . D A L Y ET A L .

“-aCH3 “‘OH

5H3

Lf

307A

3238“

R=

R=

OH 0-methyl 307A (3211

OH

0-methyl 3238

R=

OCH,

R=

‘yl, OCH,

FIG.8. Pumiliotoxin-A class alkaloids isolated as apparent artifacts from dendrobatid frogs (78). Epimerization or reaction with methanol does not occur at the allylic hydroxyl of 323A, apparently because of the additional adjacent hydroxyl group. 0-Methyl-M7A formerly was designated 321 ( I ,79). N-Oxides of pumiliotoxin 323A and allopumiliotoxin 267A have been isolated and may be artifactual (87).

223G

R=

321B

R=

H3C.

FIG.9. Structures of homopumiliotoxins from dendrobatid frogs and bufonid toads. The structure of homopumiliotoxin 2236 is based on nuclear magnetic resonance spectral analysis (77). The absolute configuration is unknown. Homopumiliotoxin 2236 occurs in dendrobatids (77), ranids (73,and bufonids (a]), while the other alkaloids have been detected only in the bufonid toads (Melanophryniscus) (81).

3. AMPHIBIAN

ALKALOIDS

217

225F. C,3H23N0,,--,--, rnlz 225 (21), 194(28), 166(79), 112(19),84(26), 70(100). Infrared spectrum (95). 2D. 237A. CI5Hz7NO,0.58, 167"C, rnlz 237(4), 236(3), 220(3), 194(12), 166(40), 70( 100). ID. H, derivative, mlz 239(5), 196(5), 168(lo), 110(30), 84(100), 70(45). 251D. C,,H,,NO, 0.52, 172"C, m k 251(5), 250(3), 236(2), 234(1), 194(5), 166(78),70(100). 1D. Infrared data(81). H,derivative, rnlz 253( l8), 110(70), 84(100), 70(55). 265D. 'c16H27Noz,'-, 191"C, m / z 265(5), 222( 14), l66( loo), 70(35). 2D. H, derivative. A structure is not proposed. 2656. 'C,7H31N0,'-,-, ion trap, mlz 265(2), 166(loo), 70(80). A keto analog of 267C (73). 267C. CI6H2,NO2,0.28, 190"C, rnlz 267(16), 194(12), 166(100), 84(18), 70(75). 2D. Infrared spectrum (86). H, derivative. 267D. Reported from nondendrobatid frogs (72), but now appears to have been identical to 267C. 277B. 'C,,H,,NO,,' 0.61, 206"C, rnlz 277(2), 206( 12), 194(48), 193(58), 17 3IS), 166(20), 163(65),84(24), 70( 100). ID. Infrared data (86). H, derivative, rnlz 283( I ) , 110(35),84(100),70(30), and, unexpectedly, a hydrogenolysis product identical to dihydro-251D (86). 281A. CI7H3,NO,,0.28, 205"C, rnlz 281(4), 280(2), 264(2), l94( 12), 166(72), 70(100). 2D. H, derivative, mlz 283( I ) , 282(2), 266(4), 208(40), 138(10), 110(lo), 84( loo), 70(85). 297B. C,,H3,NO2,0.35,222"C, mlz 297(10), 166(92),70( 100). H4derivative. A structure is not proposed. 305B. Cl9H3,NO,,-,-, ion trap, mlz 306(< l ) , 206( l6), 193(47), 166(50), 150(17),70( 100). Infrared spectrum (73). 307A. Pumiliotoxin A, CI9H3,NO,, 0.36, 216°C r n l z 307(5), 290(3), 278(4), 206(10), 194(16), 176(10), 166(85), 70(100). 2D. Infrared spectrum (73,95).H, derivative, mlz 309(3), 308(2), 210(8), 166(30), 110(45),84(loo), 70(40). H,derivative, mlz 31 1(3), I10(32), 84(IOO), 70(55). 0-Acetyl derivative. The 15-epimer 307A' corresponding to the configuration in 323A is assumed to be the natural alkaloid. Both 307A' and 307A have been isolated, presumably due to facile epimerization of 307A' (see Ref. 85). 307B. C,,H,,NO2,--, 21 I"C, rnlz 307(12), 306(4), 290(2), 194(24), 193(45), l66( IOO), 70(56). 2D. Infrared spectrum (95). H, derivative. Two diastereomers have been detected (73). Alkaloids 307B, 307F, and 3076 co-chromatograph on packed columns, but they separate on capillary columns and can be distinguished by exchangeable hydrogens and FTIR spectra. Prior studies (I) may not have correctly identified 307B, 307F, and 3076 in all extracts (see Ref. 95). In many cases both 307B and 307F alkaloids were probably present together.

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307D. ‘C18H29N03,’-, 234”C, rnlz 307(8),306(5), 292(3),290(5), 262(11), 206(1I), 194(18), 166(100), 70(85). H4 derivative, rnlz 31 1, 110, 84, 70. A structure is not proposed. 307F. C19H33N0,,’--, 21 1 ° C m/z 307(12), 194(24), 193(46), 166(100), 70(68). 1D. Infrared spectra (95). Three isomeric 307F alkaloids have been described (95). These will be termed 307F’, 307F’, and 307F”’ (see Fig. 6). The 307F alkaloids may have been incorrectly identified as 307B in some dendrobatid extracts (1). In many cases both 307B and 307F alkaloids were probably present together. 3076. ‘C19H33N02,’-,-, ion trap, mlz 307(8),262( 15),206(22),194(lo), 176(lo), 166(loo), 70(65). Infrared spectrum (73). Two diastereomers 3076’ and 3076” have been detected in a ranid frog (73). 307H. ‘C19H33N02,’-,- ion trap m/z 306(13), 206(22), 193(100), 166(45), 150(25), 84(20), 70(78). Infrared spectrum (73). Proposed to be an isomer of 307A with a 5,6 double bond (73). 309A. ‘CI9H3,NO2,’0.38, 218”C, rnlz 309(9), 308(3), 292(2), 194(1 3 , 166(100), 110(10), 84(20), 70(51). Infrared spectrum (73). H, derivative. 3WC. ‘C19H35N0,,’--, 210°C rnlz 309(3), 308(2), 292(1), 194(1 3 , 166(100), 70(90). H, derivative, rnlz 31 1(3), 110(25), 84(100), 70(30). A structure is not proposed. 321A. CzoH3,N02,--, 223”C, rnlz 321(3), 304(8), 166(65), 70(100). 1D. Now considered to be an artifact. Structure (O-methyl 307A) in Fig. 8. 323A. Pumiliotoxin B, CI9H3,NO3,0.17, 230°C mlz 323(lo), 306(5), 290(2), 278(12), 206(15), 194(26), 193(22), 176(15), 166(75), 70(100). 3D. Infrared spectrum (86,95). H, derivative, mlz 325(5), 166(25), 110(24), 84(100), 70(38). H, derivative, rnlz 327(3), 326(20), 312(I), 310(2), 282( 141, 264(12), 110(30),84( loo), 70(43). Di-O-acetyl derivatives. Threo-(15R,16R) configuration for the 15, 16-hydroxyls. The following trace diastereomer occurs in some myobatrachid and ranid frogs (73,85,86): erythro-323A. erythro-(15R,16S)-Pumiliotoxin B, CI9H3,NO3,0.17, 230°C. Mass spectra and properties nearly identical with 323A, but FTIR spectra differ (95). The two alkaloids can be separated on capillary columns as di-O-acetates and other derivatives (85). 325B. Cl,H3,N03,-, 228”C, rnlz 325(6), 309(8), 166(85), 70( 100). H2 derivative. 3D. 353. ‘C19H31N05,’- 240°C rnlz 353(4), 338( lo), 336(5), 194(20), 166(80), 70(100). 3D. A structure is not proposed. Allopumiliotoxins

2253. C,3H23N02,-,-, m/z225(40), 208(86), 182(41), 138(41), 114(34), 112(60),70( 100). 2D. Structure based on mass and proton magnetic resonance spectra (95).

3. AMPHIBIAN

ALKALOIDS

219

237B. C,,H,,NO,, 0.58, 168"C, mlz 237( 1 I), 236(2), 182(60), 114(30), 112(25), 70(100). 2D. A structure is not proposed. 2511. 'ClSH2sN02,'-, 180"C, mlz 251(7), 236(4), 210( 1 3 , 209(20), 182(1 l), 70( 100). 2D. H, derivative. A structure is not proposed. 25344. ClsH2,NO,, 0.30, 179"C, mlz 253(4), 252(1), 236(22), 182(16), 114(27), 112(26), 70(100). 2D. H, derivative, mlz 255(3), 238(8), 110(50), 84(100), 70(65). 267A. C16H,,N0,, 0.31, 186"C, mlz 267(15), 250(33), 182(18), 114(52), 112(44), 70(100). 2D. H, derivative, mlz 269(6), 252( 18), 110(50),84(loo), 70(60). 0-Acetyl derivative. 297A. CI7H3,NO3,0.13, 225"C, mlz 297(3), 296(4), 280(9), 182(21), 114(27), 112(16), 70( 100). 3D. H, derivative, mlz 299(4), 282( 12), 110(50), 84(100), 70(75). 305A. 'CI9H3,NO2,'-, 214"C, rnlz 305(23), 288( lo), 182(43), 70( 100). 2D. H6 derivative. A structure is not proposed. 307C.CI9H3,NO2,0.39,214"C, mlz 307(9), 290( 1 l), 182(62),70(100).2D. H, derivative. rnlz 31 1(3), 294(3), 110(30),84( loo), 70(35). A structure is not proposed. 309D. C,,H35N02,--, 208"C, mlz 309(13), 292(34), 182(37), 123(22), 114(26), 112(35), 70(100). 2D. H, derivative. 321C. 'C19H31N03,-,-r ion trap, mlz 322(3), 304(10), 209(55), 192(12), 182(32), 114(32), 70( 100). A structure is not proposed (7.3). 323B. Cl9H3,NO3,0.20, 228"C, mlz 323(5), 306(lo), 210(4), 209(3), 182(50), 114(20), 70( 100). 3D. Infrared spectrum (95). H, derivative. The 15-epimer 323B' corresponding to the C-15 configuration in 323A is assumed to be the natural alkaloid. Both 323B'and 323B"have been isolated, presumably due to facile epimerization of 323B'(see Ref. 85).Only 323B' was isolated in one case. 32544. CI,H3,NO3, 0.20, 232"C, mlz 325(12), 308(22), 182(100), 114(25), 112(21),70(73). 3D. Infrared spectrum (95). H, derivative. Two diastereomers 325A' and 325A" were isolated and had nearly identical mass spectra (95). 339A. CI9H3,NO4,0.07, 243"C, mlz 339(3), 322(3), 192(14), 182(75), 114(25), 70(100). 4D. H, derivative, mlz 343(3), 342(2), 324(3), 138(10), 110(25), 84( loo), 70(65). 339B. CI9H3,NO4,0.12, 243"C, mlz 339(3), 322(3), 192(lo), 182(70), 114(25), 70(100). 4D. H, derivative. The only allopumiliotoxin with cishydroxyl groups in the indolizidine ring (84). 341A. C19H3sN04,0.48, 222"C, mlz 341(4), 324(3), 182(lo), 114(lo), 112(40), 84(22), 70(100). 3D. H, derivative, rnlz 343(l ) , 342(2), 328(2), 266(10), 138(100), 84(15), 70(15). A structure is not proposed. 341B. 'CI9H3,NO4,'--, 223"C, mlz 341( l), 324(4), 182(60), 114(20), 112(20), 70(100). A structure is not proposed.

220

JOHN W. DALY E T A L .

357. C,,H3,NOS, 0.30, 240°C, mlz 357(3), 340(8), 182(20), 128(10), 110(20), 84(20), 70( 100). 4D. A structure is not proposed. Homopumiliotoxins

2076. ‘C13H2,N0,’-,-, ion trap, mlz 207(3), 180(35), 84(100). Detected in both dendrobatid and ranid frogs (31,73). A structure is not proposed. 207H. ‘C13H2,N0,’-,-, ion trap, mlz 207(3), 178(28), 84( 100). The characteristic mlz 180 fragment is not present, and the assignment to the homopumiliotoxin class is tentative. Detected with 2076 in a dendrobatid frog (31). 2236. C14H2sN0,--, 163”C, rnlz 223( 18), 190(22), 180(39), 98(27), 84(100). 1D. Infrared spectrum (73). H, derivative. 235J. ‘ClsHzsNO,’-,-, ion trap, mlz 235( 12), 138(8), 109(18), 84(100). Infrared data (73).The infrared spectrum is typical for homopumiliotoxins, as is the mlz 84 fragment, but does not have the characteristic rnlz 180 fragment. 249F. ‘c,6Hz7No,’-,-, ion trap m / z 249( 12), 220(22), 123(16),84(100). Infrared data (73). The infrared spectrum is typical for homopumiliotoxins, as is the mlz 84 fragment, but does not have the characteristic rnlz 180 fragment. 251L. ‘C16Hz,N0.’Cochromatographs with another alkaloid, and because of this a definitive mass spectrum could not be obtained. Occurs mainly as the 0-acetate (73).251L 0-acetate. ‘C18H31N02,’-r-r ion trap, mlz 293(<1), 250( 18), 222(32), 176(77), 148(40), 134(22), 84(80). Infrared data (73). The infrared spectrum is typical for homopumiliotoxins, as is the rnlz 84 fragment. 317. CzoH31N02,-,-, ion trap, mlz 318(5), 220( lo), 208( 1 3 , 207(20), 190(12), 180(100), 164(12), 148(10), 98(18), 84(72). Infrared data (73). A structure is not proposed. 319A. C20H33N02,--,--, ion trap, mlz 320(15),276(35),261(37),220(34), 208(30), 190(27), 180(190), 98(20), 84(100). ID. Infrared spectrum (81). An unconjugated ketone in the side chain. 319B. C20H33N02,--,--, ion trap, mlz 320(7),276( 12),261( 12), N O ( loo), 84033). 1D. Infrared spectrum (81).A conjugated ketone in the side chain. 321B. ‘C20H3,N02,’-r-, ion trap, mlz 322( 13), 220(22), 208(16), 190(18), 180(100), 98(12), 84(52). 2D. Infrared spectrum (81). 0-Acetyl derivative. Optical rotations of the pumiliotoxin-A class alkaloids are reported in Table V. The mass spectra have been reported in detail and summarized

3.

AMPHIBIAN ALKALOIDS

22 1

TABLE V OPTICAL ROTATIONS OF PUMILIOTOXIN-A CLASS ALKALOIDS (72,77,78)" Compound

Pumiliotoxins 209F 2251

251D 267c 307A 307A' 307A" 307F 323A

[ff1d6 - 1 I .6" (0.10, CHCII) -87.4" (0.23, CHCI3) + 17" (0.15, CHIOH) +7.2" (0.8, CHIOH) + 22.7" ( 1 .O, CH3OH) + 14.3" (0.74, CHCI,) +0.52" (0.52, CHCI,) -8.5" ( I .O, CHIOH) + 20.5" ( I .O, CHIOH) + I So ( I .O, CHCI,)

Allopumiliotoxins

267A 323B' 323B" 339A 339B

+ 24.7" (0.17, CHXOH)

+ 22.3" ( I .O, CH,OH) + 55.0" (0.I , CHIOH) + 29.4" ( I .O, CH,OH) +4.5' (0.5, CHIOH)

"Similar or identical optical rotations have been reported for synthetic 251D,W A , 3UA, 267A, 339A, and 339B (88-94).

(3,5,72-77,81,83,84,87,95,96). The proton and carbon-I3 magnetic resonance spectra have been presented and/or interpreted for pumiliotoxins and for a homopumilioand allopumiliotoxins (3,5,72,77,81,83,84,87,95), toxin (77).Vapor-phase FTIR spectra have been presented for pumiliotoxin-A class alkaloids (73,8f,86,95).In general, all pumiliotoxin A class alkaloids show a sharp hydroxyl absorption at 3544 cm-I, resulting from the 8-hydroxyl group hydrogen-bonded to the indolizidine nitrogen, a 6,lO double bond a=,- at 965 cm-I, and a characteristic Bohlmann band pattern (95). In homopumiliotoxins, the 9-hydroxyl absorption appears at 3555 cm-', and a significantly different Bohlmann band pattern is seen (73). 2. Biological Activity

Pumiliotoxin A (307A)and pumiliotoxin B (323A)are relatively toxic compounds causing death in mice at subcutaneous doses as low as 20 pg for pumiliotoxin B and 50 p g for pumiliotoxin A (see review in Ref. 5). Allopumiliotoxin 267A is less toxic, causing only locomotor difficulties at 40 p g per mouse (60).

222

JOHN W . DALY E T A L .

Pumiliotoxin B has both cardiotonic and myotonic activity in isolated atrial or rat phrenic nerve diaphragm preparations (97). The cardiotonic activity is markedly dependent on the structure of the pumiliotoxin (98). Subsequent studies on the activity of pumiliotoxin B in neuromuscular preparations were interpreted as due to an apparent facilitation of calcium translocation from internal storage sites (99; see review in Ref. 5 ) . Inhibitory effects on the calcium-dependent ATPase of sarcoplasmic reticulum were shown to be due not to pumiliotoxin B, but to phenolic impurities, namely, bis(2-hydroxy-3-tert-butyl-S-methylphenyl)methane,3,5-di-tertbutyl-4-hydroxytoluene (BHT), and nonylphenols (100). Further studies have revealed that pumiliotoxin B interacts with voltagedependent sodium channels to elicit an increased influx of sodium ions (101,102)and, in brain and heart preparations, a stimulation of phosphoinositide breakdown (101,103-1 06). The phosphoinositide breakdown can, via inositol trisphosphate, cause release of calcium from internal storage sites. The cardiotonic activity of pumiliotoxin B and various congeners and synthetic analogs correlates well with the stimulation of phosphoinositide breakdown (104,105).A number of studies on stimulation of sodium uptake by pumiliotoxin B and inhibition by local anesthetics and other agents have appeared (106-108). The effects of pumiliotoxin B on neuromuscular preparations have been reinterpreted as due primarily to effects on sodium channels, although additional direct effects on calcium mobilization remain possible (109). It has recently been proposed that pumiliotoxin B enhances the rate of activation of sodium channels (110). One characteristic effect of pumiliotoxin B is to elicit repetitive firing in neurons, apparently because of effects on sodium channel function (109-111). The effects of pumiliotoxin B on sodium channels appear to be due to interaction with a subdomain of the site at which batrachotoxin acts; scorpion toxins and brevetoxin can potentiate the effects of pumiliotoxin B and of congeneric pumiliotoxins and allopumiliotoxins (102,112).Certain pumiliotoxin congeners appear to block sodium channel activation and may act as antagonists or reverse agonists (106). Structure-activity relationships with respect to stimulation of sodium flux and phosphoinositide breakdown have been studied (106). The nature of the side chain is critical to activity. For example, whereas pumiliotoxin B is one of the most potent of these alkaloids, its 15,16-erythroisomer has much lower activity (106,112). A pumiliotoxin B-like alkaloid has been reported to be present in fractions purified from the Australian myobatrachid frog Pseudophryne coriucea (113). Effects on the cardiovascular system and cardiac, smooth, and striated muscle preparations were reported (113-1 18). The pumiliotoxin

B-like alkaloid appeared to facilitate neurotransmitter release. In these

3. AMPHIBIAN

ALKALOIDS

223

studies, the P . coriacea alkaloid appeared to be much more active than pumiliotoxin B (see Ref. 117). Ultimately, however, only pumiliotoxin B was identified in such active fractions (85,86,119),and it may be that there are factors present in such fractions that enhance the activity of pumiliotoxin B (see Ref. 86). In summary, pumiliotoxin B and congeners now appear to represent another class of alkaloids that modulate the function of voltage-dependent sodium channels. They are valuable research tools, and perhaps models, for the development of new myotonic or cardiotonic agents. Direct effects of pumiliotoxin-A class alkaloids on calcium translocation, as originally proposed (99), still remain a possibility for further pharmacological study.

3. Occurrence Pumiliotoxin-A class alkaloids, originally thought to be unique to dendrobatid frogs, have been found to have a wide distribution in amphibians, occurring in one genus of myobatrachid frogs (Pseudophryne),one genus of ranid frogs (Mantefla),and one genus of bufonid toads (Mefanophryniscus) (72,7.3,81,86). As yet, no related alkaloids have been reported elsewhere in Nature. The indolizidine trio1 swainsonine, which occurs in plants and fungi (120), is one of a number of polyhydroxyindolizidines with a hydroxyl group analogous to the 8-hydroxyl group of the pumiliotoxin-A class alkaloids, but without the analogous %methyl group or 6-alkylidene substituent. Pumiliotoxins A and B were first discovered as two of the three major alkaloids in one population on Isla Bastimentos, Panama, of the extremely variable Panamanian poison frog Dendrobates pumilio (74,75).Allopumiliotoxins 267A,323B,and 339A also occur in this frog, but as very minor alkaloids ( 1 ) . Pumiliotoxins A and B did not occur in another population of Dendrobates pumifiofrom the same small Caribbean island. This second population had a very different profile of alkaloids, wherein pumiliotoxins 251D and allopumiliotoxins 267A and 325A occur but in very minor amounts ( 1 ) . This is surprising, since in most populations of Dendrobates pumifiofrom Panama and Costa Rica pumiliotoxins and/or allopumiliotoxins are major alkaloids. Pumiliotoxins and/or allopumiliotoxins are major alkaloids in most species of the dendrobatid genus Dendrobates ( 1 ) . The exceptions are a few populations ofD. pumilio, one population of D . auratus, all the populations of D . histrionicus except those north of or in the Rio San Juan drainage, and D . occuftator and D . truncatus. In the dendrobatid genus Epipedobates, pumiliotoxins and/or allopumiliotoxins are major alkaloids in 4 of the 1 1 species examined, namely, E. erythromus, E. espinosai, E. silverstonei,

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JOHN W . DALY E T A L .

and E. tricolor (1).In E. paruulus, one population contained an allopumiliotoxin; another did not. In E. petersi, there was a trace amount of pumiliotoxin B. In the dendrobatid genus Minyobates, all species except M. opistomelus have pumiliotoxins/allopumiliotoxins as major alkaloids, often the only major alkaloids (1);M. opistomelus has pumiliotoxins as trace alkaloids. In the dendrobatid genus Phyllobates, pumiliotoxin-A class alkaloids are usually absent, the exception being a trace amount of pumiliotoxin 251D in P. aurotaenia. In dendrobatid frogs, pumiliotoxins and allopumiliotoxins usually are present together, while in nondendrobatid frogs pumiliotoxins are often found in the absence of allopumiliotoxins. In Australian myobatrachid frogs of the genus Pseudophryne, pumiliotoxins have been found in all seven species examined, three of which also contained an allopumiliotoxin (86). Pumiliotoxin B occurred in five of the seven species and was accompanied by smaller amounts of the erythro isomer in two species. One species, Ps. occidentalis, from southwestern Australia, contained only pumiliotoxin 267C in trace amounts. It is noteworthy that pumiliotoxin 267C is a common alkaloid in the myobatrachid frogs, occurring in six of the seven species, whereas it is a rare alkaloid in dendrobatid frogs. Conversely, allopumiliotoxin 267A is common in dendrobatid frogs but was not detected in myobatrachid frogs. In an initial study (72), pumiliotoxin 267C was erroneously thought to be another isomer, termed 267D, in Mantella madagascariensis and Pseudophryne semimarmorata, but subsequently it was shown to be identical with 267C. In Madagascan ranid frogs of the genus Mantella, pumiliotoxins occurred in the six species examined, accompanied by allopumiliotoxins in three species (73). Two of the species contained only trace amounts of pumiliotoxins. Pumiliotoxins A and B occurred in most species. In toads of the bufonid genus Melanophryniscus, allopumiliotoxin 323B and pumiliotoxin 267C were major alkaloids in the Brazilian species M. moreirae (71), and pumiliotoxin 251D was a major alkaloid in the Uruguayan subspecies M. stelzneri montevidensis and a minor or trace alkaloid in two populations of M. stelzneri from Argentina (81). Homopumiliotoxins, in which the indolizidine ring of pumiliotoxins has been replaced by a quinolizidine ring (Fig. 9), are relatively rare in dendrobatid frogs. Homopumiliotoxin 223G has been detected only in Dendrobates pumilio and two other dendrobatid species (1 and J. W. Daly, unpublished). Homopumiliotoxins were not detected in myobatrachid frogs (86). Homopumiliotoxin 2236 was detected in two species of the ranid genus Mantella (73). Homopumiliotoxins were not detected in two Argentine populations of Melanophryniscus stelzneri (81), nor in Brazilian M. moreirae. However, homopumiliotoxins were minor alkaloids in the bufonid Uruguayan subspecies M. stelzneri monteuidensis, where three previously unknown homopumiliotoxins were discovered (81).

3.

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225

In summary, the pumiliotoxin-A class alkaloids are unique to certain genera from four different families of amphibians. Pumiliotoxins and allopumiliotoxins often occur together. Homopumiliotoxins occur much less often. The unique and complex structures and the phylogenetic distribution of pumiliotoxin-A class alkaloids argue for the independent evolutionary development of a complex suite of biosynthetic enzymes. The branched-chain structures of the pumiliotoxin-A class also suggest the incorporation of isoprene units, unlike the preponderance of bicyclic dendrobatid alkaloids, which appear to be derived from straight-chain precursors.

D. PYRROLIZIDINES Pyrrolizidines had not been identified as alkaloids from amphibian sources until very recently. All represent minor or trace constituents. Structural definition is based on gas chromatographic-mass spectral and FTIR spectral characterization. All appear to be 3,5-disubstituted pyrrolizidines, with the exception of the tricyclic pyrrolizidine oximes (see Section IV,D). When the two side chains differ, four diastereomers are possible, namely, endo,endo and exo,exo (both also termed cis) and exo,endo and endo,exo (both also termed trans). The structure of the amphibian pyrrolizidine 2238 (see structure VII ) with an exo,exo configuration was proved through comparison of gas chromatographic and spectral properties with the known alkaloid (5Z,8E)-3-n-heptyl-5-methylpyrrolizidinefrom thief ant (Solenopsis xenovenenurn) venom (121). The exo,exo configuration also was assigned to one 223B diastereomer and one 251K diastereomer by comparison with synthetic mixtures (81).Others have the exo,endo or endo,exo configuration (81). The mass spectra of pyrrolizidines are dominated by cleavage of one or the other side chain. When the side chains differ, two major fragments are seen, although methyl loss is small, as in 2238. FTIR spectra are useful in establishing the relative configuration for pyrrolizidines (81). Only alkaloids with the endo,endo configuration having H-3, H-5, and H-8 on the same face (SZ,SZ) have appreciable Bohlmann bands, whereas in

Pyrrolizidine 223H

(VII)

226

JOHN W. DALY E T A L .

the exo,exo configuration (5Z,8E), bands are weak and in the exo,endo (5E,8Z) or endo,exo configuration (5E,8E) bands are virtually absent. The endo,endo configuration has not been detected in Nature (123-125). The alkaloids identified from frogs and toads as pyrrolizidines are listed below. Structures (some tentative) are shown in Fig. 10 or are suggested below. Pyrrolizidines 223B. ‘C15H,,N,’0.3, 159”C,m/z 223(1), 222(2), 166(100).OD. Infrared spectra (81). H, derivative. Both exo,exo (5Z,8E) and endo,exo (5E,8E) isomers occur in bufonid toads (81). 2238. CI5H2,N,-,-, m / z 223(I), 124(100). OD. Infrared spectra (81). Ha derivative. An exo,exo (5Z,8E) isomer. 2376. ‘C15H27N0,’-,-, ion trap, m / z 238(2), 124(100). OD. Infrared data (81). An exo,exo (5Z,8E) isomer. 251K. ‘C17H33Nr’-,-, ion trap, m / z 252(lo), 251(8), 194(80), 166(100), 124(12), 110(10),70(20). OD. Infrared spectra (81). Ha derivative. Both an exo,exo (5Z,8E) isomer and an exo,endo (5E,8Z)and/or endo-exo (5E,8E) isomer occur. 2658. ‘C17H3,N0,’-,-, ion trap, m/z 265(<1), 222(30), 152(100), 110(18). Infrared data (73). Tentative structure: a 3-ketoheptyl-5-propylpyrrolizidine. Two diastereomers have been detected (73). 2678. C17H33N0,-r-, ion trap, m / z 268( I), 224(35), 152(loo), 110(1 9 , 70(20). 1D. Infrared data (73). Tentative structure: a 3-hydroxyheptyl-5propylpyrrolizidine.A major and a minor diastereomer have been detected (73). The biological activity of 33-disubstituted pyrrolizidines from amphibians has not been studied. It is presumed that the pyrrolizidine alkaloids isolated from ants serve as venoms (123). 3,5-Disubstituted pyrrolizidines were first discovered in Nature as constituents from ants (121). The structure of (5Z,8E)-3-heptyl-5-methylpyrrolizidine from thief ant (Solenopsis xenouenenum) venom proved identical with the amphibian pyrrolizidine 5Z,8E-2238 (Fig. 10). Other pyrrolizidines from ants include (5E,8Z)-3,5-di(S-hexenyI)pyrrolizidine, (5Z,8E)-3-methyl-5-(8-nonenyl)pyrrolizidine,and (5E,8Z)-3-(8-nonenyl)5-[(E)-l-propenyI]pyrrolizidine from New Zealand ants of the genus Monomorium (=Chelaner) (123-128) and (5E,8E)-3-n-butyl-5-n-hexylpyrrolizidine from Venezuelan ants of the genus Megalomyrmex (126). The 3,5-disubstituted pyrrolizidines probably occur as minor or trace constituents in a number of dendrobatid frogs. However, the apparent

3.

-

(52,8E)-3,5-disubstitutedpyrrolizidines

alkaloid 52,8E-2238

(?? R'

R

227

AMPHIBIAN ALKALOIDS

5Z,8E-223H

5Z,8E-237G

R

R'

n-Bu

n-Bu

n-heptyl

Me

0

52,8E-251K

Me

n-Bu

n-hexyl

R

R'

n-Bu

n-Bu

(5E,aE)-3,5-disubstituted pyrrolizidines alkaloid 5E,8E-2238

q R'

n-Bu 5E,8E-251K

n-hexyl or

R

n-hexyl

n-Bu

~~

n-Pr 5E.8E-267H

C7H140H

or C7H140H

n-Pr

FIG.10. Structures of 3.5-disubstituted pyrrolizidines from dendrobatid and ranid frogs and bufonid toads (31,73,81,and J. W. Daly, H. M.Garraffo, and T. F. Spande, unpublished). Absolute configurations are unknown. It is assumed, based on analogy to other amphibian alkaloids, that n-alkyl side chains are present. The configurational nomenclature for these pyrrolizidines follows the system devised by Sonnet er a / . (122)for 3,5-disubstituted indolizidines where the H-5 and H-8 configurations are related to that at H-3 and are either cis ( Z ) or trans ( E ) .

distribution of pyrrolizidine 223B in dendrobatid frogs (see Ref. I for distribution) must be considered tentative until such extracts are reexamined using capillary columns and FTIR spectroscopy. Tentatively, pyrrolizidine 223B probably is present as a minor or trace alkaloid in

228

JOHN W. DALY E T A L .

Dendrobates histrionicus, D . leucomelus, D . truncatus, and D . ventrirnaculatus, and in Minyobates bombetes and M . minutus. Populations of Dendrobates auratus contain pyrrolizidines 223B, 223H,251K, and 2678 as minor or trace alkaloids (31). 3,5-Disubstituted pyrrolizidines occur as trace or minor alkaloids in several species of ranid frogs of the genus Mantefla (73).Pyrrolizidine 2678 occurs as a major alkaloid in one species. 33-Disubstituted pyrrolizidines occur in bufonid toads of the genus Melunophryniscus, where in M . stelzneri, (5Z,8E)-223H, (5Z,8E)-223B, (5E,8E)-223B, and other pyrrolizidines were detected as minor constituents (81).

E. INDOLIZIDINES Two classes of simple indolizidines have been characterized from amphibians. These are in addition to the more complex pumiliotoxin-A class of indolizidines where 8-hydroxy, 8-methyl, and 6-alkylidene substituents are present in the indolizidine ring (see Section 111,C). The simple indolizidines are either 3 3 - or 5,8-disubstituted. However, the existence of 3- or 5-monosubstituted indolizidines also has been postulated. I . 3,5-Disubstituted Indolizidines

The structure of indolizidine 223AB was determined in 1981 through gas chromatographic comparisons with the four synthetic diastereomers (127).Nuclear magnetic resonance spectral analysis confirmed the structure of 223AB isolated from Dendrobates histrionicus as (5E,9E)-3-butyI5-propylindolizidine (for nomenclature see Fig. I 1 ) and established the structures of the side-chain hydroxylated congeners as (5E,9E)-239AB and (5E,9E)-239CD(96). An enantioselective synthesis (128)indicated an absolute configuration of (3R,5R,9R), for levorotatory (5E,9E)-223ABas shown in VIII. Indolizidine 223AB isolated from Dendrobates speciosus has proved to be the 5Z,9Z isomer. It was assumed initially, based on a

lndolizidine 5E,9E-223AB

(VIII)

3.

AMPHIBIAN ALKALOIDS

229

proton magnetic resonance spectrum ( 8 7 ) , to be identical with (5E,9E)-

223AB from Dendrobates hisrrionicus. Three diastereomers of 223AB were detected in extracts of a bufonid toad, Melanophryniscus srelzneri, in the following relative amounts: 5Z,9Z > 5E,9Z > 5E,9E (81). Indolizidine 195B,isolated from D . hisrrionicus, proved to be dextroro(76), diastereomeric with motatory (5E, 9E )-3-butyl-5-methylindolizidine nomorine I (5Z,9Z) from pharaoh's ant (129). Synthesis has confirmed a 3S,5S, 9s configuration, opposite to that of 223B, for indolizidine 195B (129a,b). The 5E, 9E, the 5Z, 9Z, the 5E, 9Z, and the 5Z, 9E diastereomers of 195B were all present in a bufonid toad (81). The 3,5-disubstituted indolizidines show major fragment ions corresponding to the loss of either the 3- or 5-substituent; loss of the methyl group is much less than other substituents. A fragment at mlz 124 is often present arising from a McLafferty rearrangement during cleavage of the second side chain. It is a minor fragment in electron impact spectra but is quite significant in ion trap spectra (81). Vapor-phase FTIR spectra are diagnostic for the stereochemistry of indolizidines (81) and distinguish between 3 3 - and 5,8-disubstitution patterns. The 5,8-disubstituted class (Section 111,E,2) have an intense and sharp Bohlmann band (2785 cm-') when H-5 and H-9 are cis, while the 3,5-disubstituted class have a broader Bohlmann band pattern with weak fine structure when H-3, H-5, and H-9 are all cis (5Z,9Z). The other three 3,5-disubstituted diastereomers have Bohlmann bands decreasing in intensity in the order 5E,9Z > 5E,9E > 5Z,9E; the last isomer has virtually no Bohlmann bands. The amphibian alkaloids considered to be 3,5-disubstituted indolizidines are tabulated below. Two other bicyclic alkaloids, proposed as monosubstituted indolizidines in an earlier review, are included. These are 167B, which was considered likely to be a 5-n-propylindolizidine, based on its mass spectrum and biosynthetic considerations, and 209D, which was considered likely to be a 5-n-hexylindolizidine. These two indolizidines have been synthesized (130,131), but efforts to compare the synthetic indolizidines to natural 167B and 209D were thwarted when the natural trace alkaloids could no longer be detected in extracts. Structures of relatively well-characterized 3,5-disubstituted indolizidines from amphibians are shown in Fig. 11. 3,5-Disubstituted and 5-Substituted Indolizidines

167B.'CllH21N,'-, 151"C, mlz 167(12), 166(5), 124(100).OD. H,derivative. Tentative structure: a 5-propylindolizidine. 195B. C,,H,,N, 0.28, 156"C, mlz 195(2), 194(1), 138(100). OD. Infrared data (81). H, derivative. Various isomers occur (76,81).

230

JOHN W . DALY E T A L . (52.92)-3,5-disubstitutedindolizidines

i

5Z,9Z-l95B

(5E,gE)-3,5-disubstituted indolizidines

C-qJCqJ EH3

\

i

CH5E,9E-239AB

(5E.9Z)-3.5-disubstituted indolizidines

5E,9E-239CD

(5Z,9E)-3,5-disubstitutedindolizidines

CH,

FIG.11. Structures of 3.5-disubstituted indolizidines from amphibians. The absolute configuration of natural levorotatory (5E,9E)-223AB,(5E,9E)-239AB,and (5E,9E)-239CD,are known. Natural dextrorotatory (5E,9E)-195Bhas the opposite absolute configuration as that shown above ( 1 2 9 ~ )The . configurational nomenclature is based on that introduced by Sonnet ef al. (122). where the H-5and H-9 configurations are related to that of H-3 and are either cis ( Z ) or trans (E).

2WD. 'CI4H2,N,'--, 159"C, mlz 209(2), 124(100). OD. H, derivative. Tentative structure: a 5-hexylindolizidine. 223AB. C1SH29N, 0.30, 160"C, mlz 223( l), 222(2), 180(85), 166(100). OD. Infrared spectra (81).H, derivative. Various isomers occur (73,81,96).The relative intensities of major fragment ions differ in different isomers (127). 239AB. CIsH2,NO, 0.22, 178°C mlz 239(2), 238(3), 182(100), 180(90). 1D. H, derivative. 0-Acetyl derivative. A 5E,9E isomer has been detected (96).

3.

AMPHIBIAN ALKALOIDS

23 1

239CD. C15H2,N0,0.16, 179"C, mlz 239(4), 238(3), 196(loo), 166(60). 1D. H, derivative. 0-Acetyl derivative. A 5E,9E isomer has been detected (96). 249A. 'C17H31N,'-, 172"C, m/z 249(3), 192(loo), 180(25). OD. Infrared spectrum (73).H2derivative. A 5Z,9Z isomer has been detected in a ranid frog (73). 275C. 'C19H33N,'-,-, ion trap, m/z 275(<1), 206(23), 192(IOO), 180(15),124(22).OD. Infrared data (73).A 5Z,9Z isomer has been detected in a ranid frog (73). Optical rotations of 3,5-disubstituted indolizidines are given in Table VI. For details of the mass spectra, see Refs. 3,73,96, and 127. For proton and carbon-13 magnetic resonance spectra data, see Refs. 76 and 96 and references to synthetic material cited in Ref. 5 . For FTIR spectra, see Ref. 81. Vapor-phase FTIR spectra of the synthetic 5,9Z and 5,9E diastereomers of 5-n-hexylindolizidine (cf. 209D ) have been presented (78). Little is known of the biological activity of the 3,5-disubstituted indolizidines from amphibians. Indolizidine 239CD at a subcutaneous dose of 80 pg causes long-lasting locomotor difficulties and prostration in mice (60).Indolizidine 223AB,like many lipophilic dendrobatid alkaloids, acts as a noncompetitive blocker of nicotinic receptor-channels, both in electric ray electroplax preparations (muscle-type nicotinic receptor-channel) (64,133)and in pheochromocytoma cells (ganglionic-type nicotinic receptor-channel) (64).Indolizidine239AB appears selective for the ganglionictype channel.

OPTICAL

TABLE VI ROTATIONSOF 3,S-DISUBSTlTUTED INDOLIZIDINES(76,96)

Compound (5E,9E)-195B (5E,9E)-195B'HCI (5E,9E)-WAB (5E,9E)-223AB (SE,9E)-239AB (5E,9E)-239CD

+65" (0.41, CHJOH, 16°C)"

+ 36" (0.52, CHJOH, 24°C)

-35" (0.49, CHJOH, 16°C) -44" (1.0. n-hexane, 27"C)b -38" (1.0, CH3OH, 16°C) -52" (0.19, ChqOH, 16°C)'

a Synthetic (3S,SS,9S)-(5E,9E)-I%B had an [a]% of + 98.0" (0.3,CH,OH) (1290). Synthetic (3R,SR,9R)-(5E,9E)-WAB had an [a]; of - 101" (2.3, n-hexane) (128). Synthetic (3R,SR,9R)-(JE,9E)-2J9CD had an [a]? of -58.6" (0.21, CHjOH) (132).

232

JOHN W. DALY E T A L .

3,5-Disubstituted indolizidines, unlike many classes of amphibian alkaloids, are not unique to amphibians. 3,5-Disubstituted indolizidines such as monomorine I [(5Z,9Z)-195B]occur in ants of the genera Monomorium and Solenopsis (125,129,134).Some of the ant indolizidines are as follows: (5Z,9Z)-3-n-butyl-5-methylindolizidine(monomorine I), (5Z,9Z)-3-nethyl-5-methylindolizidine, (5Z,9Z)-3-hexyl-5-methylindolizidine,and (5E,9Z)-3-butyl-5-(4-pentenyl)indolizidine.A series of 5-substituted indolizidines, the piclavines, were reported recently from a marine tunicate (Clauelina picta) (135). Indolizidine 223AB occurs in a somewhat limited number of dendrobatid frogs. It has been found as a major alkaloid only in Dendrobates auratus, D . histrionicus, and D . speciosus, while being detected in trace amounts in D . reticulatus and D . uentrimaculatus ( 1 ) . The major isomer from D. histrionicus has the 5E,9E stereochemistry (96), while the major isomer from D . speciosus is 5Z,9Z. Indolizidines 239AB and 239CD occur as major alkaloids only in D . histrionicus and D . occultator ( 1 ) . Indolizidine 195B occurs in D . auratus, D . histrionicus, D . pumilio, and D . speciosus ( 1 ) . The major isomer isolated from D . histrionicus is 5E,9E (76). 33Disubstituted indolizidines apparently d o not occur in frogs of the dendrobatid genera Epipedobates or Minyobates ( 1 ) . Indolizidine 223AB does occur as a minor alkaloid in Phyllobates aurotaenia, but not in other species of this dendrobatid genus. Indolizidine 195B was detected in Dendrobates auratus and Phyllobates bicolor reared in an outside vivarium in Hawaii (31). In the bufonid toad Melanophtyniscus stelzneri, a range of 3,5-disubstituted indolizidines occur, including several diastereomers of 195B and 223AB (81).Ranid frogs of the genus Mantella also contain some 3,5-disubstituted indolizidines, such as 223AB, 249A, and 275C, but not 195B (73). 2. 5,8-Disubstituted Indolizidines

The isolation of indolizidine 207A in sufficient quantity for nuclear magnetic resonance spectroscopy ( 8 7 ) established a 5-(pent-4-enyl)-8methylindolizidine structure as shown in IX.The (5R,8R,9S) absolute configuration shown is the same as that of 205A, which contains a terminal

Indolizidine 207A

(IX)

3. AMPHIBIAN

ALKALOIDS

233

acetylene rather than a double bond. The (5R,8R,9S) absolute configuration of 205A and certain other congeners has been established by enantioselective syntheses (136-138). These enantiomers were levorotatory, as were the natural compounds, with the exception of 235B"and 251B which were dextrorotatory (see below). It had been apparent, based on the characteristic mass spectra of other bicyclic tertiary amine alkaloids, that there existed a large series of such 5-substituted 8-methylindolizidines in skin extracts from dendrobatid frogs. Some of these alkaloids have been isolated and their structures determined by nuclear magnetic resonance spectroscopy (77,78,87). The 5-substituted 8-methylindolizidines exhibit a mass spectral fragment of C&,,N+ (mlz 138) as the base peak and, in addition, a diagnostic fragment of C,H,,N+ (mlz 96) arising from the m / z 138 fragment by a retro-Diels-Alder process. The ion trap spectra show a much more prominent mlz 96 ion than normal electron impact-mass spectra. Several 5,8disubstituted indolizidines characterized from skin extracts of frogs of the genus Mantella (73) have 8-substituents other than methyl, and they yield major fragments at m / z 152, 176, and 178, depending on the substituent at the 8 position. The Bohlmann bands in the FTIR spectra of 5,8-disubstituted indolizidines appear diagnostic, as almost all of the alkaloids of this class encountered in frog skin extracts to date have H-5and H-9 in a cis relationship (5,9Z), and show a sharp, intense Bohlmann band at 2785 cm". The one exception is 259B from a bufonid toad, which appears to have a trans (5,9E) configuration (81). The 5-substituted 8methylindolizidines are tabulated below, followed by other 5,8-disubstituted indolizidines. Structures of relatively well-characterized 5-substituted 8-methylindolizidines and other 5,8-disubstituted indolizidines are shown in Figs. 12 and 13, respectively. Tentative structures are suggested below for several additional alkaloids. 5-Substituted 8-Methylindolizidines

167A. CIIH2,N,-, 151"C, m / z 167(1), 166(1), 138(100). OD. Ha derivative. Tentative structure: a 5-ethyl-8-methylindolizidine. 181B. CI2H2,N,--, 153"C, mlz 181(2), 180(2), 138(100). OD. Ha derivative. Tentative structure: a 5-propyl-8-methylindolizidine. 203A. C,,H,,N, 0.33 158"C, mlz 203(1), 202(2), 138(100). OD. Infrared spectrum (78).H, derivative, m / z 209(I ) , l38( 100). An isomer (203A') has been detected at 157°C. 205A. C,,H,,N,--, 158"C, mlz 2 0 3 I), 204(2), l38( 100). OD. H, derivative, mlz 209, 138. An isomer (205A') has been detected at 156°C.

234

JOHN W . DALY E T A L .

~

~

203A

205A

\

\

/

207A

&)cTc,bl$ 2098

233D

2358'

OH

FIG. 12. Structures of 5-substituted 8-methylindolizidines from amphibians. The absolute configurations of natural levorotatory 205A and 235B' are known. The rotation of 207A is unknown: 203A and 233D are levorotatory, and 235B" and 2518 are dextrorotatory. All are shown with the same absolute configuration as 205A and 235B'. All have the 5,9Z configuration except for 259B from a bufonid toad, which has the 5,9E configuration (81). The configuration at the 8 position could not be determined from FTIR spectra and, thus, is unknown for several of these alkaloids.

207A. C,,H,,N, 0.35, 158"C, mlz 207(1), 206( I ) , l38( 100).OD. H, derivative, mlz 209, 138. An isomer (207A') has been detected at 156°C. An isomer from a bufonid toad (207A') has a cis double bond in the fivecarbon side chain (81). Infrared spectrum of 207A (81). 2WB. 'C,4H27N,'-, 162"C, mlz 209(5), l38( 100). OD. H, derivative. 221A. 'C,SH27N,'-r 162°C mlz 221(<1), 220(2), 138(100).OD. H, derivative, mlz 223, 138. Tentative structure: a 5-hexenyl-8-methylindolizidine.

3. AMPHIBIAN

ALKALOIDS

235

FIG.13. Structures of other 5,8-disubstituted indolizidines from amphibians. Most have been characterized from ranid frogs of the genus Mnnrella (73). Absolute configurations are unknown, All have the 5,9Z configuration. The configuration at the 8 position could not be determined from FTIR spectra.

223D. 'C15H2,N,'0.3,159"C,mlz223(2), 222(1), 138(100). OD. H,derivative. Tentative structure: a 5-hexyl-8-methylindolizidine. 225D. Cl,H2,N0,--, 164"C,rnlz 225(< I ) , l38( 100). ID (not on nitrogen). H, derivative. Tentative structure: a 5-hydroxypentyl-8-methylindolizidine. 231c. C I ~ H ~ 0.38, ~ N , 171"C, m/z 231(3), 138(100). OD. H6 derivative, mlz 237, 138. Tentative structure: a 5-(heptenynyl)-8-methylindolizidine. 233D. c16H2,N,-,-, m/z 233(5), 232(2), 176(2), 164(8), 151(22), 138 (100). OD. H, derivative. W5B. CI6H2,N,-, 166"C, m/z 235(1), 234(1), 138(100). OD. Infrareddata (73). H2 derivative. Two isomers have been isolated, 235B' and 235B" (formerly 235B) (see Ref. 78). 237D. 'C16H31N,'-, 163°C mlz 237(1), 236(2), 138(100). OD. Hoderivative. 239G. C15H2,N0,--, 178"C, rnlz 239(1), 238(3), 138(100). ID (not on nitrogen). H, derivative. Tentative structure: a 5-hydroxyhexyl-%methylindolizidine .

236

JOHN W. DALY E T A L .

S I B . ‘CI~H~,NO,’--,184”C, m/z 251(2), 234(4), l38( 100). ID (not on nitrogen). H2derivative, m/z 253, 138. Structure based on nuclear magnetic resonance spectra (78). 253B. ‘C16H31N0,’-, 192”C,mlz 253( I ) , 138(100). ID (not on nitrogen). Infrared data (73). H, derivative. Tentative structure: a 5-hydroxyheptyl8-methylindolizidine. 257C. ‘cl,H27N,’-, 190”C, m/z 257(<1), l38( 100). OD. H, derivative, m / z 265, 138. Tentative structure: a 5-nonadienynyl-8-methylindolizidine. 259B. ‘cl,I-Iz,N,’-,-, m/z 259(3), l38( 100). OD. Infrared spectrum (81). Only an atypical 5,9E isomer has been detected (81). 279D. ‘C18H33N0,’--,--, m/z 279(<1), 278(<1), l64( 12), 151(20), 138(IOO), 96(32). 1D. Infrared data (73).Tentative structure: a 5-hydroxynonen yl-8-meth ylindolizidine. 295B. ‘C18H33N02,’-,-, m/z 295(< I), l80( l7), 167(20), 154(IOO), 112(20), 94( 13). Infrared data (73). Di-0-acetate. Provisionally proposed as a 5-substituted 8-methylindolizidine having a ring hydroxyl group and a nine-carbon 5-substituent with a second hydroxyl group and a double bond (73). Other 5,8-Disubstituted Indolizidines 217B.C15H23N,--,--, ion trap, m/z218(1), 152(100),96(48).OD. Infrared spectrum (73). 219F. ‘ClsH25N,-,-, ion trap, m/z 2l9(< I ) , l52( 100). Tentative structure: a dihydro-217B (73). 2231. ‘CIsH2,N,’-,-, ion trap, m/z 224(4), 223(3), l80( IOO), 96(42). Tentative structure: an 8-butyl-5-propylindolizidine, formerly referred to as 223A’ (31). 237H. ‘c1&31N,’--,-, ion trap, m/z 237(< I ) , l52( IOO), 96(62). Tentative structure: an 8-ethyl-5-hexylindolizidine (73). 24lF.C17H23N,-,-, ion trap, m / z 241(<1), 176(IOO), 96(50). OD. Infrared data (73). 243B. CI7Hz5N,-, 178”C, mlz 243(<1), 176(100), 96(62). OD. Infrared data (73). 243C. C17H25N,-,-, ion trap, mlz 243(<1), 178(100),96(55). OD. Infrared data (73). 243D. ‘C,7H25N,’-,-, ion trap, m/z243(<1), 242(17), 176(18),164(12), 152(32), 122(44),96(45), 90(50), 70( 100). OD. Infrared data (73).Provisionally proposed as a 5,8-disubstituted indolizidine with 8-ethyl and 5trans,cis-CH=CHCH,CH=CHG=CH substituents (73). 245B. C17H27N,-,-, ion trap, m/z 245(<1), 178(100),96(62). OD. From a ranid frog (73).

3. AMPHIBIAN

ALKALOIDS

237

245C. 'C,7H27N,'-,-, ion trap, mlz 245(<1), 244(19,216(20), 206,204,202(-lo), 188(15), 175(15), 174(17), 164(15), 152(32), 134(15), 132(1% 122(35), 96(45), 91(48). Infrared data (73). Provisionally proposed as a 5,8-disubstituted indolizidine with 8-ethyl and 5-transCH=CH(CH,),C=CH substituents (73).

Optical rotations of 5,8-disubstituted indolizidines are given in Table VII. Proton and carbon-13 magnetic resonance spectral data have been reported (77,78,87). The proton assignments for 235B' reported in Ref. 87 are for the hydrochloride salt, and the chemical shift for H-15is 5.31 ppm (not 5.81 as reported). Impurities were included in the integration between 1 .O and 2.15 ppm. Vapor-phase FTIR spectra of 5,8-disubstituted indolizidines have been reported (73,78,81). Little is known of the biological activity of 5-substituted 8-methylindolizidines. Like many lipophilic dendrobatid alkaloids, indolizidines 205A, 207A, and congeners appear to be noncompetitive blockers of nicotinic receptor-channel complexes, both in electric ray electroplax and pheochromocytoma cells (139). Indolizidine 205A is remarkable in enhancing binding of [3H]perhydrohistrionicotoxinto a noncompetitive blocker site on the nicotinic receptor-channel of electric ray electroplax (139). The 5-substituted 8-methylindolizidines appear to be unique to amphibians. However, a 5-(3-furyl)-8-methylindolizidinehas been reported as a

OPTICAL

TABLE VII ROTATIONSOF 5,8-DISUBSTITUTED INDOLIZIDINES (77,78,87Y

Compound U)3A 205A 233D.HCI 235B' 235B"

251B

- 23.3" (0.30, CHCI3) -35" (0.24, CH3OH)" -3.4" (0.16, CH3OH) -61" (0.50, CH3OH. 25°C) + 11.3" (1.0. CH30H, 25°C)b + 25.9" (0.8, CHCI,)

Synthetic (SR,8R,9S)-U)SA had [alDvalues of -83.5" (0.30, CHIOH) (137) and -74.2" (0.82. CH,OH) (138). Synthetic (SR,8R,9S)-M7A had an [a]: of -86.5" (0.95, CHCIJ (138). Synthetic (SR,8R,9S)-USB"(formerly U S B ) had [alDvalues of -94.3" (1.85, CHIOH) (137)and -85.4"(0.79, CHIOH) (138). The positive rotations of natural USB" and 2518 are anomalous; either these are diastereomers of the (SR,8R,9S) enantiomers, or there were strongly dextrorotatory impurities present.

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JOHN W . DALY E T A L .

trace component in extracts from a mammalian scent gland (140) and has been synthesized (141). It is closely related in structure to certain Nuphar quinolizidine alkaloids. 5-Substituted 8-methylindolizidines occur in a wide range of dendrobatid species. In the dendrobatid genus Dendrobates, 5-substituted 8-methylindolizidines are major alkaloids in D . arboreus, D . auratus, D . new species (Panama), D . pumilio, and D . speciosus ( 1 ) . 5-Substituted 8-methylindolizidines are present as trace alkaloids in D . azureus, D . histrionicus, D . lehmanni, and D . truncatus, but have not been detected in D . granuliferus, D . leucomelus, D . quinquevittatus, D . reticulatus, D . tinctorius, and D . ventrimaculatus. In the dendrobatid genus Epipedobates, 5-substituted 8methylindolizidines are major alkaloids in E. erythromus, E. siluerstonei, and E. tricolor; trace alkaloids in E. espinosai, E.femoralis, and E. trivittatus; and absent in E. anthonyi, E. myersi, E. parvulus, E. petersi, and E . pictus (I). In the dendrobatid genus Minyobates, 5-substituted 8-methylindolizidines are major alkaloids in M . fulguritus, M . new species (Panama); minor or trace in M . minutus and M . opistomelus; and absent in M . abditus, M . altobueyensis, M . bombetes, M . steyermarki, and M . viridis. In the dendrobatid genus Phyllobates, 5-substituted 8-methylindolizidines have not been detected with the possible exception of a trace of 223D in P . vittatus ( 1 ) . Two 5-substituted 8-methylindolizidines, 207A"and 259C, occur in a bufonid toad, Melanophryniscus stelzneri (81). Several 5,8disubstituted indolizidines occur in ranid frogs of the genus Mantella. These include the 5-substituted-8-methylindolizidines 205A and 207A (72,73) but also several 5,8-disubstituted indolizidines with an apparent 8-substituent other than methyl (73). Such 5,8-disubstituted indolizidines also are present in dendrobatid frogs (31 and J. W. Daly, unpublished).

F. QUINOLIZIDINES The occurrence of quinolizidines in skin extracts from dendrobatid frogs has been suspected for some time (3, but only recently have sufficient mass and FTIR spectral data been obtained to permit assignment of structures to quinolizidines from dendrobatid and ranid frogs and bufonid toads. As yet, none of these quinolizidines has been isolated in quantities sufficient for nuclear magnetic resonance spectral characterization. The amphibian quinolizidines appear to be 1,Cdisubstituted ring homologs of the 5,8-disubstituted indolizidines. A characteristic Bohlmann band pattern in their FTIR spectra, which is broader and less intense than that

3.

239

AMPHIBIAN ALKALOIDS

from 5,8-disubstituted indolizidinesfor some, but not all, of the amphibian quinolizidines (73,supports a cis relationship between H-4 and H-10 (i.e., 4,102). The configuration at the 1 position is unknown. The mass spectra show a base peak corresponding to loss of the 4-substituent. In addition, there is a characteristic fragment of C7H12N+(mlz 110) analogous to the C,H,,N+ (mlz 96) fragment of the 5,8-disubstitutedindolizidines that arises from a similar retro-Diels-Alder cleavage. This cleavage is more pronounced with the ion trap technique than in conventional electron impact-mass spectra. Over 30 amphibian alkaloids from dendrobatid frogs, bufonid toads, or ranid frogs now are assigned to the 1,4-disubstituted quinolizidine class. For many of these, FTIR data are not yet available. Thus, structural and stereochemical assignments to this class, and the proposed 4,lOZ configuration, should be considered tentative for many of these alkaloids. A 4,6-disubstituted quinolizidine class, considered likely by analogy to the 3,5-disubstituted indolizidines and biosynthetic considerations, has yet to be conclusively demonstrated in amphibians, although several unclassified alkaloids may possess this structure. The following alkaloids appear to belong to a 1,Cdisubstituted quinolizidine class of amphibian alkaloids. Structures of three relatively well-characterized quinolizidines from amphibians are shown in Fig. 14. Tentative structures are suggested below for several additional alkaloids. 1,CDisubstituted Quinolizidines

181A. ‘C12H23N,’-, 152”C,mlz 181(2),180(1), 152(100).OD. H, derivative. Tentative structure: a 4-ethyl-I-methylquinolizidine.

b b < < < 217A

231A

233A

FIG. 14. Structures of 1.4-disubstituted quinolizidines from amphibians. All have been characterized only by mass and FTIR spectral properties (73.81).All have the 4, IOZ configuration. The configuration at the 1 position is as yet unknown.

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JOHN W. DALY E T A L .

195D. ‘CI3H2,N,’-, 158”C, mlz 195(2), 166(100). OD. H, derivative. Tentative structure: a 1,4-diethylquinolizidine. 207C. ‘CI4H2,N,’-, 155”C, mlz 207( l6), l52( 100). OD. H, derivative. Tentative structure: a 4-butenyl- I-methylquinolizidine. 207I. ‘CI4H2,N,’-,-, ion trap, mlz 207(<1), 206(4), l66( loo), 1 lO(27). Infrared data (73). Tentative structure: a 4-allyl-1-ethylquinolizidine (73). 209C. C14H27N,-r 164“C, mlz 209( 1 I ) , l52( 100). OD. H, derivative. Tentative structure: a 4-butyl- 1-methylquinolizidine. 209E. ‘C14H27N,’-, 158”C, mlz 209( 17), 166(100). OD. H, derivative. Tentative structure: a 1-ethyl-4-propylquinolizidine. 217A. CI5H2,N,0.40, 166”C, mlz 217(2), 216(3), 152(100). OD. Infrared spectrum (73). H, derivative, mlz 223, 152. Trace amounts of another isomer were also detected in a ranid frog (73). 219B. ‘CI5H2,N,’-, 162”C,mlz 219(1), 218(2), 152(100). OD. H, derivative, rnlz 223, 152. Tentative structure: a I-methyl-4-pentadienylquinolizidine. 219E. ‘C15H25N,’-, 161”C, mlz 219(1), 218(3), 166(100).OD. Tentative structure: a 4-butynyl-I-ethylquinolizidine. 223A. ‘C,,H,,N,’ 0.3, 159”C, mlz 223(10), 222(2), 180(100), ion trap, mlz 223(<1), 180(100), 124(30). OD. H, derivative. Tentative structure: a 1,4-dipropylquinolizidine.An alkaloid (223A‘) has been detected at 163°C ( I ) , but appears to be a 5,8-disubstituted indolizidine and is now named 2231 (31). 223C. ‘C,,H,,N,’ 0.3, 159°C mlz 223(1), 222(2), 152(100). OD. H, derivative. Tentative structure: a I-methyl-4-pentylquinolizidine. 231A. C,,H,,N, 0.30, 166”C,mlz 231(2), 230(1), 166(100). OD. Infrared data (73). H, derivative, rnlz 237, 166. An apparent isomer (231A’) has been detected at 171°C (I). 231B. C,,H,,N, 0.30, 166“C, mlz 231(2), 230(1), 152(100), IlO(1O). OD. H, derivative, rnlz 237, 152. An apparent isomer (231B’) has been detected at 168°C (1). Tentative structure: a 4-hexenynyl-I-methylquinolizidine. 233A. ‘c16H27N,’-, 167°C m/z 233(2), 232(2), 166(100). OD. Infrared data (73). H, derivative, mlz 237, 166. W5E. ‘C,&&,,N,’-, 168”C,mlz 235(3), 152(100), I lO(15). OD. H,derivative, mlz 237, 152. Structure: a 4-hexenyl-I-methylquinolizidine.An isomer (235E’) from a bufonid toad has an interior double bond in the sixcarbon side chain (81). Infrared data on 2353’ (81). 237C. ‘c&3,N,’-, 168”C,rnlz 237(2), 236(1), 180(100). OD. H, derivative. Tentative structure: a 4-butyl-l -propylquinolizidine. 249C. ‘C17H31N,’-,-r mlz 249(<1), l52( IOO), 1 lO(30). OD. Tentative structure: a 4-heptenyl-I-methylquinolizidine(73).

3. AMPHIBIAN ALKALOIDS

24 1

263A. ‘C,8H33N,’-, 174”C, rnlz 263(2), 152(100). OD. Tentative structure: a 1-methyl-4-octenylquinolizidine. 273A. ‘C19H31N,’-,-, ion trap, mlz 273(<1), 234(5), 152(100), 1 lO(37). OD. Tentative structure: a 1-methyl-4-nonenynylquinolizidine. Infrared spectrum indicates an unconjugated double bond and a terminal acetylene (73). 275A. C,,H3,N, 0.28, 198”C, rnlz 275(3), 274(2), 260(5), 152(100). OD. H, derivative, rnlz 279, 278, 152. Tentative structure: a l-methyl-4nonadien y lquinolizidine. 277A. ‘C,9H3sN,’-, 186”C, rnlz 277(5), 152(100). OD. H, derivative, rnlz 279. Tentative structure: a 1-methyl-4-nonenylquinolizidine. 289A. ‘C2,,H3sN,’-, 216”C, rnlz 289(2), 287(2), 274(3), 152(100). OD. H, derivative, rnlz 293, 152. Tentative structure: a 4-decadienyl- or 4-decynyl1-methylquinolizidine. There have been no studies on the biological activity of the above quinolizidine alkaloids. It is expected that they, like the 5,8-disubstituted indolizidines and other lipophilic dendrobatid alkaloids, will be noncompetitive blockers of nicotinic receptor-channels. Quinolizidine alkaloids occur widely in plants, but none with the 1,4dialkyl substituents of the amphibian alkaloids. Certain Nuphar plant alkaloids, such as deoxynupharidine, are substituted in the 4 position with a 2-fury1 moiety and in the 1 position with a methyl group (142). The 1,4-dialkyl-substituted quinolizidines apparently are unique to amphibians, occurring in dendrobatid frogs (31), ranid frogs of the genus Mantella (73), and bufonid toads of the genus Melanophryniscus (81). Their distribution in dendrobatids would appear widespread (cf. listing of above alkaloids in Ref. I), but detailed confirmation of the distribution of individual quinolizidines will require further mass and FTIR spectral studies. In the dendrobatid genus Dendrobates, quinolizidines appear likely to be major alkaloids in 12 of the 16 species examined, being absent in only D. quinqueuittatus and D. uentrimaculatus ( I ) . In the dendrobatid genus Epipedobates, quinolizidines appear likely to be significant alkaloids in only 2 of 10 species, namely, E. espinosai and E. tricolor, and appear to be absent in the other species ( I ) . In the dendrobatid genus Minyobates, quinolizidines appear likely to be major alkaloids in 5 of 9 species, while being absent in 2 species (11). In the dendrobatid genus Phyllobates, quinolizidines appear to be absent (1). A quinolizidine is a major alkaloid in the bufonid toad Melunophryniscus stelzneri, but not Melanophryniscus moreirae (81). Other quinolizidines occur as trace alkaloids in Melunophryniscus. Quinolizidines are well represented in six species of ranid

242

JOHN W . DALY E T A L .

frogs of the genus Mantella, 217A and 231A being major alkaloids in Mantella madagascariensis (73).

IV. Tricyclic Alkaloids A. GEPHYROTOXINS There are four classes of tricyclic alkaloids that have been detected in amphibians. The first tricyclic alkaloid from amphibians to be structurally defined was gephyrotoxin (287C) (58). However, there remains a controversy as to the absolute configuration of natural gephyrotoxin. The structure depicted in X is that derived by X-ray analysis (58) of a small crystal of natural gephyrotoxin, whose rotation was not determined (57), from material isolated from frogs collected in 1971. This also is the structure of d-gephyrotoxin synthesized by an unambiguous enantioselective route (143). However, the rotation of another sample of gephyrotoxin isolated from frogs of the same population of Dendrobates histrionicus collected 3 years later indicated that natural gephyrotoxin is the I enantiomer, not the d enantiomer! The rotations of synthetic d-gephyrotoxin and natural gephyrotoxin isolated from the later extracts are given in Table VIII. No satisfactory explanation for this discrepancy can be advanced. It is clear that the major natural isomer, at least in the later extract, is the 1 enantiomer; therefore, if the synthesis of d-gephyrotoxin did not involve an unexpected inversion, the X-ray analysis must have been of a crystal d-gephyrotoxin, perhaps present in only small amounts with the major

Gephyrotoxin 287C

(XI

3.

243

AMPHIBIAN ALKALOIDS

TABLE VIII PHYSICAL A N D SPECTRAL PROPERTIES OF GEPHYROTOXIN (287C) (57,143) mp: 23 1-232°C Ultraviolet (C2HSOH):A,,, 225 nm, E 8400 Optical rotation (natural): [a]; - 5 1.5" (1 .O, C2HSOHY Optical rotation (synthetic): [a]::+50.0" (1 .O, C~HSOH) "Rotation is for gephyrotoxin isolated from extracts of frogs collected in 1974 (143). Rotation was not determined on gephyrotoxin isolated from extracts of frogs collected in 1971 (57). Gephyrotoxin was referred to as HTXD in that report (57). The 1971 material was the source of a crystal whose structure was revealed by X-ray crystallography (58) to be that shown in X.

enantiomer, I-gephyrotoxin. The structures of the two known gephyrotoxins shown in Fig. 15 are based on this rather tenuous argument (see discussion in Ref. 3,while the structure X above is that shown by the original X-ray analysis. Properties of the two gephyrotoxins are tabulated below. The name gephyrotoxin was originally also applied to indolizidine 223AB,but that designation has been discontinued because the structures are not closely related. Gephyrotoxins 287C. Gephyrotoxin. C,,H,,NO, 0.20, 218"C, rnlz 287(5), 286(3), 242(100), 222(45), 122(14). 1D. H, derivative, mlz 293(5), 292(30), 250(16), 248( loo), 222(32). 0-Acetyl derivative, mlz 329(3), 264(45), 242( 100). 289B.Dihydrogephyrotoxin. C,,H,,NO, 0.25,217"C, rnlz 289(4), 288(3), 244( loo), 223(49). ID. H, derivative.

(

287C

)

HO

)

HO

FIG.15. Structures of gephyrotoxins from dendrobatid frogs. The absolute stereochemistry remains in question (see text).

I:

1400-

3

1200-

a

l

E

Y

Q,

I

1000-

I l

0

c

8

800-

a

600:

5: a

1II I aD-

400 m

200 -

:

N (u

FIG.16. Vapor-phase FTIR spectrum of gephyrotoxin (287C). The spectrum was obtained with a Hewlett-Packard Model 5965A FTIR instrument with a narrow band (4000-750 cm-') detector with a 59970 IRD ChemStation to generate the F'TlR spectra of gas chromatographic peaks. An HP-5 (bonded 5% diphenylsiloxane-95% dimethylsiloxane) fused silica capillary column (25 m x 0.32 mm) was used with a program from 100 to 280°C at IO"C/min. FTIR spectra, such as this, can be obtained routinely with less than I Fg of a compound.

3.

AMPHIBIAN ALKALOIDS

245

Further properties of gephyrotoxin are presented in Table VIII. The proton magnetic resonance spectrum of gephyrotoxin has been presented (58,143). Proton and carbon-13 assignments for gephyrotoxin have been reported (144). The vapor-phase FTIR spectrum of gephyrotoxin is shown in Fig. 16. The biological activity of gephyrotoxin has been examined in several systems (see review of Ref. 5 ) . Gephyrotoxin is relatively nontoxic; a subcutaneous dose of 80 pg in mice causes only a reduction in spontaneous activity (60). Gephyrotoxin has weak activity as a muscarinic antagonist (97) and acts as a noncompetitive blocker of nicotinic receptor-channels of muscle (145,146),electric ray electroplax (133),and pheochromocytoma cells (64). Gephyrotoxins are unique in Nature to amphibians and, indeed, occur only in certain populations of the extremely variable Colombian poison frog, Dendrobates histrionicus ( 1 ) . Gephyrotoxin might be derived by cyclization of a cis-decahydroquinoline having a trans orientation of the 2- and 8a-substituents in the piperidine ring, such as found in decahydroquinolines cis-219A and cis-243A.

B. COCCINELLINES The coccinelline class of tricyclic alkaloids was first detected from ladybug beetles (family Coccinellidae), and structures have been established for several of these insect alkaloids (147,148). Coccinelline, the archetype for the class, is an N-oxide of the alkaloid precoccinelline. Recently, precoccinelline (193C) and related tricyclic alkaloids have been detected in skin extracts of dendrobatid frogs and bufonid toads (31,81). Alkaloid 193C was identified by comparison with authentic insect-derived precoccinelline. A related tricyclic alkaloid, namely, 205B, had been isolated from extracts of the Panamanian poison frog Dendrobates pumilio (77). A tentative structure for 205B was proposed based on nuclear magnetic resonance spectral analysis. Further evaluation of this tentative structure is in progress. The mass spectra of coccinellines are not dominated by one favored fragmentation pathway and, therefore, are complex, with a large M - 1 + fragment and many hydrocarbon-like fragmentations (loss of ethyl, propyl, butyl, pentyl, etc.) (80). Precoccinelline and tricyclic alkaloids that are apparently closely related in structure to coccinellines are tabulated below. The structure of precoccinelline and a tentative structure for 205B are shown in Fig. 17.

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JOHN W . DALY ET A L .

193C 2058 Precoccinelline FIG.17. Structures of a coccinelline and a related tricyclic alkaloid from amphibians. The absolute configuration of 193C is unknown. The structure of 2058 is tentative (77) and under investigation.

Coccinellines and Related Alkaloids

191. 'CI3H2,N,'-, -, ion trap, mlz 192(43), 191(28), 163(58), 152(20), 148(30), 134(33), 120(loo), 106(33),95(50). Tentative structure: a dehydro193C (73). 193C.Precoccinelline, 'C,3H23N,'-, -, m/z 193(45), 192(95), 178(35), 164(55), 151(80), 150(100), 137(40), 136(50), 122(40), I10(15), 108(30), 96(20), 94(30), 82(20), 80(20). OD. Infrared spectrum (81). 205B. C1&3N, -, 158"C, mlz 205(38), 204(54), 190(100). OD. H,derivative. The tentative structure (Fig. 17) reported in Ref. 77 is under further study. 2075. 'C14H25N,' -, -, ion trap, mlz 207(18), 192(20), 178(28), 164(30), 152(28), 150(28), 136(100), 122(18), 110(35), 108(53),96(18). A structure is not proposed (73). 2351. C16H29N, -, -, m/z 235(63), 234(100), 220(61), 208(12), 206(21), 192(26), 178(1l), 150(20), 138(18), 136(50), 122(21), 110(20),96(20), 84(27), 70(30). OD. Infrared spectrum (81). A structure is not proposed. D 5 K . C16H29N,-,-, ion trap, mlz 235(21), 234(100), 220(7), 206(28), 192(52), 178(10), 150(60), 136(62), 122(28). Detected in ranid frog (73). Little is known of the biological activity of such alkaloids. The coccinellines do appear to serve ladybug beetles and their eggs as repellents, particularly against ants (see Ref. 149). A number of coccinellines also have been detected in the Australian soldier beetle, where they may serve in chemical defense (150). Precoccinelline(193C)and related tricyclic alkaloids have been detected only rarely in dendrobatid frogs and bufonid toads. Precoccinelline is a minor alkaloid in one Central American population of Dendrobates auratus (31), but it was a major alkaloid in an introduced population of D.

3. AMPHIBIAN

ALKALOIDS

247

auratus in Hawaii (31). The tricyclic alkaloid 205B is a trace alkaloid in one population of Dendrobates pumilio (77). Precoccinelline is a minor alkaloid in the bufonid toad, Melanophryniscus stelzneri (81).

c. CYCLOPENTA[b]QUINOLIZIDlNES A major alkaloid (251F)in the small Colombian poison frog Minyobates bombetes was unusual in exhibiting a base peak at an odd mass (mlz I I I ) in its mass spectrum (151). It was clearly unrelated to other dendrobatid alkaloids and appears to be unique to this species. The structure of alkaloid 25lF has been elucidated by nuclear magnetic resonance and FTIR spectroscopy (152); it is a cyclopenta[b]quinolizidine as shown in XI. The cyclopenta[b]quinolizidines are tabulated below. Mass, proton, and carbon-13 magnetic resonance and vapor-phase FTIR spectral data have been presented for 251F and its 0-acetate (152). The structure of 251F and tentative structures of the nine congeners also detected in Minyobates bombetes are shown in Fig. 18. Most show a base peak or major fragment at an odd mass (mlz 109, 111, 125, or 139).

Cyclopenta[b]quinolizidines 2358. fC16H29N,' -, -, mlz 235(60), 234(68), 220(28), 178(48), 164(20), 152(28), 150(32), 112(50), 1 1 1(100), 98(52), 96(40). OD. USA. fC16H2,N0,'-, -, m / z 245(20), 109(100), 108(55), 107(60), 94 (30). OD. 247. Cl&,NO, -, 175°C m / z 247(15), 110(37), 109(100). 1D. Infrared data (152). H, derivative, mlz 251, 232, I l l , 70. 249B. 'C16H2,N0,' -, -, m / z 249(18), 248(34), 222(26), 221(98), 220(100), 206(30), 192(28), 186(l3), 178(20), 172(63), 168(30), 166(30), 164(48), 152(100), 136(18), 124(15), 114(53), 1 1 1(66), 98(30). OD. Em.C,,H2,N0, 0.25, 184"C, m / z 251(54), 250(65), 236(27), 222(28), 221(30), 220(68), 194(62), 164(19), 152(35), 150(17), 112(43), 11 1(100),

"3C

251F

2498

235H

R=R'=H

245A

R'=CHO

251F

R = H , R'=OH

247

R'=CH20H

2658

R=CH,,

2798

R=C2H5, R = O H

R'=OH

H3CH*

R

H3C 3 :c *

CH3

CH3

251J

R=H

279C

R=C2HS

251F'

FIG. 18. Alkaloids of the cyclopenta[blquinolizidineclass from the dendrobatid frog, Minyobaies bombeies. The structure of 251F was determined by nuclear magnetic resonance spectroscopy (152). but the absolute configuration of 251F is unknown. The other structures are tentative and are based primarily on analogy and mass spectra.

3.

AMPHIBIAN ALKALOIDS

249

98(35). 1D. Infrared spectrum (152). H, derivative. 0-Acetyl derivative. An isomer (251F') emerges before 251F and has a nearly identical mass spectrum; it is proposed to be a diastereomer of 25lF (152). 25U. 'C,,H,,NO,' -,-, r n / z 251(92), 250(86), 236(24), 234(63), 222(23), 195(23), 194(23), 178(27), 164(28), 152(100), 150(85), 112(38), 1 1 1(82), 98(42). ID. Emerges before 251F. 265B. C,,H,,NO, 0.28, 193"C, rnlz 265(18), 264(22), 250(12), 236(17), 234(20), 194(16), 166(25), 126(30), 125(100), 112(28). ID. H, derivative. 279B. 'CI8H3,NO,' -, -, r n / z 279(85), 278( loo), 264(40), 250(45), 249(45), 248( IOO), 236(28), 233(40), 222(20), 210(50), 194(58), 180(35), 139(40), 117(42). ID. 279C. 'C,,H,,NO,' -, -, m/z 279( loo), 278(95), 264(43), 262(64), 250(21), 243(23), 236(23), 222(22), 195(22), 180(64), 140(23), 139(33), 126(28). 1D. Nothing is known of the biological activity of 251F.The alkaloid fraction from Minyobares bornbetes, containing 251F as a major alkaloid, caused severe locomotor difficulties, muscle spasms, and labored breathing, followed by minor convulsions, on injection into a mouse. However, these effects could be due to the pumiliotoxin B and congeners also present in this alkaloid fraction (151). Alkaloid 251F and its congeners have been detected from only one source, a small Colombian poison frog, Minyobares bornbetes (I).It occurs in two populations of this species (151) along with major amounts of alkaloids of the pumiliotoxin-A class, which are typical of dendrobatid frogs of the genus Minyobates. Alkaloid 251F is a major alkaloid in a population from a montane forest island, while being a trace alkaloid in a population from a streamside gallery forest (151).

D. PYRROLIZIDINE OXIMES Three novel tricyclic alkaloids (222,236,and 252A)were isolated from skin extracts of a Panamanian population of the poison frog Dendrobates purnilio. Amidine structures were proposed for these alkaloids in 1987 based on mass spectral and nuclear magnetic resonance spectral analyses (77). However, recent gas chromatographic-FTIR spectra showed that these three alkaloids could not be amidines, since they had no absorption around 1630 cm-' where amidines show an intense absorption. A reexamination of the nuclear magnetic resonance spectral data and acquisition of new data led to revised structures (153). The simplest member (222) is a spiropentanopyrrolizidine oxime, whereas 236 is the corresponding

250

JOHN W. DALY E T A L .

0-methyl oxime and 252A a hydroxy 0-methyl oxime, as shown in Fig. 19. The configuration of the 0-methyloxime is not rigorously established, but it appears to be as shown for 236 and 252A (153). Reduction of 236 with sodium in butanol afforded a primary amine, consonant with the oxime structure of 236 (153). Reduction of synthetic nitropolyzonamine (see below) yields the same primary amine (K. Hutchinson, H. M. Garraffo, T. F. Spande, and J. W. Daly, unpublished). The pyrrolizidine oximes detected from amphibians are tabulated below. The proton magnetic resonance spectra for 222,236, and 252A have been presented (77).Detailed analysis of proton and carbon- 13 nuclear magnetic resonance spectral data have been presented (153).The vapor-phase FTIR spectra for 222, 236, and 252 have been presented (153). Absorptions typical of oximes (222)o r 0-methyloximes (236,252A) are seen. The optical rotations are as follows: 236, [aID+ 55.6" (1 .O, CH,OH); 252A, [aID + 18.4" (0.47, CH,OH), -4.3" (0.47, CHCl,) (76). Pyrrolizidine Oximes

222. C13H22N20,-, 18O"C, mlz 222(1), 221(2), 112(100). 1D. Infrared spectrum (153). 236.CI4H2,N20,-, 172"C, mlz 236(12), 126(100). 1D. Infrared spectrum (153). 252A.C14H24N202, -, 179"C, mlz 252(4), 251(3), 142(100). 1D. Infrared spectrum (153). 252B. 'C14H24N202,'-, 185"C, mlz 252(1), 221(5), 126(100). 1D. A hydroxyl group appears to be located either in the spiro system or at C7 rather than at C-3 as in 252A. A minor alkaloid in a myobatrachid frog (86).

222

R=R'=H

236

RsCH,, R ' = H

252A

R=CH3, R'=OH

,,lllR

FIG. 19. Structures of pyrrolizidine oximes from dendrobatid frogs. Absolute configurations are unknown.

3.

AMPHIBIAN ALKALOIDS

25 1

Nothing is known of the biological activity of these spiropentano-pyrrolizidine oximes. A bicyclic spiropentano-pyrroline, polyzonimine from a millipede is a potent topical irritant to insects (154). The pyrrolizidine oximes are unknown in Nature except in amphibians. There are, however, two closely related natural products, nitropolyzonamine (the oxime of 236 is replaced by a p-nitro substituent in nitropolyzonamine) and its proposed bicyclic precursor polyzonimine, both of which were isolated from defensive secretions of the millipede Polyzonium rosalbum (154,155). Nitropolyzonamine, like the amphibian pyrrolizidine oximes, is dextrorotatory (155). Neither nitropolyzonamine nor polyzonimine has been detected in amphibians. At present, pyrrolizidine oximes have been detected only in certain populations of Dendrobates pumilio (1) and in the Argentine toad Melanophryniscus stelzneri (81).An apparent isomer of 252A was detected as a trace alkaloid (252B) in one population of a myobatrachid frog, Pseudophryne coriacea (86).A remarkable aspect of the occurrence of pyrrolizidine oximes in dendrobatid frogs should be noted. Pyrrolizidine oximes 236 and 252A were first detected as significant, albeit minor, components in alkaloid fractions obtained from one population of Dendrobates pumilio in 1981, and again in extracts collected in 1983, 1984, and 1986 (68 and J. W. Daly, unpublished data). Neither 236 nor 252A, however, was detectable in alkaloid fractions from the same population obtained nearly a decade earlier in 1971 and 1972.

V. Monocyclic Alkaloids The monocyclic 2,6-disubstituted piperidines have been considered as possible precursors for dendrobatid alkaloids containing piperidine rings, such as the histrionicotoxins, decahydroquinolines, and gephyrotoxins (see Ref. 5). Similarly, the monocyclic 2,5-disubstituted pyrrolidines have been considered as possible precursors for dendrobatid alkaloids containing pyrrolidine rings, such as the pumiliotoxins, the indolizidines, and now the pyrrolizidines (see Ref. 5 ) . It should be noted that 2,6disubstituted piperidines and 2,5-disubstituted pyrrolidines occur only rarely in dendrobatid frogs, while in ants they appear as major venom constituents, along with pyrrolizidines and indolizidines. It has been proposed that the monocyclic piperidines and pyrrolidines may serve as biosynthetic precursors of the bicyclic alkaloids in ants (125,134).

252

JOHN W . DALY E T A L .

A. PYRROLIDINES

As yet, the structure of only one pyrrolidine from a dendrobatid frog has been firmly established (96). This is pyrrolidine 197B, whose structure was established by comparison with synthetic trans-2-n-butyl-5-npentylpyrrolidine. The structure of 197B is shown in Fig. 20, along with tentative structures for two other dendrobatid pyrrolidines. The absolute stereochemistry of 197B was established as (2S,5S) by comparison on chiral gas chromatographic columns of the N-benzamide of 1WB with Nbenzamides of synthetic d- and I-enantiomers (156,157). The amphibian pyrrolidines are tabulated below. None have been isolated for further spectral characterization. It should be noted that Bohlmann bands in the FTIR spectra of N-methyl derivatives of 2,5-disubstituted pyrrolidines appear to be diagnostic for cis or trans configurations, with the cis derivative having a somewhat more pronounced Bohlmann band pattern than the trans (H. M. Garraffo, L. D. Simon, T. H. Jones, T. F. Spande, and J. W. Daly, unpublished). Pyrrolidines

183B. 'C12H25N,' -, 151"C, mlz 183(10), 126(100). 1D. H, derivative. 197B. CI3H2,N,0.35, 163"C, mlz 197(1), 196(2), 140(78), 126(100). 1D. H, derivative. 2232. C15H3,N,0.4, 172"C, mlz 225(1), 224(2), 168(70), 126(100). ID. H, derivative. 2,5-Disubstituted pyrrolidines from ants have insecticidal activity and, as venom constituents, presumably serve the ants in a defensive repellant function (149). Some also may have trail pheromone activity. Synthetic cislrrans-pyrrolidine 197B acted as a noncompetitive blocker of nicotinic

225c R = n-butyl, R' = n-heptyl FIG.20. Structures of pyrrolidines from dendrobatid frogs. The absolute configuration of natural frans-lWBis shown.

3. AMPHIBIAN

253

ALKALOIDS

receptor-channels in electric eel electroplax and pheochromocytoma cells

(64). The 2,5-disubstituted pyrrolidines (and pyrrolines) are well known as constituents of ant venoms, particularly ants of the myrmicine genera Solenopsis, Monomorium, and Megalomyrmex (158). More than 20 such compounds have been identified in myrmicine ants (134,149,158-161). These include the trans-pyrrolidine 197B. Incidentally, all ant pyrrolidines so far detected are of the trans configuration. A trans-Zn-butyl-5-nheptylpyrrolidine (cf. 225C) occurs in ants, but a 2,5-di-n-butylpyrrolidine (cf. 183B) has not been reported. In dendrobatid frogs, pyrrolidines have been detected only in certain populations of Dendrobates histrionicus and Dendrobates pumilio ( 1 ) . Pyrrolidine trans-197B is the only pyrrolidine to occur as a major alkaloid, and it appears only in the most northern populations of Dendrobates histrionicus. In nondendrobatid frogs, 2,5-disubstituted pyrrolidines have not been detected.

B. PIPERIDINES Simple piperidines occur only rarely in amphibians; thus far they have been found only in dendrobatids. As yet, the structure of only one piperidine from a dendrobatid frog has been firmly established (87). This is piperidine U l D , whose structure, determined to be a cis,cis-2-methyl-6n-nonyl-4-hydroxypiperidine by nuclear magnetic resonance spectroscopy, is shown in Fig. 21. A congener (255) which appears to have a keto group in the nonyl side chain also occurs in the same dendrobatid frog (87). Two other alkaloids from dendrobatid frogs appear to be 2,6-disubstituted

241D

2258

R = R' = n-pentyl

2391 R = n-butyl, R' = n-heptyl FIG.21. Structures of piperidines from dendrobatid frogs. The absolute configuration of 241D is unknown. A side-chain keto congener (255) of 241D also occurs.

254

JOHN W. DALY E T A L .

piperidines (1). The tentative structures of these two alkaloids are presented in Fig. 21. The amphibian piperidine alkaloids are tabulated below. Piperidines

225B. C,,H,,N, 0.4, 174"C, mlz 225(1), 224(1), 154(100). ID. Ha derivative. 2391. CI6H3,Nr-, 170"C, mlz 239(3), 162(40), 140(100). ID. Ha derivative. 241D. CISH3,N0,-, 188"C, mlz 241(2), 240(3), 114(100),70(62). 2D. H, derivative. 255. Cl,H,,NO2, -, 195"C, rnlz 255(3), 114(100). 2D. Ha derivative. An analog of 241D with an apparent keto group in the nonyl side chain (87). The proton magnetic resonance assignments for 241D have been preis + 39" (0.2, CH30H).The strucsented (87).The optical rotation, [a],25, ture of 241D has been confirmed by synthesis (M. W. Edwards, personal communication, 1990). None of the other amphibian piperidines has been isolated for further spectral analysis. However, Bohlmann bands in FTIR spectra will allow assignment of cis or trans configurations to such 2,6disubstituted piperidines (see Section 111,B). 2,6-Disubstitutedpiperidines (and piperideines)from ants have insecticidal activity and, as venom constituents, serve the ant in a defensive, repellant function (149). 2,6-Disubstituted piperidines are potent noncompetitive blockers of nicotinic receptor-channels in neuromuscular preparations (162) and in electric eel electroplax (163). Synthetic 241D was a potent noncompetitive blocker of nicotinic receptor-channels in both electric eel electroplax and pheochromocytoma cells (64). 2,6-Disubstituted piperidines (and piperideines) are well known as constituents of myrmicine ant venoms, particularly in fire ants of the genus Solenopsis (125,134,149,161,164).Both cis and trans isomers occur. Cisand/or trans-2-methyl-6-nonylpiperidinesare prominent ant alkaloids. These ant alkaloids have not been detected in amphibians, but the 4hydroxy piperidine analog (241D) has. Piperidines, like pyrrolidines, appear to have a very limited distribution in dendrobatid frogs. Piperidine 241D occurs as a major alkaloid in one population of Dendrobates speciosus and as a trace alkaloid in a population of Dendrobates pumilio (1). 2,6-Disubstituted piperidines 225B and 2391 have been detected as trace alkaloids in Dendrobates histrionicus (two populations) and Epipedobates triuittatus (one population), respectively (1). In nondendrobatid frogs, 2,6-disubstituted piperidines have not been detected.

3. AMPHIBIAN ALKALOIDS

255

VI. Pyridine Alkaloids

A. EPIBATIDINE A trace alkaloid from the Ecuadoran poison frog Epipedobates tricolor had potent analgetic activity and apparently represented a new class of amphibian alkaloids (60). The structure of this novel chlorine-containing alkaloid (208/210)has been determined by analysis of mass, FTIR, and nuclear magnetic resonance spectra (165). It was shown that the chlorine was not introduced artifactually during isolation. The name epibatidine has been coined for this alkaloid to reflect its origin from a frog of the dendrobatid genus Epipedobates. It has the structure exo-2-(6-chloro-3pyridyl)-7-azabicyclo[2.2.llheptane shown in XII. A key to the structure elucidation after partial purification was conversion of epibatidine to the N-acetyl derivative in order to facilitate separation from small amounts of accompanying tertiary amines, namely, allopumiliotoxins 247A and 323B. Less than 500 pg of N-acetylepibatidine was finally obtained from the skin extracts of 750 frogs. Analysis of the proton magnetic resonance spectra of N-acetylepibatidine provided the structure. Epibatidine is accompanied in extracts of E. tricolor by very trace amounts of an N-acylated 308/310 alkaloid. The properties of epibatidine, formerly termed alkaloid 208/210, and of 308/310are as follows. Epibatidines

2081210. Epibatidine, C,,H,,N,CI, 0.25, 177"C, mlz 210(4), 208( 12), 181(1),179(3), 175(5), 142(3),140(9),69( 100). ID. Infrared spectrum (165). N-Acetyl derivative. 3081310. 'Cl,H2,N2O2CI,' -, -, m/z 310(<1), 308(<1), 207(25), 169(40), 143(5), 142(8), 141(lo), 140(20),69(100). ID. This trace alkaloid appears to be an N-hydroxyacyl derivative of epibatidine.

Epibatidine 2081210

(XI11

256

JOHN W. DALY E T A L .

The proton magnetic resonance spectra and FTIR spectra of epibatidine and N-acetylepibatidine have been presented (165). The ultraviolet spectrum (CH,OH) has a maximum of 217 nm and a broad shoulder at 250280 nm (absorbance ratio 2: 1) (265). Hydrolysis of N-acetylepibatidine to epibatidine proved very difficult, proceeding in poor yield only under very rigorous conditions (165). The biological activity of epibatidine has not been studied in any detail because of the minute quantities available from the frog Epipedobates tricolor. Its presence as a trace alkaloid and its chromatographic purification were based on the Straub tail response that epibatidine causes in mice. The Straub tail response is characteristic of opiate alkaloids; however, unlike that caused by morphine and other opiates, the response caused by epibatidine was not reversed by the opiate antagonist naloxone (165). Epibatidine proved to be a potent analgetic, being manyfold more potent than morphine in the hot plate analgetic assay (165). Epibatidine had very low affinity for opioid receptors (165). Epibatidine is a unique alkaloid with a nitrogen-bridged six-membered ring system previously not known in Nature. The overall structure is reminiscent of nicotine alkaloids, while the bridged ring system is reminiscent of the tropane alkaloids. Epibatidine has been detected only in frogs of the dendrobatid genus Epipedobates (165). In E. tricolor, epibatidine occurs in variable amounts in different populations. It also has been found in one population of E. anthonyi, but not in any other populations of that frog, and at very low levels in E . espinosai and E . pictus. About 1 pg per frog occurred in certain populations of E. tricolor and in one population of E. anthonyi, while lesser amounts occurred in the other populations of E. tricolor and in populations of E . espinosai and one population of E. pictus.

B. NORANABASAMINE One other pyridine alkaloid has been detected in dendrobatid frogs. The structure of this minor alkaloid, noranabasamine (XIII), was established by proton and carbon-13 magnetic resonance spectroscopy (14). The ultraviolet spectrum was as follows:h,,, (CH,OH) 244 nm, E I 1,000, 275 nm, E 10,000. The optical rotation, [a]i5, was - 14.4" (CH,OH). Anabasamine, a plant alkaloid, also is levorotatory, but it is unknown whether noranabasamine, now given a code number 2395, has the same 2s configuration. 2395. Noranabasamine, C,5H,,N,, 0.21, 204°C mlz 239(75), 238(30), 210(25), 183(20), 182(35), 157(80), 84(100). ID. H, derivative.

3. A M P H I B I A N

ALKALOIDS

257

Noranabasamine (XIII)

The biological activity of noranabasamine has not been assessed. Anabaseine and other bipyridyl alkaloids are toxic and have nicotine-like effects (166,167). Noranabasamine apparently has not been detected elsewhere in Nature, although anabasamine and related compounds are a well-known class of plant alkaloids. Anabaseine [2-(3-pyridyl)-l,2-dehydropiperidine]is a constituent of the venom glands of myrmicine ants of the genus Aphaenogaster, where it appears to serve as an attractant to foraging ants of this genus (168). Anabaseine and related bipyridyl and tetrapyridyl alkaloids occur in marine hoplonemertine worms (166). Noranabasamine occurs as a minor or trace constituent in the three western Colombian species of Phyllobates, but not in the two Central American Phyflobates, nor has it been detected in other genera of dendrobatid frogs or in other amphibians (1).

VII. Indole Alkaloids A. PSEUDOPHRYNAMINES AND RELATEDALKALOIDS A major class of amphibian alkaloids was recently discovered in frogs of the myobatrachid genus Pseudophryne (86,119). These represent the unidentified indolic compounds first noted by Erspamer and co-workers in 1976 (169). Isolation of two of these indole alkaloids from partially purified extracts of Pseudophryne coriacea allowed structure elucidation of pseudophrynaminol (XIV)and pseudophrynamine A (XV) by nuclear magnetic resonance spectral analysis (119). Methoxide cleavage of pseudophrynamine A yielded pseudophrynaminol and a methyl ester

258

JOHN W . DALY E T A L . 0

I

I

I

I

H

CH,

CH,

H

Pseudophrynamine A 512 <

;

C H @ ,

N Q

I

H

t

CH,

Pseudophrynarninol 258

(XIV)

(XV) QN;co/cH3

I

H

I

CH,

286A

(XVU

identical with another pseudophrynamine alkaloid, namely, 286A (XVI ). Pseudophrynamine 286A could be an artifact derived from methanolysis during extraction of a labile pseudophrynamine ester, but it does not appear to have formed by methanolysis of pseudophrynamine A (119). The strong negative extremes at 242 and 295 nm in the CD spectra of pseudophrynaminol (XIV) suggest the same absolute configuration as in I-physostigmine. The structures of other alkaloids of the pseudophrynamine class (3aprenylpyrrolo[2,3-b]indoles)were deduced from mass spectral analysis (86). The pseudophrynamines are tabulated below. In addition to the listed pseudophrynamines, other trace analogs of pseudophrynamine A (XV) with molecular ions at m/z 526,540, and 542 were detected. The structures of the alkaloids of the pseudophrynamine class from frogs of the genus Pseudophryne are shown in Fig. 22.

3.

258

259

AMPHIBIAN ALKALOIDS

R=CH,OH

302

286A R = COZCH3

'i' H

256

R=CHO

RB'

CH,

I

I

H

CH,

R=H, R'=OH

316

R = H , R'=OCH3

332

R = O H , R'=OCH3

346A R = R' = OCH3

2728 R

528 R

I

300

R=H

330

R=OCH3

CH,

FIG.22. Structures of pseudophrynamines from myobatrachid frogs of the genus Pseudophryne. Structures of 258, %A, and 5U were determined by nuclear magnetic resonance spectroscopy ( 1 19). Structures of the other pseudophrynamines are tentative. The absolute configuration of 258 is uncertain but is presumably the same as I-physostigmine (119) as shown in structure XIV.

Pseudophrynamines

256. 'C16H20N20r' -, 204"C, m/z 256(22), 228(10), 227( lo), 199( lo), 185(28), 173(100), 171(20), 156( lo), 144(28), 130(95), 110(25), 109(30). 1D. Infrared data (86). 0.30,206"C, m/z 258(25), 183 18), 258. Pseudophrynaminol, CI6H2,NZO, 173(100), 130(90). 2D. Infrared data (86) and in CHCl, (119). 272A. 'C1,Ht4N20,'-, 204"C, mlz 272(50), 2 5 3 12), 199(27), 187(80), 144(loo), 143(23), 130( 18). 1D. Tentative structure: an N8-methyl derivative of 258. 272B. 'C,6H20N20Z,' -, 208"C, m/z 272(50), 255(15), 199(33), 187(54), 144(100), 143(25). 2D.

260

JOHN W . DALY ET A L .

286A. C,7H22N202, 0.53, 21 1"C, mlz 286(38), 199(20), 185(32), 173(100), 157(62), 156(52), 130(97). ID. Infrared data (86) and in CHCI, (119). 286B. 'C17H22N202,' -, 211"C, mlz 286(<1), 199(100), 173(7), 156(10), 130(7),70(50).OD. Infrared data (86). Tentative structure: an unconjugated double bond isomer of 286A. 300. 'C17H20N203rf -, 219"C, mlz 300(100), 269(23), 241(40), 240(43), 225(10), 213(20), 198(16), 187(47), 185(30), l70( l9), 159(25), 106(34). OD. A trace isomer [mlz 300(78), 269(22), 195(20), 187(100), 144(75), 122(25), 85(57); OD] also occurs, but rarely. The structure (Fig. 22) of 300 was shown incorrectly in Ref. 86. 302. C,7H22N203,-, 234"C, mlz 302(66), 300(24), 190(35), 189(IOO), 173(20), 146(30). 2D. A trace isomer 302' with only one exchangeable hydrogen also occurs. 316. 'C,8H24N203,'-, 232"C, m / z 316(90), 215(22), 203( loo), 188(37), 174(16), 160(85), 146(18). ID. 330. CISH22N204, 0.61, 237"C, mlz (direct probe) 300( IOO), 302(45), 296(90), 282(32), 243(32), 217(30), 215(30), 199(25),189(87).OD. Red color. 332. C18N24N204, -, 239"C, m/z 332(40), 282(10), 219(100), 217(30), 189(25), 176(35), 161(15). 2D. Infrared data (86). Two isomers were detected by gas chromatography (86). 346A.'C19H26N204r' -, 235°C mlz 346(60), 233( loo), 218(22), 190(60), 17325). ID. 512. Pseudophrynamine A, C32H40N402, 0.38, - (not detected by gas chromatography), rnlz (direct probe) 5 12(56), 456(23), 455( I3), 340(55), 338(100), 273(20), 241(40), 21 1(17), 197-199(20-23), 182-185(22-25), 173(80), 172(60), 144(20), 130(38). 2D. Infrared data in CHCI, (119). 524. C3,H4,N4O2,-, - (not detected by gas chromatography), m/z (direct probe) 524. OD. Tentative structure: an N8-methyl-dehydro-derivative of 512. 528. 'C32H40N403,f -, - (not detected by gas chromatography), m/z (direct probe) 528( lo), 472(5), 356(15), 354(25), 173(100), 130(95). 3D. Proton and carbon- 13 assignments for pseudophrynamines 258, 286A, and 512 have been reported, and the proton magnetic resonance spectrum of pseudophrynamine A (512)has been presented (119). The ultraviolet spectra of 258 and 286A with maxima at about 240 and 295 nm have been reported (119). Hydrogenation, while useful in characterizing many amphibian alkaloids, gave ambiguous results because of hydrogenolysis when applied to the pseudophrynamine 258 (86). One other indole alkaloid, reminiscent in structure of the pseudophrynamines, has been isolated from amphibians, namely, chimonanthine (14) (XVII ). Chimonanthine was accompanied in the dendrobatid poison-dart

3. AMPHIBIAN CH3

I

I

H

ALKALOIDS

26 1

H

I

I

CH,

H3C

H

d-Chimonanthine 3468

Kalycanthine 346C

(XVII)

(XVIII)

frog Phyllobates terribilis by calycanthine (14) (XVIII),another “dimeric” alkaloid that in plants is formed by rearrangement of chimonanthine (170). The structures were established from proton and carbon- 13 magnetic resonance spectra (14). The code numbers 346B and 346C are assigned to these “dendrobatid alkaloids.” Remarkably, the chimonanthine and calycanthine from the dendrobatid frog were the optical enantiomers of the corresponding plant alkaloids: frog chimonanthine, [ ( Y I +280” ~ ~ (CH,OH); frog calycanthine, [a]i5-570” (CH,OH). Dimeric “Indole” Alkaloids

346B. d-Chimonanthine, C22H26N4, 0.18, 192”C, m / z 346, 173(100). 346c. I-Calycanthine, C22H26N4, 0.18, 195”C,m / z 346, 173(100). The biological properties of pseudophrynamines have not been explored. The structures are reminiscent of physostigmine, which is a potent inhibitor of cholinesterases. The pseudophrynamines have not been detected elsewhere in Nature. In myobatrachid frogs of the genus Pseudophryne, they occur in varying amounts in all species examined (86). The pair of dimeric “indole” alkaloids, chimonanthine/calycanthine,have been detected in amphibians only in the dendrobatid frog Phyllobates terribilis and, tentatively, as a trace alkaloid in Phyllobates bicolor (14).

B. INDOLE AMINES Many frogs and toads contain high levels of N- and 0-methylated amines derived from the indolic biogenic amines serotonin and tryptamine, as well as other amines, such as the tyramines and catecholamines. Such simple

262

JOHN W. DALY ET AL.

methylated derivatives can be considered alkaloids, but they will not be treated as such in this review (see Refs. 1 and 5 for references, structures, and tabulation of occurrence in amphibians).

C. DEHYDROBUFOTENINE An unusual derivative of serotonin, dehydrobufotenine (XIX), occur in large amounts in parotoid glands of the bufonid toad Bufo marinus. Analysis of a proton magnetic resonance spectra led to the correct zwitterionic structure in 1961 (171,172). Dehydrobufotenine is moderately toxic, with a lethal dose in mice on subcutaneous injection being about 120 pg (173). Death occurs with clonic convulsions. The pharmacological activity has not been investigated. Dehydrobufotenine occurs in several species of bufonid toads of the genus Bufo (174), but it has not been detected elsewhere in Nature. The 0-sulfate (bufothionine)also occurs in the toad Bufo marinus.

H

Dehydrobufotenine (XIX)

D. TRYPARGINE Trypargine represents another class of indole alkaloids from amphibians. Trypargine was isolated from a hyperolid frog (175). Structure XX was defined from chemical and spectral properties (175) and confirmed by synthesis (176). Trypargine is moderately toxic, causing paralysis, respiratory failure, and death in mice at an intravenous dose of about 200 p g (175). The pharmacological activities of trypargine do not appear to have been reported. Trypargine was first isolated from Kassina senegalensis (175). Trypargine, or a closely related compound, was stated to occur in large amounts in another hyperolid frog, Kassina macufata (formerly Hylambates maculatus).

3.

263

AMPHIBIAN ALKALOIDS

Trypargine

H

(XX)

VIII. Imidazole Alkaloids Many frogs contain high levels of N-methylhistamine and NNdimethylhistamine in their skin. Although such simple methylated derivatives can be considered alkaloids, they will not be treated as such in this review (see Refs. 1 and 5 for structures and tabulation of occurrence in amphibians). Spinceamine (XXI ) and 6-methylspinceamine (XXII ) occur in amphibians (1 77-180) and represent cyclized analogs of histamine and N-methylhistamine, respectively. The spinceamines readily form by the reaction of formaldehyde with histamine or N-methylhistamine (see Ref. 5 ) . Spinceamine has no histamine-like activity in a variety of systems (181,182) but does have bacteriostatic activity (183). Spinceamines have been reported from frogs of the leptodactylid genus Leptodactylus and from frogs of the hylid genera Litoria and Nictimystes (177-180).

f i \

Spinceamine

R=H

(XXI) 6-Methylspinceamine

R =C H ~

(XXII)

IX. Morphine Morphine (XXIII), a well-known plant alkaloid, has been detected in trace amounts in the skin of the bufonid toad, Bufo morinus (184). It is possible that this, and perhaps other trace alkaloids found in amphibian

264

JOHN W. DALY E T A L .

Morphine

(XXIII)

skin, originate via sources in the food chain of plant + insect + amphibian. Some frogs have efficient mechanisms for concentrating dietary alkaloids into skin (see Section XII).

X. Guanidinium Alkaloids The highly toxic guanidinium alkaloid tetrodotoxin was first isolated from the Japanese puffer fish, Fuga rubipes. In the early 1960s, a guanidinium alkaloid was isolated from eggs of the California newt Taricha torosa and named tarichatoxin (185). It proved identical with tetrodotoxin from puffer fish (186; see Ref. 5 for a historical review). Tetrodotoxin and other guanidinium toxins, namely, chiriquitoxin and the zetekitoxins, have been shown to occur in other amphibians. A. TETRODOTOXIN In 1964, the structure of tetrodotoxin (XXIV)was finally elucidated by analysis of a variety of chemical, X-ray crystallographic, and spectral data (187-190). One key reaction was the facile conversion to 2-amino6-hydroxymethyl-8-hydroxyquinazoline (XXV).Natural tetrodotoxin is levorotatory, with an [a]& of -8.5"(3.3, D,O). Tetrodotoxin is accompanied in certain salamanders and newts of the family Salamandridae by 4,9and 1 l-deoxyanhydrotetrodotoxin, 4-epitetrodotoxin,6-epitetrodotoxin, tetrodotoxin, as well as trace amounts of 4,9-anhydro-4-epitetrodotoxin, 4,9-anhydro-6-epitetrodotoxin,4,9-anhydro- 1 1-deoxytetrodotoxin, and 1 1-deoxy-9-epitetrodotoxin (191-193). An 1 1-nortetrodotoxin-(6R)-olhas been isolated from the puffer fish Fuga niphlobles (194), but apparently has not been detected in amphibians.

3. AMPHIBIAN

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265

0 -

CTetrodotoxin

(XXIV)

OH

I

Tetrodotoxin is an extremely toxic substance: the LD,, dose on intraperitoneal injection in mice is about 0.2 pg. Structural modifications of tetrodotoxin result in marked reductions in toxicity (195). Among the congeners of tetrodotoxin that occur in Nature, 4-epitetrodotoxin,6-epitetrodotoxin, and 1 1-deoxytetrodotoxinare very toxic, but all are at least 7fold less toxic than tetrodotoxin (191,193). 4,9-Anhydrotetrodotoxin is virtually inactive (195). The toxicity and pharmacological actions of tetrodotoxin are caused by blockade of voltage-dependent sodium channels in nerve and muscle (for a review, see Ref. 196). Tetrodotoxin has no antibiotic activity. Newts and salamanders of the salamandrid genera Taricha, Cynops, and Notophthalamus that contain tetrodotoxin are resistant to its toxic and pharmacological effects (see Ref. 5 and references therein). However, the newt Ambystoma tigrinurn of the family Ambystomatidae is known to contain tetrodotoxin (192), and embryos of this newt are sensitive to tetrodotoxin (197). Tetrodotoxin has a wide distribution among marine organisms, and a bacterial origin has been postulated (198). In the octopus Hapalochhenu

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maculosa, tetrodotoxin occurs in the venom gland and apparently serves as a venom (199) rather than as a passive poison. Tetrodotoxin occurs both in tailed (order Caudata) and tailless (order Anura) amphibians. In the order Caudata, tetrodotoxin and congeners have been detected in newts and salamanders of the family Salamandridae, namely, in species of the genera Taricha, Cynops, Triturus, Notophthalmus, and Paramesotriton (see Refs. 5,191,192,200 and references therein). One species (Salamandra salamandra) of this family apparently does not contain tetrodotoxin (200), but rather samandarines (see Section 11,B). Tetrodotoxin occurs in the eggs, skin, muscles, ovaries, and blood of newts of the genus Taricha (190,200). In an early study, tetrodotoxin was not detected in species from six other families of Caudata (201). However, recently, tetrodotoxin and 6-epitetrodotoxin were identified in the newt Ambystoma tigrinum of the family Ambystomatidae (192). In the same early study, tetrodotoxin was not detected in species from four families of the order Anura, namely, Bufonidae ( B u f i boreus), Hylidae (Hyla cinerea), Ranidae ( R a m pipiens), and Pipidae (Xenopus laeuis) (201). Tetrodotoxin has been found, however, in certain species of the neotropical bufonid genus Atelopus. Tetrodotoxin was identified in skin extracts of two subspecies of Central American Atelopus uarius, namely, A . uarius uarius and A . varius ambulatorius, and, along with a congener, chiriquitoxin (see Section X,B), in A . chiriquiensis (202). Tetrodotoxin and chiriquitoxin also cooccurred in eggs of A . chiriquiensis (203). Tetrodotoxin was not detected in A . zeteki, where instead another guanidinium alkaloid, atelopidtoxin, now referred to as zetekitoxin (see Section X,C), was the major guanidinium toxin (204,205). Whether the toxin in A . cruciger (204) is tetrodotoxin or zetekitoxin is unknown. Only low levels of toxin occurred in A . planispinu (204), and its nature is unknown. It should be noted that atelopid frogs also contain toxic bufadienolides (206), which would contribute to the toxicity of the extracts. Recent studies indicate that tetrodotoxin does occur as a minor toxin in extracts from A . zeteki and A . ignescens and as a major toxin in A . spurelli (207). In addition, the watersoluble toxin reported to be present in skin extracts from the dendrobatid frog Colostethus inguinalis (207) has been shown to be tetrodotoxin (208). 4-Epitetrodotoxin and 4,9-anhydrotetrodotoxin also were present in this dendrobatid frog. A guanidinium toxin that appears in several chromatographic systems to be identical with tetrodotoxin has been reported from the toad Brachycephalus ephippium of the family Brachycephalidae (209). The biological activity appeared similar to that of tetrodotoxin, but it was judged to be less potent. The name ephippiotoxin was coined, but the toxin may prove to be identical to tetrodotoxin.

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Inhibition of binding of [3H]saxitoxin to sodium channels in brain membranes has been used as a screen for tetrodotoxin-like compounds in skin extracts of frogs and toads (208). Inhibitory activity was high in bufonid toads of the genus Atelopus (A.ignescens, A. spurelli, A. zeteki) and in the dendrobatid frog Colostethus inguinafis, whereas tetrodotoxins appeared absent in other bufonid toads (Melanophryniscus moreirae, Dendrophryniscus minutus, Bufo regularis) and other dendrobatid frogs (Colostethus herminae, Colostethus riueroi, Aromabates nocturnus, Dendrobates pumilio, Phyllobates bicolor). Binding assays indicated the apparent absence of tetrodotoxin in skin extracts from a wide range of anurans: Hylidae (Cyclorana australis, Hemiphractus fasciatus, Litoria albuguttata, Nictomystes tympanocrystis, Osteocephalus taurinus, Phrynohyas uenulosa), Leptodactylidae (Eleutherodactylus gollmeri), Microhylidae (Scaphiophryne marmorata, Otophryne robusta, Phrynomerus bifaciatus), Myobatrachidae (Heleioporus albopunctatus, Notaden nichollis, Pseudophryne corroboree), and Ranidae (Mantella aurantiaca, Rana rugulosa, Rana septentrionalis) (208). Nothing is known of the biosynthesis of tetrodotoxin, although the involvement of arginine and an isoprenoid has been postulated (1 91). Radiolabeled acetate, arginine, citrulline, or glucose were not incorporated into the tetrodotoxin of newts of the genus Taricha (210). B. CHIRIQUITOXIN Chiriquitoxin is the major guanidinium toxin of the bufonid frog Atelopus chiriquiensis (202,203,21I ) . Structure XXVI has been elucidated by analysis of the nuclear magnetic resonance spectra of chiriquitoxin and its 0-

Chiriquitoxin

(XXVI)

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JOHN W . DALY E T A L .

6,13-lactone (212). Chiriquitoxin has the 11-CH,OH group of tetrodotoxin replaced by an erythro-(1lR, 12s)-CHOHCHNH,COOH group. The proton resonance spectrum of chiriquitoxin has been presented (203). Chiriquitoxin is nearly as toxic as tetrodotoxin, having an LD,, in mice of about 0.3 pg. Pharmacologically,chiriquitoxin, like tetrodotoxin, blocks voltage-dependent sodium channels, but in addition it apparently also blocks potassium channels (211,213). The effects of chiriquitoxin on potassium currents were prevented by tetrodotoxin. Chiriquitoxin has been reported only from the skin of one species, Atelopus chiriquiensis, where it occurs with tetrodotoxin in a ratio of 7 parts chiriquitoxin to 3 parts tetrodotoxin (202). Both toxins also occur in eggs (203). Further studies will be required to determine whether this unusual congener of tetrodotoxin occurs elsewhere in Nature. C. ZETEKITOXIN

Another guanidinium alkaloid was detected in skin extracts from the Panamanian golden frog Atelopus zeteki, where it was first termed atelopidtoxin (204). The name was later changed to zetekitoxin (205) to indicate that it had been detected only from this species of Atefopus. The infrared spectrum was stated (214) as showing strong peaks at 3000-3600 cm-' (NH,OH), multiple peaks from 1540 to 1680 cm-', and broad absorption from 1000 to 1250 cm-' (carbon-oxygen bonds). The "region through 1500 cm-'" was stated to be similar to that of saxitoxin. At least two toxins were present that could be separated by electrophoresis (205).The major one was termed zetekitoxin AB, since it was uncertain whether it consisted of two very similar toxins. The minor one was termed zetekitoxin C. The major toxin(s) had the same electrophoretic mobility as tetrodotoxin, and the minor toxin, a slightly higher mobility. It was suggested that zetekitoxin AB was probably zwitterionic at pH 7. Gel-exclusion chromatography suggested a molecular weight of 500 k 100. A californium plasma desorption mass spectrum was inconclusive, with ions detected at mlz 496,411,407,393, 323, and 281. Under these conditions, tetrodotoxin exhibited a protonated parent ion at mlz 320, and chiriquitoxin, a protonated parent ion at mlz 392. An infrared spectrum of zetekitoxin AB was apparently similar to that reported previously for the mixture of zetekitoxins (see above). A strong triple peak between 1600 and 1700 cm-' would be consonant with the guanidinium group that was detected by the Weber test. A shoulder at 1760 cm-' was interpreted as possibly due to a lactone. The peaks of the fingerprint region for zetekitoxin AB were listed. It was stated that virtually all the proton resonances (3.8-6 ppm) were in the region associated with protons attached to carbons bearing a hetero atom. The coupled doublets at approximately 5.9 and

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2.8 ppm, assigned to the 4 and 4a protons of tetrodotoxin, respectively, were absent. The structure of zetekitoxin remains obscure, but it seems likely that it will prove to be related to the tetrodotoxin/chiriquitoxin present in other species of Atelopus. Zetekitoxins are very toxic compounds, causing convulsions and, like tetrodotoxin, locomotor difficulties, hypotension, and respiratory failure (200,205).Zetekitoxins cause terminal ventricular fibrillation in dogs (205). Zetekitoxin AB is about as toxic as tetrodotoxin in mice, whereas zetekitoxin C is about 8-fold less toxic (205). The pharmacological effects of zetekitoxin (atelopidtoxin) do appear to differ from those of tetrodotoxin, in particular with respect to “bizarre” cardiac rhythms and to a very low activity in blockade of conduction in sciatic nerves (204,205,215). Zetekitoxin has no effect on spontaneously beating chick cardiocytes (216). Zetekitoxin does block the action of veratridine in brain slices, a property it shares with tetrodotoxin (217). The effects of zetekitoxin in certain preparations (205) were suggested to be reminiscent of those of another guanidinium compound, guanethidine, an agent that blocks postganglionic adrenergic neurons by interfering with release of norepinephrine. In an early publication it was stated that the toxins in Atelopus varius varius, A . varius ambulatorius, and A . cruciger elicited “symptoms identical with those produced by atelopidtoxin” (204). Later the major toxin in the two Atelopus varius subspecies was shown to be tetrodotoxin (202). Zetekitoxin has been reported from only one atelopid species, Atelopus zeteki. Tetrodotoxin and chiriquitoxin, if present, were stated to be at levels less than 3% of the zetekitoxin (202). Tetrodotoxin has now been shown to be present in skin extracts of A . zeteki by a more sensitive assay (M. Yotsu and T. Yasumoto, personal communication, 1992). Further studies are needed to determine if zetekitoxin is present in other species of Atelopus. Alternative sources of zetekitoxin would be desirable since A. zeteki has been declared an endangered species in spite of its abundance in some montane areas of Panama (see Ref. 5 ) .

XI. Other Alkaloids There remain a large number of alkaloids, usually only trace constituents in alkaloid fractions from anuran skin extracts, that have been characterized only by gas chromatography-mass spectrometry (see Appendix for methodology). Most of these are from dendrobatid frogs, and many will undoubtedly prove to be members of “izidine” (pyrrolizidine, indolizid-

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JOHN W . DALY E T A L .

ine, quinolizidine) or decahydroquinoline classes of amphibian alkaloids. Some may prove to be related to the pumiliotoxin-A class. Unfortunately, many of the trace alkaloids are no longer detectable in alkaloid fractions that have been stored for years or even decades, and thus FTIR data cannot be obtained. Certain alkaloids seem likely to represent new classes. One apparently new class of alkaloids has been detected only in Madagascan ranid frogs of the genus Mantella (72,73). The major alkaloid (235C)has a molecular formula of CI5H2,NO,one exchangeable hydrogen (hydroxyl), and reduces to a tetrahydro derivative whose mass spectrum and infrared Bohlmann bands resemble those of a quinolizidine (73). The mass spectrum has a base peak of C,,H,,N+ (mlz 162). Certain properties of 235C were analogous to those of the pumiliotoxin-A class alkaloids, which suggested that 235C might be a dehydro analog of a homopumiliotoxin. A tentative formulation for 235C is shown in XXVII.There are several congeners that occur in frogs of the genus Mantella, and their properties are as follows.

221F. 'CI4H2,NO,'-,-, ion trap, mlz 221(<1), 220(40), 176(10), 162(100), 160(40), 148(18), 134(42), 120(38), 91(35). A lower homolog of 235c. 233F. CI5H2,NO,--,--, ion trap, mlz 233(<1), 162(100), 160(60), 120(40). Infrared data (73). A ketone related to 235C. 235C. C15H2,N0,--, 166"C, mlz 235(28), 234(53), 220(20), 162(100), 160(40). 1D. Infrared spectrum (73). H2 and H4 derivatives. 0-Acetyl derivative. Two further isomers occur (73). 2516. C,,H2,N02,--, 178"C, mlz 251(26), 250(45), 162(loo), 160(40). 2D. H2derivative. Reduction may have been incomplete. Tentative structure: a congener of 235C with an additional side-chain hydroxyl group. 265F. Cl6H2,No2,-,-, ion trap, m/z 265(5), 206(18), 194(100), 192(40), 166(10), 148(15), 136(37), 134(50), 127(37). Infrared data (73). H, derivative. 0-Acetyl derivative. A structure is not proposed (73). Isolation of sufficient 235C for nuclear magnetic resonance spectral analysis will be necessary to confirm the proposed structure. Alkaloid 235C is a minor alkaloid in Mantella aurantiaca and Mantella crocea (73). A second group of alkaloids that may represent a distinct new class is comprised of two isomeric alkaloids 283B and 283C (5). The molecular formula for both is C17H,,N02.Both have one exchangeable hydrogen (hydroxyl), and because neither is reduced catalytically, each has two rings. Mass spectral analysis (5) indicated that alkaloid 283B loses a C5H,, side chain to afford a major fragment ion at mlz 212 (C,2H22N02+), while the base peak is at mlz 140 (C,H,,N+). Alkaloid 283C loses a C4H9side

3. AMPHIBIAN

ALKALOIDS

27 1

235C Tentative structure

(XXVII)

chain to afford a major fragment ion at mlz 226 (C,3H24N02+), while the base peak is at mlz 126 (C,H,,N+). The properties are as follows.

283B. 152(lo), 283C. 224( lo),

CI7H3,NO,, 0.36, 197°C rnlz 283(<1), 282(1), 254(2), 212(40), 140(100). ID. H, derivative. 0-Acetyl derivative. CI7H3,NO2,0.40, 195°C mlz 283(<1), 282(1), 240(5), 226(28), 166(60), 126(100). 1D. H, derivative. 0-Acetyl derivative.

At present there are not sufficient data to formulate structures for 283B and 283C, which were major alkaloids in one population of Dendrobates histrionicus, and in a sympatric sister species, Dendrobates occultator (60). Nearly 70 unclassified alkaloids are tabulated below, along with the correction of an earlier erroneous identification of a contaminant as alkaloid 265A. All were trace alkaloids unless otherwise noted. Empirical formulas within single quotes are tentative. For the occurrence of these alkaloids in dendrobatid frogs, see Ref. 1 . The listing is updated from that in Ref. 1 to reflect addition or removal of alkaloids that subsequently have been detected, identified, or classified. Unclassified Alkaloids

151. ‘CIOHI7N,’-, 152”C, rnlz 151(100), 150(25). OD. l53B. ‘CIOHI9N,’--, 151”C, mlz 153(45), 152(100). OD. H, derivative. 161. C&IllN3,-,-, ion trap, rnlz 161(76), 160(100), 133(10), 107(22). OD. Infrared data (73).An aromatic system was proposed (73). 167C. CIIHZIN,-, 152”C, rnlz 167(100), 166(55). OD. H, derivative. 181C. ‘Cl2HZ3N,’-, 153”C, mlz 181(100), 180(68). OD. H, derivative. 183A. CH2,N,--, 154”C, rnlz 183(3), 154(100). 1D. H, derivative. A possible structure would be a 2-ethyl 3-, 4-, or 5-pentylpiperidine. A minor alkaloid in Dendrobates speciosus. 185. ‘C,lH,3N0,’ 0.2, 153”C, rnlz 185(1), 170(100). 1D. H, derivative. 193A. Cl3HZ3N,--, 152”C, mlz 193(100), 192(65). OD. H, derivative.

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193B. ‘CI3H2,N,’-, 158”C,rnlz 193(23), 192(13), 150(100).OD. H,derivative. A major alkaloid in one population of Dendrobates pumilio. A minor alkaloid in a population of Dendrobates auratus. 195C. C,,H,,N,--, 153”C,mlz 195(65), 152(100).OD. Infrared data (73). H, derivative. Alkaloid 195C may prove to represent different compounds in different extracts. Major alkaloid in one population of Dendrobates speciosus; minor alkaloid in certain populations of D. leucomelus, D. pumilio, and D . speciosus and in D . reticulatus and Minyobates steyermurki. Detected in a ranid frog (73). 195E. ‘CI3H2,N,’-, 158”C, rnlz 195(45), 194(100). OD. H, derivative. 197A. ‘C,,H,,NO,’--, 160”C, rnlz 197(1), 180(100), 126(35). 1D. H, derivative. 201. ‘C14H19N,’-, 167”C, rnlz 201(<1), 200(2), 136(100). OD. 203B. C,,H,,N,--, 159”C, mlz 203(<1), 166(100). OD. 207D. ‘CI4H2,N,’-, 159”C, rnlz 207(58), 180(loo), OD. H, derivative, mlz 209, 180. 207E. ‘CI4H2,N,’-, 158”C, rnlz 207(12), 164(loo), 84(35). OD. 207F. ‘CI4H2,N,’-, 157”C, rnlz 207(7), 192(100). OD. H, derivative, mlz 209, 194. 209A. ‘C,3H,,N0,’--, 162”C, rnlz 209(5), 168(100). H, derivative, rnlz 211, 168. 211B. ‘C13H2,N0,’--, 168”C,rnlz 21 1(4), 160(100). 1D. 2196. ‘Cl5H2,N,’-,-, ion trap, mlz 220(20), 164(100),162(46),120(30). OD. Infrared data (81). H4 derivative. 221B.‘C,,H2,N0,’--, 173”C,mlz 221(2), 192(100). 1D. H, derivative. 221E. ‘CISH27N,’-,-r ion trap, rnlz 222(45), 220(8), 152(loo), 148(28), 134(10). OD. Detected in a bufonid toad (81). 2233. ‘C,,H,,NO,’--, 163”C,rnlz 223(2), 222(3), 168(100). 1D. H, derivative, mlz 225, 168. 225A. ‘C,4H27N0,’--, 164”C, mlz 225(3), 224(6), 208(2), 168(loo), 152(25). ID. H, derivative. A minor alkaloid in Dendrobates speciosus. 231D. ‘CI5H,,NO,’--, 168”C, m / z 231(1), 154(100). 1D. H, derivative. WIF. ‘CI,~H~~N,’-, 164”C, m / z 231( 1), 180(100). OD. H,j derivative. 233B. ‘C,,H,,NO,’-, 180”C, rnlz 233(<1), 168(100). ID. A minor alkaloid in Minyobates opistomelas. 233C. ‘C,,H,,NO,’-, 173”C,rnlz 233(1), 192(60), 96(100). 1D. 235D. ‘C,5H2,N0,’--, 182”C, rnlz 235(<1), 196(20), 170(100). 1D. H6 derivative. A minor alkaloid in Minyobates new species (Panama). DSF. ‘ C I ~ H ~ ~ N , ’170”C, -, mlz 235(5), 234(3), 166(36), 138(100). OD. 235G. ‘C16H29N,’-, 172°C m / z 235(2), 206( loo), 194(65). 237E. ‘C,,H27N0,’0.25, 180”C,rnlz 237(1), 236(3), 208(70), 152(100).H, derivative, mlz 239, 152.

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239A. ‘C,,H,,NO,’--, 178”C, rnlz 239(2), 238(3), 182(100). H, derivative. An isomer 239A’ has been detected at 174°C. 239B. ‘CISH2,N0,’--, 178”C,rnlz 239(2), 238(3), 180(100). ID. H, derivative. A minor isomer in Minyobates bombetes. A trace isomer 239B’ has been detected at 174°C. 239C. ‘C,,H2,N0,’--, 179”C,mlz 239(2),238(3), 196(100). H, derivative. An isomer 239C’ has been detected at 174°C. 239D. T,,H2,N0,’--, 179’32, rnlz 239(2), 238(3), 166(100). H, derivative. An isomer 239D‘ has been detected at 174°C. 2393. ‘C,,H,,NO,’--, 176”C,rnlz 239(2), 238(3), 210(40), 152(100). 1D. H, derivative. 239F. ‘C,,H,,NO,’ 0.30, 176°C rnlz 239(1), 168(100). ID. H, derivative. 0-Acetyl derivative. 241A. ‘CI4H2,NO2,’-, 180”C, rnlz 241(2), 240(3), 166(loo), 126(48). 241B. C,,H,,N,--, 167”C,rnlz 241(15), 125(45),58( 100). OD. H, derivative. Detected as a major alkaloid in Mantella madagascariensis obtained through the pet trade (72), but not in wild-caught M . madagascariensis (73). The empirical formula and mass spectrum suggest an aliphatic acyclic tertiary amine. Aliphatic acyclic amines have been reported in certain ponerine ants of the genus Mesoponera (2f8). 241C. ‘C14H2,N02,’--, 177”C,mlz 241(1), 152(100).A minor alkaloid in one population of Dendrobates pumilio. 241E. ‘CI4H2,NO2,’-, 174”C, rnlz 241(3), 222(62), 152(100). 2D. 251C. ‘C&,,NO,’--, 190”C,mlz 251(2), 234(4), 154(100).1D. H, derivative, rnlz 253, 154. A minor alkaloid in one population of Minyobates minutus. 251E. ‘C,,H,,NO,’--, 175”C,mlz 251(3), 250( l), 234(2), 168(30),84(18), 70( 100). A minor alkaloid in one population of Minyobates minutus. 251H. ‘C,,H2,NO,’--, 170°C, mlz 251( l), 178(30), 150(100). 253C. ‘C,,H,lNO,’--, 175”C,m / z 253(3), 192(100). ID. 257A. ‘C,,H2,N,’ 0.30, 188”C, mlz 257(1), 256(2), 216(100). H, derivative, rnlz 265, 222. 257B. ‘C,,H,,N,’ 0.35, 192”C, rnlz 257(60), 256( loo), 152(20). ID. 257D. ‘c18H2,N,’-, 190”C,rnlz 257(5), 256(3), 190(100). OD. H, derivative. A minor alkaloid in two populations of Dendrobates pumilio. 263B. ‘c18H33N,’-, 170”C, mlz 263(3), 198(100). OD. 265A. Nonalkaloidal, 0.35, 198”C, mlz 265(50), 264( loo), 222(58), 180(72). This proved to be octadecenoic acid methyl ester with a true parent ion of rnlz 296. Fatty acid methyl esters do not afford a major protonated parent ion with NH, chemical ionization-mass spectrometry. Such fatty acid methyl esters frequently represent trace contaminants of alkaloid fractions.

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265C. CI7H3,NO,-, 182”C,mlz 2 6 3 14), 236( 10) 210(C13H,,NO+, loo), 192(10), 138(21),84(C5HloN+, 45). 1D. H, derivative. A minor alkaloid in Dendrobates new species (Panama). 267B. ‘C16H29N02,’-, 208”C, mlz 267(7), 266(4), 250( I ) , 170(100), 152(4), 112(13). 2D. 2673. ‘CI7H3,NO,’--, 182”C,mlz 267(18), 266(1 I ) , 196(loo), 96(58). OD. H, derivative. 26m.‘ C I ~ H ~ ~ N @ , ’198”C, -, mlz 267( 12), 250(20), 178(100). 2676. ‘CI6H2,NO2,’-, 19O”C, rnlz 267(4), 152(100). 2D. H, derivative. A minor alkaloid in one population of Dendrobates pumilio. 269C. ‘C&~INO,,’-, 207”C, mlz 269(2), 98(100). 2D. A minor alkaloid in one population of Dendrobates pumilio. 269D. ‘C19H27N,’-, 199”C, mlz 269(2), 176(100). OD. HI, derivative. 271A. ‘C,9H29N,’--, 198”C, rnlz 271(3), 178(100). OD. H, derivative. 271B. ‘C19H29N,’-,-, ion trap, mlz 271(5), 228(CI6H2,N+,100) (73). 279A. ‘CI8H3,NO,’--, 190°C,mlz 279(35), 210(90), 190(75),84( 100). 1D. H, derivative. 281B. ‘cl,H35N0,’- 200”C, rnlz 281(4), 264( 12), 208(25), 206(20), 150(65), 98(5), 96(20), 70( 100). H, derivative. Minor alkaloid in Dendrobates new species (Panama). 281C. ‘C17H31N02,’-, 195”C,mlz 281(25), 208( 100). 2D. H, derivative. Minor alkaloid in one population of Dendrobates pumilio. 281D. ‘CI7H3,NO2,’-, 196”C,mlz 281(16), 210(100). 2D. H, derivative. Minor alkaloid in Dendrobates quinquevittatus. 281E. ‘C,7H31N0,,’--, 195”C,mlz 281(8), 196(100). 2D. Minor alkaloid in Dendrobates quinquevittatus. 281F. ‘C17H31N02,’-r-r ion trap, mlz 280(2), 222(23), 84(55), 70(100). 2D. Infrared data (73). Tentatively proposed to be of the pumiliotoxin A class, but with the 6,lO double bond reduced (73). 285D.‘C,,H27N0,’--, 190”,mlz 285(3),270(2),256(2), 180(35), 140(100). 291B. ‘C,,H,,NO,’ 0.12,221°C, mlz 291(2), 290(3), 276(6), 168(100). 1D. H, derivative, mlz 295, 168. 291C. ‘Cl,H33N0,’ 0.20, 220”C, mlz 291(1), 290(2), 276(4), 210(10), 152(100). 1D. H, derivative, mlz 295, 152. 291D. C,,H,,NO,-, 201”C, mlz 291(<1), 168(100). ID. H, derivative. Minor alkaloid in Dendrobates new species (Panama). 293B. ‘C19H35N0,’--,-, ion trap, mlz 292( I), 150(33), 95(35), 81(70), 67(100). ID. Infrared data (73).Tentatively proposed based on the infrared spectrum to be related to the pumiliotoxin-A class (73). 295A. ‘CI9H3,NO,’0.09, 224”C, mlz 295(3), 278(4), 138(100). 301. ‘C21H3SN,’-, 213”C, mlz 301(<1), 260(100). 309B. ‘C2,H,,NO,’ 0.09, 220°C mlz 309(1), 152(100).

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275

W E . ‘C,8H31N03,’--,235”C, mlz 309(32), 266( l3), 240( IOO), 205(22), 124(35), 114(25). 3D. H, derivative. Major alkaloid in one population of Dendrobates auratus. W F . ‘C19H35N02,’-, 206”C, mlz 309(2), l52( 100). 2D. H, derivative. 351. ‘C21H37N03,’0.15, 230°C mlz 351(6), 350(2), 336(4), 152(38), 138(65), 70(100). 4D. 384A.C26H44N2r-,-r mlz 384(5), 342(28), 341(100), 136(10). 2D. Infrared data (73). Proposed to be a “dimer” related to the octahydroquinoline 193C with which it occurs in a ranid frog (73). 384B. C26H44N2,-,-, mlz 384(2), 342(24), 341(100). 2D. Infrared data (73). Proposed to be a dimer related to the octahydroquinoline 193C (73).

X11. Summary Amphibians have elaborated an astonishing array of alkaloids, most of which are known only from the skin or skin glands of adult amphibians. These substances, in most cases, are probably released defensively against predators andlor microorganisms onto the skin surface from the cutaneous granular “poison” glands that develop during metamorphosis. Such glands are present even in frogs that do not seem to have elaborated a defensive substance (207). The question of antipredator or antimicrobial roles for the alkaloids remains unresolved (see Ref. I ) . Perhaps some alkaloids (batrachotoxin, tetrodotoxin) play only an antipredator role, while others also may serve an antimicrobial role. Alkaloids generally are bitter substances, and thus all amphibian alkaloids could be noxious, although in many cases not particularly toxic to predators. The marked effects of most amphibian alkaloids on the membrane function of nerve and muscle strongly suggest that buccal tissue could be a major target. The origin of amphibian alkaloids is even less clear. Many represent unique structural classes, while others occur in certain insects. Ants contain simple pyrrolidines, piperidines, pyrrolizidines, and indolizidines; beetles contain tricyclic coccinellines; and a millepede contains a possible precursor for the pyrrolizidine oxime class. However, the other classes of amphibian alkaloids have no known dietary source, plant or animal. Biosynthetic studies on amphibian alkaloids have either been negative or unconvincing. The phylogenetic distribution of alkaloids in amphibians (Table IX) argues strongly for genetic control through specific sets of biosynthetic enzymes.

TABLE IX OCCURRENCE OF LIPOPHILIC ALKALOIDS I N AMPHIBIANS" Order. family.and genus Caudara Salamandridae Solomondm Anura Dendrobatidaeb Phyllobates Dendroborrr Minyobares Epipedoborrs Bufonidae Melanophyrnisrus Myobatrachidae Pwudophrynr Ranidae Monrella

Pumiliotoxin-A Class(PTX) Batracho- Saman- Hisrrionitoxins darines cotoxins PTX AlloPTX

-

+

-

-

+

+

+

-

-

+

+

+

HomoPTX

-

-

+

+ +

+ +

+ +

-

-

+

+

+

-

-

+

+

-

(+)<

+

+

+

-

-

-

Tricyclics Pyrrolizi-. Pseudo- F'iperidines dines. lndolmdines QumoliziDecahydro3.5dines. Pyr. Coccine- Epibali- phrynand quinolines disub. 3.5-Disub. 5.8-Disub. 1.4dirub. GTX oximes llines dme amines pyrrolidines

+ + + +

+

+

+

+

+ +

-

-

+ -

+ -

+

+

+ + +

+

+

-

+

+

+

-

+ t

+

+

+

-

+

+

-

-

+

+

-

+

+

-

'I See Refs. 1 and 86 for listings of amphibians in which alkaloids have not been detected using the protocol described in the Appendix, and Ref. 1 for listings of the occurrence of other "noxious" skin components in amphibians. bThe two other dendrobatid genera Colosrethus and Aromabates (219) do not contain lipophilic alkaloids. Histrionicotoxins were detected in skin extracts of M .madagascariensis obtained from a commercial dealer (72) but not from wild-caught specimens (73).

3.

AMPHIBIAN ALKALOIDS

277

Nonetheless, the involvement of symbiotic microorganisms, dietary precursors or cofactors, or other environmental factors in alkaloid production in amphibians cannot be excluded, although genetic factors must play a role. Most remarkably, dendrobatid frogs reared in captivity do not contain any skin alkaloids (29,31).Thus, something is missing under these conditions, even though wild-caught frogs appear to maintain alkaloids in captivity for years (29,31). A variety of environmental manipulations, including stress, frequent changes in terraria, increased fluorescent lighting, and modification of diets, have not caused the accumulation of alkaloids in captive-raised frogs (31). Feeding experiments, however, indicate that captive-raised frogs of the alkaloid-producing genera Dendrobates and Phyllobates have the ability to accumulate dietary alkaloids quite efficiently into the skin (220). Remarkably, this process can be most selective. Thus, feeding Dendrobates auratus with pharaoh's ant, Monomorium pharaonis, which contains the indolizidine monomorine I and trans-5-(5-hexen-1-yl)-2-pentylpyrrolidine in a 1:2 ratio, resulted in a large accumulation of monomorine I in the frog skin, while not a trace of the pyrrolidine was detected (220).The naturally alkaloid-free dendrobatid frog Colostethus talamancae does not accumulate any alkaloids from the diet into skin. Alkaloids that had been accumulated into the skin of dendrobatid frogs (Dendrobates auratus, Phyllobates bicolor) from dietary sources were maintained in skin for months. These results indicate a genetically programmed mechanism in certain frogs, which can accumulate dietary alkaloids or salvage skin alkaloids when the frogs shed and ingest their skin. The source of amphibian alkaloids thus remains an unresolved and challenging question.

Appendix The investigation of dendrobatid alkaloids at the National Institutes of Health has continued for nearly three decades. During this time gas chromatography-mass spectral analyses have played a major role, as also gas chromatography-FTIR spectral analyses do now. The sensitivity and resolution of such gas chromatographic techniques have increased markedly over the years, particularly through the use of capillary columns. The sensitivity and capabilities of nuclear magnetic resonance spectral analyses have increased even more markedly, with the structures of complex tricyclic amphibian alkaloids having been obtained with less than a 500-pg sample of isolated alkaloid (151,165).

A

B

FIG.23. Flame ionization-gas chromatograms of alkaloids from skin extracts of dendrobatid frogs. The chromatograms were obtained with a 6-ft (2 mm i.d.) 1.5% OV-I-packed column with a flame ionization detector and a flow rate of 30 mllrnin helium. A sample of 2 pI of a methanolic alkaloid fraction equivalent to 2 mg wet weight skin was injected at a column temperature of 150°C. After the solvent maximum had passed (0.3 min), the column was programmed to 280°C at 10°C per minute. The major and minor alkaloids are indicated by code number. (A) Phyllobates bicolor, 10-skin sample, Santa Cecilia, Rio San Juan, Risaralda, Colombia, Sept. 1977. Other Phyllobates species show much less or only trace amounts of alkaloids on gas chromatograms. The batrachotoxins do not chromatograph even on packed columns, but a pyrolysis product (molecular ion mlz 399) is sometimes seen at 278

C

D

280°C. (B) Dendrobates histrionicus, 10-skin sample, Guayacana, Narino, Colombia, Oct. 1972. This is the reference sample that has been used since 1978 to adjust the sensitivity for flame ionization-gas chromatograms of other alkaloid fractions. Many other Dendrobates species show lower amounts of alkaloids. (C) Epipedobares tricolor, 10-skin sample, 16 km west of Santa Isabel, Azuay, Ecuador, Nov. 1979. Most Epipedobates species have levels of alkaloids similar to or less than this sample, but two Epipedobates species (E. myersi. E . fernoralis) had virtually no alkaloids. (D) Minyobares sreyermurki, 10-skin sample, Cerro Yapacana, Amazonas, Venezuela, Feb. 1978. Other species of Minyobates have similar or somewhat greater levels of alkaloids. 279

280

JOHN W . DALY E T A L .

In the expectation that the comprehensive survey of alkaloids in dendrobatid frogs and now in frogs and toads of the myobatrachid, bufonid, and ranid families would be useful to biologists for taxonomic purposes, the protocols for extraction and partitioning to obtain an alkaloid fraction have been kept relatively constant, and an initial gas chromatographic profile of the alkaloid fraction with a flame ionization detector has been obtained under conditions as constant as possible so that such profiles provide an indication of the amounts of alkaloids present per 2 mg of skin of each species, population, or individual. Flame ionization-gas chromatograms have been published for a number of species and populations as follows: the neotropical dendrobatid genera Dendrobates (31,60,71,87,96,221), Epipedobates (60,222), Minyobates (60,151), and Phyllobates (31: a captive-raised specimen); the Australian myobatrachid genus Pseudophryne (72,86); the Madagascan ranid genus Mantella (72,73);and the South American bufonid genus Melanophryniscus (72). A flame ionization-gas chromatogram for one species representative of each of four dendrobatid genera is presented in Fig. 23. Marked species and population differences in profiles and amounts have been observed. For some populations, individual variations in amount, and to a lesser degree in profile, have been noted, whereas for other populations, amounts and profiles for individual frogs are very constant. For some populations or species, profiles of samples comprising 5 to 20 skins have been very constant over the years, whereas for other populations or species there have been significant variations. Such variations are inexplicable given the present state of our knowledge of the source of amphibian alkaloids. Briefly, our current protocol for analysis of alkaloids in skin extracts is as follows. Frogs are skinned and the skins stored in methanol ( 1 part skid2 or more parts methanol). Skins are cut into small pieces and macerated two or three times with separate portions of methanol ( 1 part skins/ 4-20 parts methanol). The combined methanol extracts are diluted with an equal volume of water. In some cases, for large skin samples, the combined methanol extracts are first concentrated at 30°C in uacuo. The aqueous methanol extract is then extracted three times, each time with 1 volume of chloroform. The combined chloroform layers are dried over Na2S0, and concentrated to a small volume (1-10 ml). The concentrated chloroform is restored to the original volume with n-hexane. The n-hexane solution, containing a small amount of chloroform, now is extracted three times, each time with one-half volume ofO. 1 N HCI. Extraction of alkaloids is much more complete from the n-hexane phase than had been the case in the original procedure in which the alkaloids were directly extracted from the combined chloroform layers. Significant amounts of more hydrophobic alkaloids (as hydrochloride salts) were found to remain in the chloroform layer with the original procedure used until 1989.

3. AMPHIBIAN

ALKALOIDS

28 1

The combined 0.1 N HCI fractions are adjusted to pH 9 with 1 N aqueous ammonia followed by reextraction of the alkaloids three times into chloroform, each time with one-half volume of chloroform. The combined chloroform layers are dried over Na,SO, and then evaporated to dryness at 30°C in uucuo with a water aspirator. Many of the dendrobatid alkaloids have appreciable volatility, and evaporation in uucuo must be done carefully. The resulting alkaloid residue is dissolved in methanol so that 100 p1 corresponds to 100 mg of original wet weight of skin, then stored at -20°C in glass vials with Teflon-lined caps. This alkaloid fraction contains mainly alkaloids, but traces of fatty acid methyl esters, steroids, and environmental artifacts, such as phthalates and phenolic antioxidants, often are present as minor contaminants. The alkaloid fractions are analyzed primarily by gas chromatography in conjunction with mass spectrometry and infrared spectroscopy. An initial profile of alkaloids is obtained on a short (5-6 ft) 1.5% OV-1-packed column programmed at 10"C/minfrom 150 to 280°C with a flame ionization detector and a flow rate of 30 ml/min of helium (see Fig. 23). The conditions and column have varied somewhat over the years so that emergent temperatures have varied. Further analysis now is carried out with microbore or capillary columns using a mass spectrometer or a Fourier transform infrared detector. The capillary column currently in use is the following: HP5 (bonded 5% diphenylsiloxane-95% dimethylsiloxane) fused silica column (25 m x 0.32 mm) programmed from 100 to 280°C at 10"Clmin. Often some of the more polar alkaloids do not elute with capillary columns used in these studies. Mass spectral analysis uses high- and low-resolution electron impact spectrometry (-70 eV), the latter occasionally with D,O exchange to determine number and site of exchangeable hydrogens. A Finnigan Model 800 ion trap mass detector is used for pseudo electron impact spectra. Chemical ionization-mass spectrometry, with NH, or isobutane reagent gases, is used to detect protonated molecular ions; ND, is used to determine the number of exchangeable hydrogens. Additional data on alkaloid fractions can be obtained after perhydrogenation, acetylation, butylboronation, or base-catalyzed exchange of hydrogens (see Refs. 1,60,78,81,86). REFERENCES

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101. F. Gusovsky, E. B. Hollingsworth, and J. W. Daly, Proc. Narl. Acad. Sci. U.S.A. 83, 3003 (1986). 102. F. Gusovsky, D. P. Rossignol, E. T. McNeal, and J. W. Daly, Proc. Narl. Acad. Sci. U.S.A. 85, 1272 (1988). 103. F. Gusovsky and J. W. Daly, Neuropharmacology 27, 95 (1988). 104. J. W. Daly, E. T. McNeal, and F. Gusovsky, Biochim. Biophys. Acfa 930,470 (1987). 105. J. W. Daly, E. T. McNeal, F. Gusovsky, F. Ito, and L. E. Overman, J . Med. Chem. 31, 477 (1988). 106. J. W. Daly, F. Gusovsky, E. T. McNeal, S. Secunda, M. Bell, C. R. Creveling, Y. Nishizawa, L. E. Overman, M. J. Sharp, and D. P. Rossignol, Biochem. Pharmacol. 40,315 (1990). 107. F. Gusovsky, E. T. McNeal, and J. W. Daly, Mol. Pharrnacol. 32, 479 (1987). 108. D. C. Deecher and D. M. Soderlund, Pestic. Biochem. Physiol. 39, 130 (1991). 109. K . S. Rao, J. E. Warnick, J. W. Daly, and E. X. Albuquerque, J . Pharrnacol. Exp. Ther. 243, 775 (1987). 110. R. E. Sheridan, S. S. Deshpande, F. J. Lebeda, and M. Adler, Brain Res. 556, 53 (1991). 11 I . G. G. Schofield, F. F. Weight, and S. R. Ikeda, Eur. J . Pharrnacol. 147, 39 (1988). 112. F. Gusovsky, W. L. Padgett, C. R. Creveling, and J. W. Daly, Mol. Pharmacol. in press (1992). 113. V. Erspamer, G. F. Erspamer, P. Melchiom, and G. Mazzanti, Neuropharmacology 24, 783 (1985). 114. G. F. Erspamer, and G. Farruggia, Neuropharmacology 25, 803 (1986). 115. G. F. Erspamer, V. Erspamer, and P. Melchiorri, Neuropharrnacologv 25,807 (1986). 116. G. F. Erspamer and C. Severini, Arch. Inr. Pharmacodyn. Ther. 285, 324 (1987). 117. G . F. Erspamer, C. Severini, V. Erspamer, and P. Melchiorri, Neuropharmacology 28, 319 (1989). 118. G. Bagetta, G. DeSarro, M. T. Corasanti, D. Rotiroti, and G. Nistico, Toxicon 30, 197 (1992). 119. T. F. Spande, M. W. Edwards, L . K. Pannell. J. W. Daly, V. Erspamer, and P. Melchiorri, J . Org. Chem. 53, 1222 (1988). 120. S. M. Colegate, P. R. Dorling, and C. R. Huxtable, Ausr. J. Chem. 37, 1503 (1984). 121. T. H. Jones, M. S. Blum, H. M. Fales, and C. R. Thompson, J. Org. Chem. 45,4778 ( 1980). 122. P. E. Sonnet, D. A. Netzel, and R. Mendoza, J. Hererocycl. Chem. 16, 1041 (1979). 123. T. H. Jones, R. J. Highet, A. W. Don, and M. S. Blum, J. Org. Chem. 51,2712 (1986). 124. T. H. Jones, S. M. Stahly, A. W. Don, and M. S. Blum, J. Chem. Ecol. 14,2197 (1988). 125. T. H. Jones, A. Laddago, A. W. Don, and M. S. Blum, J. Nur. Prod. 53, 375 (1990). 126. T . H. Jones, M. S. Blum, H. M. Fales, C. R. F. Brandgo, and J. Lattke,J. Chem. Ecol. 17, 1897 (1991). 127. T. F. Spande, J. W. Daly, D. J. Hart, Y.-M. Tsai, and T. L. MacDonald, Experenria 37, 1242 (1981). 128. I. Royer and H.-P. Husson, Tetrahedron Lerr. 26, 1515 (1985). 129. F. J. Ritter, 1. E . M. Rogans, E . Talman, P. E. J. Verwiel, and F. Stein, Experienfia 29, 530 (1973). 129a. N. Yamazaki and C. Kibayashi, J. Am. Chem. Soc. 111, 1396 (1992). 129b. N. Machinga and C. Kibayashi, J. Org. Chem. 57, 5178 (1992). 130. R. P. Polniaszek and S. E. Belmont, J. Org. Chem. 55, 4688 (1990). 131. C. W. Jefford, Q. Tang, and A. Zaslona, J . Am. Chem. Soc. 113, 3513 (1991). 132. N. Machinaga and C. Kibayashi, J. Chem. Soc., Chem. Commun. 405 (1991).

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3.

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287

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Y. H. Kim, G. B. Brown, H. S. Mosher, and F. A. Fuhrman, Science 189, 151 (1975). L. A. Pavelka, Y. H. Kim, and H. S. Mosher, Toxicon 15, 135 (1977). F. A. Fuhrman, G. J. Fuhrman, and H. S. Mosher, Science 26, 1376 (1969). G . B. Brown, Y. H. Kim, H. Kiintzel, H. S. Mosher, G. J. Fuhrman, and F. A. Fuhrman, Toxicon 15, 115 (1977). J. Flier, M. W. Edwards, J. W. Daly, and C. W. Myers, Science 208, 503 (1980). M. Neuwirth, J. W. Daly, C. W. Myers, and L. W. Tice, Tissue Cell 11, 755 (1979). J. W. Daly, F. Gusovsky, C. W. Myers, M. Yotsu, and T. Yasumoto, Toxicon in preparation (1992). A. Sebben, C. A. Schwartz, D. Valente, and E. G. Mendes, Toxicon 24, 799 (1986). Y. Shimizu and M. Kobayashi, Chem. Pharm. Bull. 31, 3625 (1983). C. Y. Kao, P. N. Yeoh, M. D. Goldfinger, F. A. Fuhrman, and H. S. Mosher, J . Pharmacol. Exp. Ther. 217, 416 (1981). M. Yotsu, T. Yasumoto, Y. H. Kim, H. Naoki, and C. Y. Kao, Tetrahedron Leu. 31, 3187 (1990). C. Y. Kao, Fed. Proc. 40, 30 (1981). J. Shindelman, H. S. Mosher, and F. A. Fuhrman, Toxicon 24, 135 (1969). B. K. Ranney, G. J. Fuhrman, and F. A. Fuhrman, J . Pharmacol. Exp. Ther. 175,368 ( 1970). J. S . Roseen and F. A. Fuhrman, Toxicon 9,411 (1971). M. Huang and J. W. Daly, J. Neurochem. 23, 393 (1974). H. M. Fales, M. S. Blum, Z. Bian, T. H. Jones, and A. W. Don, J. Chem. Ecol. 10, 651 (1984). C. W. Myers, A. Paolillo, and J. W. Daly, A m . Mus. Nouir. No. 3002 (1991). J. W. Daly, H. M. Garraffo, T. F. Spande, S. I. Secunda, A. Wisnieski, and J. Cover, Toxicon in preparation (1992). C. W. Myers, J. W. Daly, and V. Martinez, A m . Mus. Nouir. No. 2783 (1984). C. W. Myers and J. W. Daly, A m . Mus. Novir. No. 2674 (1979).

CUMULATIVE INDEX OF TITLES

Aconitum alkaloids, 4, 275 (1954). 7, 473 ( 1 9 6 0 ~34, 95 (1988)

C19diterpenes, 12, 2 (1970) Czoditerpenes, U ,136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, experimental antitumor activity of acronycine 21, 1 (1983) N-Acylirninium ions as intermediates in alkaloid synthesis, 32, 271 (1988) Ajmaline-Sarpagine alkaloids, 8, 789 (1965), 11, 41 (1968) Alkaloid production, plant biotechnology of 40, 1 (1991) Alkaloid structures spectral methods, study, 24, 287 (1985) unknown structure, 5, 301 (1955). 7, 509 (1960). 10, 545 (1967). 12, 455 (1970). 13, 397 (1971), 14, 507 (1973), 15, 263 (1975). 16, 511 (1977) X-ray diffraction, 22, 51 (1983) Alkaloids forensic chemistry of, 32, 1 (1988) histochemistry of, 39, 165 (1990) in the plant, 1, 15 (1950). 6, I (1960) Alkaloids from Amphibians, 21, 139 (l983), 43, 185 (1993) Ants and insects, 31, 193 (1987) Chinese Traditional Medicinal Plants, 32, 241 (1988) Mammals, 21, 329 (1983). 43, 119 (1993) Marine organisms, 24, 25 (1983, 41, 41 (1992) Mushrooms, 40, 189 (1991) Plants of Thailand, 41, 1 (1992) Allelochemical properties or the raison d’@treof alkaloids, 43, I (1993) A110 congeners, and tropolonic Colchicum alkaloids, 41, 125 (1992) Alsfonia alkaloids, 8, 159 (1965). 12, 207 (1970), 14, 157 (1973) Amaryllidaceae alkaloids, 2, 331 (1952), 6, 289 (1960). 11, 307 (1968). 15, 83 (1975). 30, 251 ( 1987) Amphibian alkaloids, 21, 139 (1983). 43, 185 (1993) Analgesic alkaloids, 5, I (1955) Anesthetics, local, 5, 21 1 (1955) Anthranilic acid derived alkaloids, 17, 105 (1979). 32, 341 (1988), 39, 63 (1990) Antifungal alkaloids, 42, I17 (1992) Antimalarial alkaloids, 5, 141 (1955) Antitumor alkaloids, 25, 1 (1985) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4, 119 (1954), 9, I (1967), 24, 153 (1985) Arisrolochia alkaloids, 31, 29 (1987) Arisrofeliu alkaloids, 24, 113 (1985) 289

290

CUMULATIVE INDEX OF TITLES

Aspergillus alkaloids, 29, 185 (1986) Aspidosperma alkaloids, 8, 336 (1965). 11, 205 (1968), 17, 199 (1979) Azafluoranthene alkaloids, 23, 301 (1984)

Bases simple, 3, 313 (1953), 8, 1 (1965) simple indole, 10, 491 (1967) simple isoquinoline, 4, 7 (1954). 21, 255 (1983) Benzodiazepine alkaloids, 39, 63 (1990) Benzophenanthridine alkaloids, 26, 185 (1985) Benzylisoquinoline alkaloids, 4, 29 (1954), 10, 402 (1967) Betalains, 39, 1 (1990) Biosynthesis, isoquinoline alkaloids, 4, I (1954) Bisbenzylisoquinoline alkaloids, 4, 199 (l954), 7,429 (1960).9, 133 (1967). 13,303 (l97l), 16, 249 (1977). 30, I(1987) synthesis, 16, 319 (1977) Bisindole alkaloids, 20, 1 (1981) Bisindole alkaloids of Carharanrhus, C-20’ position as a functional hot spot in, 37, 133 (1990) isolation, structure elucidation and biosynthesis, 37, I (1990) medicinal chemistry of, 37, 145 (1990) pharmacology of, 37, 205 (1990) synthesis of, 37, 77 (1990) therapeutic use of, 37, 229 (1990) Buxus alkaloids, steroids, 9, 305 (l967), 14, 1 (1973). 32, 79 (1988) Cactus alkaloids, 4, 23 (1954)

Calabar bean alkaloids, 8, 27 (1%5), 10, 383 (1967). 13, 213 (1971). 36, 225 (1989) Calabash curare alkaloids, 8, 515 (1965), 11, 189 (1968) Calycanthaceae alkaloids, 8, 581 (1965) Camptothecine, 21, 101 (1983) Cancentrine alkaloids, 14, 407 (1973) Cannabis sariua alkaloids, 34, 77 (1989) Canthin-6-one alkaloids, 36, 135 (1989) Capsicum alkaloids, 23, 227 (1984) Carbazole alkaloids, 13, 273 (1971). 26, 1 (1985) Carboline alkaloids, 8, 47 (1965). 26, 1 (1985) P-Carboline congeners and Ipecac alkaloids, 22, I (1983) Cardioactive alkaloids, 5, 79 (1955) Celastraceae alkaloids, 16, 215 (1977) Cephaloraxus alkaloids, 23, 157 (1984) Cevane group of Verarrum alkaloids, 41, 177 (1992) Chemotaxonomy of Papaveraceae and Fumaridaceae, 29, 1 (1986) Chinese medicinal plants, alkaloids from, 32, 241 (1988) Chromone alkaloids, 31, 67 (1987) Cinchona alkaloids, 3, 1 (1953), 14, 181 (1973), 34, 332 (1989) Colchicine, 2, 261 (1952). 6, 247 (1960), 11, 407 (1968). 23, I (1984) Colchicum alkaloids and allo congeners, 41, 125 (1992)

CUMULATIVE INDEX OF TITLES

29 1

Configuration and conformation, elucidation by X-ray diffraction, 22, 51 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4, 249 (1954). 10, 463 (1967), 29, 287 (1986) Curare-like effects, 5, 259 (1955) Cyclic Tautomers of Tryptamine and Tryptophan, 34, 1 (1989) Cyclopeptide alkaloids, 15, 165 (1975) Daphniphyllum alkaloids, 15, 41 (1975). 29, 265 (1986) Delphinium alkaloids, 4, 275 (1954). 7, 473 (1960) Clo-diterpenes,12, 2 (1970) Cz0-diterpenes, 12, 136 (1970) Dibenzazonine alkaloids, 35, 177 (1989) Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8, 336 (1965) Diterpenoid alkaloids Aconirum, 7, 473 (1960). U,2 (1970). l2, 136 (1970). 34, 95 (1989) Delphinium, 7, 473 (1960). 12, 2 (1970). 12, 136 (1970) Garrya, 7, 473 (1960), 12, 2 (1960), 12, 136 (1970) chemistry, 18, 99 (1981), 42, 151 (1992) general introduction, 12, x v (1970) structure, 17, 1 (1970) synthesis, 17, 1 (1979)

Eburnamine-vincamine alkaloids, 8, 250 (1965), 11, 125 (1968). 20, 297 (1981). 42, 1 (1992) Elueocurpus alkaloids, 6 , 325 (1960) Ellipticine and related alkaloids, 39, 239 (1990) Enamide cyclizations in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in uiro, 18, 323 (1981) Ephedra alkaloids, 3, 339 (1953) Ergot alkaloids, 8,726 (1965). 15, 1 (1975). 39, 329 (1990) Eryrhrina alkaloids, 2, 499 (1952), 7, 201 (1960). 9, 483 (1967). 18, 1 (1981) Erythrophleum alkaloids, 4, 265 (1954), 10, 287 (1967) Eupornaria alkaloids, 24, I (1985) Forensic chemistry, alkaloids, l2,514 (1970) by chromatographic methods, 32, 1 (1988) Galbulirnima alkaloids, 9, 529 (1967). 13, 227 (1971) Gardneria alkaloids, 36, 1 (1989) Garrya alkaloids, 7, 473 (1960), 12, 2 (1970). 12, 136 (1970) Geissospermum alkaloids, 8, 679 (1965) Gelsernium alkaloids, 8, 93 (1965), 33, 84 (1988) Glycosides, monoterpene alkaloids, 17, 545 (1979) Guarreria alkaloids, 35, 1 (1989) Haplophyron cimicidum alkaloids, 8, 673 (1965) Hasubanan alkaloids, 16, 393 (1977), 33, 307 (1988)

292

CUMULATIVE INDEX OF TITLES

Histochemistry of alkaloids, 39, 165 (1990) Holarrhena group, steroid alkaloids, 7, 319 (1960) Hunteria alkaloids, 8, 250 (1965) Iboga alkaloids, 8, 203 (1965), 11, 79 (1968)

lmidazole alkaloids, 3, 201 (1953), 22, 281 (1983) Indole alkaloids, 2, 369 (1952). 7, 1 (1960), 26, 1 (1985) distribution in plants, 11, I (1968) simple, 10, 491 (1967), 26, I (1985) Reissert synthesis of, 31, I (1987) Indolizidine, simple and quinolizidine alkaloids, 28, 183 ( 1986) 2,2’-lndolylquinuclidinealkaloids, chemistry, 8, 238 (1965). 11, 73 (1968) Ipecac alkaloids, 3, 363 (1953), 7, 419 (1960), 13, 189 (1971). 22, 1 (1983) Isolation of alkaloids, 1, I (1950) Isoquinoline alkaloids, 7, 423 (1960) biosynthesis, 4, 1 (1954) I3C-NMR spectra, 18, 217 (1981) simple isoquinoline alkaloids, 4, 7 (1954). 21, 255 (1983) Reissert synthesis of, 31, 1 (1987) lsoquinolinequinones, from Actinomycetes and sponges, 21, 55 ( 1983) Khat (Catha edulis) alkaloids, 39, 139 (1990) Kopsia alkaloids, 8, 336 (1965) Lead tetraacetate oxidation in alkaloid synthesis, 36, 70 (1989) Local anesthetics, 5, 211 (1955) Localization in the plant, 1, 15 (1950). 6, 1 (1960) Lupine alkaloids, 3, 119 (1953), 7, 253 (1960), 9, 175 (1967). 31, 16 (1987) Lycopodium alkaloids, 5, 265 (1955), 7, 505 (1960). 10, 306 (1967), 14, 347 (1973). 26, 241 ( 1985)

Lythraceae alkaloids, 18, 263 (1981). 35, 155 (1989) Mammalian alkaloids, 21, 329 (l983), 43, 119 (1993) Marine alkaloids, 24, 25 (1985). 41, 41 (1992) Maytansinoids, 23, 71 (1984) Melanins, 36, 254 (1989) Melodinus alkaloids. 11, 205 (1968) Mesembrine alkaloids, 9, 467 (1967) Metabolic transformation of alkaloids, 27, 323 (1986) Microbial and in uifro enzymatic transformation of alkaloids, 18, 323 (1981) Mifragyna alkaloids, 8, 59 (1963, 10, 521 (1967). 14, 123 (1973) Monoterpene alkaloids, 16, 431 (1977) glycosides, 17, 545 (1979) Morphine alkaloids, 2, 1 (part I , 1952). 2, 161 (part 2, 1952). 6, 219 (1960), 13, 1 (1971) Muscarine alkaloids, 23, 327 (1984) Mushrooms, alkaloids from, 40, 190 (1991) Mydriatic alkaloids, 5, 243 (1955)

CUMULATIVE INDEX OF TITLES

a-Naphthophenanthridine alkaloids, 4, 253 (1954), 10, 485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (1986) Narcotics, 5, 1 (1955) Nuphar alkaloids, 9, 441 (1967), 16, 181 (1977), 35, 215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11, 205 (1968) Ourouparia alkaloids, 8, 59 (1963, 10, 521 (1967) Oxazole alkaloids, 35, 259 (1989) Oxaporphine alkaloids, 14, 225 (1973) Oxindole alkaloids, 14, 83 (1973)

Papaveraceae alkaloids, 19, 467 (1967), 12, 333 (1970). 17, 385 (1979) pharmacology, 15, 207 (1975) toxicology, 15, 207 (1975) Pauridiunfha alkaloids, 30, 223 (1987) Pavine and isopavine alkaloids, 31, 3 17 (1987) Penfaceras alkaloids, 8, 250 (1965) Peptide alkaloids, 26, 299 (1985) Phenanthrene alkaloids, 39, 99 (1990) Phenanthroindolizidine alkaloids, 19, 193 (1981) Phenanthroquinolizidine alkaloids, 19, 193 (1981) P-Phenethylamines, 3, 313 (1953), 35, 77 (1989) Phenethylisoquinoline alkaloids, 14, 265 (1973), 36, 172 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7, 433 (1960), 9, 117 (1967). 24, 253 (1985) Picralima alkaloids, 8, 119 (1965). 10, 501 (1967), 14, 157 (1973) Piperidine alkaloids, 26, 89 (1985) Plant biotechnology, for alkaloid production, 40, 1 (1991) Plant systematics, 16, I (1977) PIeiocarpa alkaloids, 8, 336 (1965), 11, 205 (1968) Polyamine alkaloids, 22, 85 (1983) Pressor alkaloids, 5, 229 (1955) Protoberberine alkaloids, 4, 77 (1954), 9, 41 (1967). 28, 95 (1986) transformation reactions of, 33, 141 (1988) Protopine alkaloids, 4, 147 (1954), 34, 181 (1989) Pseudocinchona alkaloids, 8, 694 (1965) Purine alkaloids, 38, 226 (1990) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960). 11, 459 (1968). 26, 89 (1985) Pyrrolidine alkaloids, 1, 91 (1950), 6, 31 (1960). 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6, 35 (1960). 12, 246 ( 1 9 7 0 ~26, 327 (1985) Quinazolidine alkaloids, see Indolizidine Alkaloids Quinazoline alkaloids, 3, 101 (1953), 7, 247 (1960), 29, 99 (1986) Quinazolinocarbolines, 8, 55 (1965), 21, 29 (1983) Quinoline alkaloids related to anthranilic acid, 3, 65 (1953). 7, 229 (1960). 17, 105 (1979). 32, 341 (1988) Quinolizidine alkaloids, and indolizidine, 28, 183 ( 1985)

293

294

CUMULATIVE INDEX OF TITLES

Rauwolfia alkaloids, 8, 287 (1965) Reissert synthesis of isoquinoline and indole alkaloids, 31, I (1987) Reserpine, chemistry, 8, 287 (1965) Respiratory stimulants, 5, 109 (1955) Rhoeadine alkaloids, 28, I (1986)

Salamandra group, steroids, 9, 427 (1967) Sceletium alkaloids, 19, I (1981) Secoisoquinoline alkaloids, 33, 23 1 (1988) Securinega alkaloids, 14, 425 (1973) Senecio alkaloids, see Pyrrolizidine alkaloids Simple indole alkaloids, 10, 491 (1967) Simple indolizidine alkaloids, 28, 183 (1986) Sinomenine, 2, 219 (1952) Solanurn alkaloids chemistry, 3, 247 (1953) steroids, 7, 343 (1960), 10, I (1967), 19, 81 (1981) Sources of alkaloids, 1, 1 (1950) Spectral methods, alkaloid structures, 24, 287 (1985) Spermidine and related polyamine alkaloids, 22, 85 (1983) Spermine and related polyamine alkaloids, 22, 85 (1983) Spirobenzylisoquinoline alkaloids, 13, 165 (1971), 38, 157 (1990) Sponges, isoquinolinequinone alkaloids from, 21, 55 (1983) Sternona alkaloids, 9, 545 (1967) Steroid alkaloids Apocynaceae, 9, 305 (1967). 32,79 (1988) Bums group, 9, 305 (1967). 14, 1 (1973). 32, 79 (1988) Holarrhena group, 7, 319 (1960) Salarnandra group, 9, 427 (1967) Solanurn group, 7, 343 (1960). 10, I (1967). 19, 81 (1981) Verutrum group, 7, 363 (1960), 10, 193 (1967). 14, I (1973),41, 177 (1992) Stimulants respiratory, 5, 109 (1955) uterine, 5, 163 (1955) Structure elucidation, by X-ray diffraction, 22, 5 1 (1983) Strychnos alkaloids, 1, 375 (part I , 1950), 2, 513 (part 2, 1952). 6, 179 (1960). 8, 515, 592 (1963, 11, 189 (1968), 34, 211 (1989). 36, 1 (1989) Sulfur-containing alkaloids, 26, 53 (1985), 42, 249 (1992) Synthesis of alkaloids, Enamide cyclizations for, 22, 189 (1983) Lead tetraacetate oxidation in, 36, 70 (1989) Tabernaernontana alkaloids, 27, 1 (1983) Taxus alkaloids, 10, 597 (1967). 39, 195 (1990) Thailand, alkaloids from the plants of, 41, 1 (1992) Toxicology, Papaveraceae alkaloids, 15, 207 (1975) Transformation ofalkaloids, enzymatic, microbial and in vitro. 18. 323 (1981) . , Tropane alkaloids, chemistry, 1,271 (1950), 6, 145 (1960). 9, 269 (1967), 13, 351 (1971), 16, 83 (1977). 33, 2 (1988) Tropoloisoquinoline alkaloids, 23, 301 (1984)

CUMULATIVE INDEX OF TITLES

Tropolonic Colchicum alkaloids, 23, 1 (1984), 41, 125 (1992) Tylophora alkaloids, 9, 517 (1967) Uterine stimulants, 5, 163 (1955) Veratrum alkaloids cevane group of, 41, 177 (1992) chemistry, 3, 247 (1952) steroids, 7, 363 (1960). 10, 193 (1967), 14, 1 (1973) Vinca alkaloids, 8, 272 (1965), 11, 99 (1968). 20, 297 (1981) Voacanga alkaloids, 8, 203 (1965), 11, 79 (1968)

X-ray diffraction of alkaloids, 22, 51 (1983) Yohimbe alkaloids, 8, 694 (1965), 11, 145 (1968), 27, 131 (1986)

295

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INDEX

N-Acetyldopa ethyl ester, two-electron oxidation, 169-170 Alkaloids biosynthesis after wounding or elicitation, 9 1-92 as waste products, 6 Allelochemicals, 1-103 action at organ level, 58-61 allelopathic properties, 82-86 antiviral properties, 79-82 cellular targets biomembranes and transport processes, 55-57 cytoskeleton, 57 electron chains, 55 enzyme inhibition, 57-58 nucleic acids, 51-55 protein biosynthesis, 55 concentrations in plants and allelochemical activities, 87-89 criteria for alkaloids, 6-7 cytotoxic activity, 32-39 importance in plant fitness, 92-96 molecular targets, 40-50 mutagenic or carcinogenic activity, 52-54 plant-herbivore interactions, 8-61 invertebrates, 8-22 mode of action, 23, 32-61 vertebrates, 22-31 plant-microbe interactions, 61-78 antibacterial activity, 61-70 antifungal activity, 70, 72-78 presence at right site and right time, 89-92 production by animals, 101-103 toxicity in vertebrates, 24-31 Allelopathic properties, allelochemicals, 82-86 Allergenic effects, allelochemicals, 60-61 Allopumiliotoxins occurrence, 223-224 spectral properties, 218-220 structures, 213, 215 297

3-Amino-P-carbolines, 136 2-Arnino-6-hydroxymethyl-8hydroxyquinazoline, 264-265 Amphibian alkaloids, 185-281, see also Bicyclic alkaloids analytical protocol, 280-281 flame ionization-gas chromatograms. 278-280 guanidinium alkaloids, 264-269 imidazole alkaloids, 263 lipophilic alkaloids, occurrence, 275-276 Manrella, 270 monocyclic, 25 1-254 morphine, 263-264 origin, 275 steroidal alkaloids, 187-199 batrachotoxins, 187-194 samandarines, 194-199 tricyclic alkaloids, 242-251 unclassified alkaloids, 271-275 Animals, alkaloid production, 101-103 Antibacterial activity, allelochemicals, 61-70 Antifungal properties, allelochemicals, 70. 72-78 Antiviral properties, allelochemicals, 79-82 Atelopus, 266-269

Azaspiro[5.5]undecanols,see Histrionicotoxins Batrachotoxins, 187-194 biological activity, 189, 191 occurrence, 192-194 structures, 187-190 Benzylisoquinolines, 142-144 biosynthesis, 153 Berbines, acetaldehyde, 153, I55 Betaine, fluorescent. 135 Bicyclic alkaloids, 199 decahydroquinolines, 206-2 12 histrionicotoxins. 200-206

298

INDEX

indolizines, 228-238 pumiliotoxins, 2 12-225 pyrrolizidines, 225-228 quinolizidines, 238-242 Biomembranes, allelochemicals and, 55-57 Blood, allelochemicals action, 60 Bufo marinus, 263

Calycanthine, 261 Carbinolamines, optically active, 156 P-Carbolines, 122- 136 analytical methods, 132, 134-135 P-carboline-3-carboxylicacid esters, 130-1 3 1 chemistry, 123- 126 conversion of N-hydroxytryptophan, 170-17 I metabolic transformations, 131-133 monoamine oxidase inhibition, 134-136 nomenclature, 122 occurrence, 128-130 optically active tetrahydro-P-carbolines, 126-128 pharmacological effects, 134-136 reactions, 125-126 synthesis, 123-124 Carcinogenicity, allelochemicals, 52-54 Catechol 0-methyltransferase. 165, 167 Caudata, tetrodotoxin, 266 Central nervous system, allelochemicals action, 58-59 Chimonanthine, 260-26 I Chiriquitoxin, 267-268 Circulatory system, allelochemicals action, 60 Coccinellines, 244, 246-247 Constitutive compounds, 71 Cycloneosamandarine, 196 Cycloneosamandione, 195-196 Cyclopenta[b]quinolizidines, 247-249 Cytoskeleton, allelochernicals and, 57 Cytotoxic activity, allelochemicals, 32-39

Decahydroquinoline alkaloids, 206-212 biological activity, 21 I occurrence, 21 1-212 physical and spectral properties, 208-21 I structures, 206-21 I

Dehydrobufotenine, 262

(S)-4’-Demethylreticuline,165-166 Dendrobates histrionicus, 242, 244,253-254 Dendrobates pumilio, 244, 247. 249, 251, 253-254 Dendrobatid alkaloids, see Histrionicotoxins 1 I-Deoxytetrodotoxin, 264-265 3’.4’-Dideoxynorlaudanosoline1carboxylic acid, 146-147 Dimeric “indole” alkaloids, 261 L-Dopa, optically active tetrahydroisoquinolines from, 149- 150

Electron chains, allelochemicals and, 55 Elicitors, 71 Enzymes, inhibition, allelochemicals, 57-58 Eosinophilia syndrome, 136-137 Epibatidines. 255-256 Epipedobates, 255-256 4-Epitetrodotoxin, 264-265 6-Epitetrodotoxin, 264-266 I , 1’-Ethylidenebis(trypt0phan). 136- I37

Gephyrotoxins, 242-245 Guanidinium alkaloids, 264-269 chiriquitoxin, 267-268 tetrodotoxin, 264-267 zetekitoxin, 268-269

Harmala alkaloids, UV spectra, 134 Herbivores insect, adapted specialist allelochemicals, 97-100 vertebrate, adapted specialist allelochemicals, 100- 101 Histrionicotoxins, 200-206 biological activities, 203-205 occurrence, 205-206 physical and spectral properties, 202-204 structures, 200-204 Homobatrachotoxin, 189 Homopumiliotoxins occurrence, 224-225 spectral properties, 22 structures, 213, 216 4P-Hydroxybatrachotoxin, 189 4~-Hydroxyhomobatrachotoxin, 189

INDEX

4-Hydroxytetrahydroisoquinolines,143, 145 N-Hydroxytryptophan, conversion to Pcarbolines, 170-171 lmidazole alkaloids, 263 Indole alkaloids, 257-263 dehydrobufotenine, 262 dimeric, 261 indole amines, 261-262 pseudophrynamines, 257-261 trypargine, 262-263 lndole amines, 261-262 lndolizidines 3,5-disubstituted, 228-232 occurrence, 232 optical rotations, 231 spectral properties, 229-23 I 5,8-disubstituted, 232-238 optical properties, 237 spectral properties, 236-237 5-substituted, 229-23 1 (4R)-2-(3-Indolylmethyl)1,3-thiazolidine-4carboxylic acid, 140 Insects, herbivores, adapted specialist allelochemicals, 97-100 Invertebrates, plant interactions, 8-22 Isatin, 139-140 monoamine oxidase inhibition, 139-140 Isoquinolines, see Mammalian isoquinoline alkaloids

Kidney function, allelochemicals action, 59

Lipophilic alkaloids, occurrence, 275-276 Liver function, allelochemicals action, 59 Lupines, quinolizidine alkaloids, 92-95

Mammalian alkaloids, 119-172, see also Mammalian isoquinoline alkaloids formation as therapeutic concept, 168-169 Pictet-Spengler cyclization, 120-121, 123- 124 structural comparisons with plant alkaloids, 119-120 Mammalian indole alkaloids 3-amino-P-carbolines, 136

299

P-carbolines, 122-136 I,1'-ethylidenebis(tryptophan), 136- I37 isatin, 139-140 novel metabolites, 140 Mammalian isoquinoline alkaloids, 141-162 analytical methods, 158-159 biological activities, 159- 160 chemistry, 141-145 fluorescent, 159 metabolism, 153-157 occurrence, 150- 153 pyridoxal-derived isoquinolines, 160-162 tetrahydroisoquinoline- 1-carboxylic acids, 143, 146-148 tetrah ydroisoquinoline-3-carbox ylic acids, 147, 149-150 tetrahydroisoquinolines,I4 1- 145, 150- 160 Mammalian morphine, 162-168 biosynthetic pathways, 164-168 Mantella. 270 Methylation, tetrahydroisoquinoline, 153-154 8-Methylindolizidines, 5-substituted, 233-238 occurrence, 237-238 spectral properties, 233-236 6-Methylspenceamine, 263 Microbes, plant interaction, 61-78 Microorganisms adapted specialist allelochemicals, 96-97 plant defenses, 2-5 Minyobutes bombetes, 247, 249 Monoamine oxidase. inhibition by P-carbolines. 134-136 by isatin, 139-140 Monocyclic alkaloids, 251-254 piperidines, 253-254 pyrrolidines, 252-253 Morphine, 263-264 biosynthesis, 162- 163 mammalian, 162-168 Mutagenicity, allelochemicals, 52-54

Neuromuscular junction, allelochemicals action, 58-59 Neurotransmitters, allelochemicals and. 56-57 Noranabasamine, 256-257

300

INDEX

(S)-Norreticuline. 167-168 Nucleic acids, allelochemicals and, 5 1-55

Oxazolidine ring, samandarines, 194-195 Oxidation, salsolinol, 170, 172

Phyllohares, 187, 192-193

Phytoalexins, 71 Pictet-Spengler cyclization, 120-121, 123- 124 Piperidines. 253-254 Pitohui, batrachotoxins, 193- 194 Plants alkaloid structural comparison with mammalian alkaloids, 119-120 alkaloid toxicity toward other, 86 allelochemicals concentrations and activities, 87-89 biosynthesis induction after wounding or elicitation, 91-92 defense against microorganisms, 2-5 fitness, alkaloid importance, 92-96 herbivore interactions, 8-61 invertebrates, 8-22 mode of action, 23, 32-61 vertebrates, 22-31 invertebrate interactions, 8-22 microbe interactions, 61-78 antibacterial activity, 61-70 antifungal activity, 70, 72-78 mode of action of alkaloids in animals, 23, 32-61 vertebrate interactions, 22-3 I Precoccinelline. 244. 246-247 Protein biosynthesis, allelochemicals and, 55 Pseudophrynamines, 257-261 spectral properties, 259-260 structures, 257-259 Pumiliotoxin A class, 212-225 biological activity, 221-223 occurrence, 223-225 optical rotations, 221 spectral properties. 212, 217-218 structures, 212-221 Pumiliotoxin B, biological activity. 221-223 Pyridine alkaloids, 255-257 epibatidine, 255-256 noranabasamine, 256-257

Pyridoxal-derived isoquinolines, 160- 162 Pyrrolidines, 252-253 Pyrrolizidine oximes, 249-251 Pyrrolizidines, 225-228 biological activity, 226 3,5-disubstituted, 226-227 spectral properties, 226 structures, 225-227 Pyruvic acid pathway, 157

Quinolizidine alkaloids inhibitory action, 86 in lupines, 92-95 organ-specific concentrations, legumes, 87-88 Quinolizidines. 238-242 1,4-disubstituted, 239-242 occurrence, 241-242 spectral properties, 239-241 structures, 239

Reproduction, allelochemical action, 59-60 Reticuline conversion to salutaridine, 164 morphine precursor, 165 (R)-Reticuline, biosynthetic pathways. 167-168

Salsolinol optically active, 156-157 oxidation, 170, 172 Salutaridine, conversion of reticuline to, 164 Samandarines, 194-199 biological activity, 198 occurrence, 198-199 physical and spectral properties. 196-198 structure, 194- 196 synthetic, 196 Spinceamine, 263

Tetrahydro-0-carboline (IS,3S)dicarboxylic acid, 127-128 Tetrah ydro-0-carbolines optically active, 126-128 reaction. 125- 126

INDEX

Tetrah ydroisoquinolines methylation, 153- 154 occurrence, 150, 152-153 optically active, biosynthesis, 156-157 rat testicular endocrine function effects, 169 Tetrahydropapaverine, condensation, 153, 155-156 Tetrahydropapaveroline, 143-144 occurrence, 150-152 (R)-Tetrahydropapaveroline, 167-168 Tetrodotoxin, 264-267 1,3-Thiazolidines, formation, 168-169 Tricyclic alkaloids, 242-25 1 coccinellines, 245, 246-247 cyclopenta[b]quinolizidines,247-249 gephyrotoxins, 242-245 pyrrolizidine oximes, 249-25 1 Trypargine, 262-263

30 1

Tryptamine. condensation products, 138 L-Tryptophan condensation products, 138 peak E in, 136-137 Tryptophans. Pictet-Spengler reaction, 124, 126

uv spectra, Harmala alkaloids, 134 Vertebrates herbivores, adapted specialist allelochemicals, 100- 10 1 plant interactions, 22-31

Zetekitoxin. 268-269

I S B N 0-12-qb95'43-4