The Biochemistry of Alkaloids 978-3-540-04275-4, 978-3-662-01015-0

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Molecular Biology Biochemistry and Biophysics Molekularbiologie Biochemie und Biophysik

3 Editors: A. Kleinzeller, Philadelphia . G. F. Springer, Evanston H. G. Wittmann, Berlin Advisory Editors: F. Cramer, Gottingen . F. Egami, Tokyo· M. Eigen, Gottingen F. Gros, Paris· H. Gutfreund, Bristol . B. Hess, Dortmund Munich . R. W. jeanloz, Boston . E. Katchalski, H. ｪ｡ｨｲｭｫ･Ｌ＠ Rehovoth . B. Keil, Prague . M. Klingenberg, Munich 1. M. Klotz, Evanston . F. Lynen, Munich . W. T. j. Morgan, London . K. Muhlethaler, Zurich . S. Ochoa, New York R. R. Porter, Oxford . W. Reichardt, Tubingen . H. Tuppy, Vienna J. Waldenstrom, Malmo . R. J. Winzler, Buffalo

Springer-Verlag Berlin Heidelberg GmbH

Trevor Robinson

The Biochemistry of Alkaloids

With 37 Figures

Springer-Verlag Berlin Heidelberg GmbH

Trevor Robinson Associate Professor of Biochemistry Department of Biochemistry, University of Massachusetts Amherst, Massachusetts, USA

ISBN 978-3-540-04275-4 ISBN 978-3-662-01015-0 (eBook) DOI 10.1007/978-3-662-01015-0 AII tights reservcd. No part of this book may be translated or reproduced written permissîon from ｓｰｲｩｮｧ･ｾｖｬ｡Ｎ＠

ln

any form without

© by Springer-Verlag Berlin Heidelberg 1968

Originally published by Springer·Verlag Berlin Heidelberg New York 1968 Softcover reprint of the hardcover lst edition 1968 Library of Congress Catalog Card Number 68·22463. The use of general descriptive names, trade names, tradc marks etc. in thIS publication, even if the former are not especially indentified 15 not to be taken as a slgn that such namcs, as undcrstood by the Tradc Marks and Merchandise Marks Act, may accordingly be used freely by anyone.

Talc No. 3803

Preface

The alkaloids were of great importance to mankind for centuries, long before they were recognized as a chemical class. The influence they have had on literature is hinted at by some of the quotations I have used as chapter headings. Their influence on folklore and on medicine has been even greater. The scientific study of alkaloids may be said to have begun with the isolation of morphine by SERTURNER in 1804. Since that time they have remained of great interest to chemists, and now in any month there appear dozens of publications dealing with the isolation of new alkaloids or the determination of the structures of previously known ones. The area of alkaloid biochemistry, in comparison, has received little attention, and today is much less developed. There is a certain amount of personal arbitrariness in defining "biochemistry", as there is in defining "alkaloid", and this arbitrariness is doubtless compounded by the combination. Nevertheless, it seems to me that in any consideration of the biochemistry of a group of compounds three aspects are always worthy of attentionpathways of biosynthesis, function or activity, and pathways of degradation. For the alkaloids, treatment of these three aspects is necessarily lopsided. Much has been learned about routes of biosynthesis, but information on the other aspects is very scanty. It would be possible to enter into some speculation regarding the biosynthesis of all the more than 1,000 known alkaloids. I have for the most part limited consideration to those alkaloids for which there is experimental information. In a few cases, however, rigid adherence to this principle would have resulted in the exclusion of important compounds that seemed worthy of mention even if only in a purely speculative way. The alkaloids are best known for their pharmacological effects, but most of these effects are only slightly interpretable in terms of biochemistry. It is hoped that this monograph, besides summarizing some past findings in alkaloid biochemistry, will point out areas in need of attention from biochemists. I thank all those who helped in the preparation of this book - some who helped with their criticism and positive suggestions, others who helped with their forbearance in putting up with my withdrawal from many other endeavors. The literature has been reviewed through August 1967, with a few later references. I shall appreciate having any errors or omissions called to my attention. Amherst, Massachusetts June 1968

TREVOR ROBINSON

Table of Contents

Chapter 1 Chapter 2 Chapter 3 Chapter 4

Introduction . . . . . . . . . . . . Bibliography . . . . . . . . . . . .

6

General Theories of Alkaloid Biosynthesis Bibliography. . . . . . . . . . . .

13

Simple Amino Acid Derivatives and Protoalkaloids Bibliography . . . . . . . . . . . . . .

23

1 8

15

Pyrrolidine, Piperidine, and Pyridine Alkaloids Piperidine Alkaloids Pyridine Alkaloids Trigonelline . . . Ricinine . . . . . Tobacco Alkaloids Nicotinic Acid . . Bibliography . . .

24

Chapter 5

Tropane Alkaloids Bibliography . .

41 46

Chapter 6

Lupine Alkaloids Bibliography . .

48 53

Chapter 7

Isoquinoline Alkaloids Bibliography . . . .

54

Chapter 8

Aporphine and Morphinan Alkaloids Bibliography . . . . . . . . . .

63 70

Chapter 9

Amaryllidaceae Alkaloids and Colchicine Bibliography . .

72

25 28 29 29 31

36 38

62

76

77

Indole Alkaloids Ergot Alkaloids Bibliography . .

90

Chapter 11

Quinolines and Other Alkaloids Related to Anthranilic Acid Bibliography . . . . . . . .

92 96

Chapter 12

Some Miscellaneous Alkaloids . Pyrrolizidine Alkaloids. Purine Alkaloids . . . Benzoxazoles. . . . . Lycopodium Alkaloids.

Chapter 10

87

97 97 99 101 101

x

Table of Contents Betacyanins and Betaxanthins Imidazole Alkaloids. . . . Bibliography. . . . . . .

102 103 104

Terpenoid and Steroid Alkaloids. Diterpenoids . Steroids . . . . . . . . . . Bibliography . . . . . . . .

105 106 108

Chapter 14

Metabolism of Alkaloids by Bacteria and Animals. Animals Microorganisms Higher plants . Bibliography. .

115 115 120 122 123

Chapter 15

Biochemical Pharmacology of Alkaloids . Bibliography . . . . . . . . . . .

125 134

Chapter 13

113

Addendum . .

136

Subject Index .

141

Chapter 1

Introduction Glory to God for dappled things All things counter, original, spare, strange; Whatever is fickle, freckled (who knows how?) With swift, slow; sweet, sour; adazzle, dim; He fathers forth whose beauty is past change: Praise Him. Pied Beauty,

GERARD MANLEY HOPKINS*

The alkaloids are a group of naturally occurring organic compounds containing nitrogen. Their name, meaning "alkali-like", was given them because most of them are basic in nature and form salts with acids. Basicity is a common characteristic of organic nitrogen compounds, which can be regarded as derived from ammonia by the substitution of organic radicals for hydrogen. The simple amines are considered a class apart from the alkaloids, although with increasing complexity of structure the dividing line between amine and alkaloid tends to become indistinct. According to one definition, alkaloids contain nitrogen in heterocyclic rings while the nitrogen of amines is aliphatic. Compounds such as colchicine and mescaline would be excluded from the alkaloids by such a definition, but the exclusion seems inadvisable on the basis of historical usage. Introduction of the term "protoalkaloid" [1] for a group of borderline compounds leaves very few compounds unclassified. A few otherwise impeccable alkaloids, such as rutaecarpine and ricinine, that have electron-withdrawing functional groups either adjacent to or conjugated with their nitrogen atoms, do not show the characteristic of basicity. The chemical classification of alkaloids is based on their carbon-nitrogen skeletons. Some of the commonest skeletons are shown in Fig. 1-1. The chemistry of alkaloids is outside the scope of this book. Many textbooks of organic chemistry and heterocyclic chemistry offer an adequate introduction to the general chemical properties of alkaloids. Except for some increase in complexity, alkaloids show no striking peculiarities to set them apart from simpler compounds with the same functional groups. Readers interested in the proof of structure and synthesis of alkaloids are referred to the comprehensive treatise of MANSKE and HOLMES [ 1a] or the book of BOIT [2]. These works are also a source of much of the information about alkaloid distribution which is given below. Physically, most alkaloids are colorless, crystalline solids slightly soluble in neutral or alkaline aqueous solution but readily soluble in acid or in organic solvents such as ether, chloroform, or ethanol. A few alkaloids (e.g., nicotine and coniine)

* Reproduced

by kind permission of Oxford University Press from the "Poems of

GERARD MANLEY HOPKINS".

1 Robinson, The Biochemistry of Alkaloids

Introduction

2

are liquid at room temperature, and some (e.g., berberine and sanguinarine) are colored. Many alkaloids are optically active, and the fact that they rarely occur as racemic mixtures is taken as evidence that they are synthesized at least partially by enzymatic catalysis; In some cases both enantiomorphs are known to be naturally occurring, but each from a different source. Because of their polar, basic nature most alkaloids occur dissolved in plant saps as cations which on evaporation of the sap form salts with the organic acids that are also present. Just as some plants are noted for containing certain alkaloids, they may be noted for containing a preponderance of certain acids, and therefore a particular alkaloid may occur most often as the salt of

H

0

Pyrrolidine

Piperidine

Q

0

N"

H

W

CO

CO

Quinoline

Isoquinoline

ｾ＠

Indole

rn

Tropane

Pyridine

N"-:

ｾ＠

..-:N

ｬＺｲｾＩ＠ Purine

Benzylisoquinoline Fig. 1-1. Some common alkaloid ring skeletons a particular acid. However, there appears to be no necessary correlation between the biosynthesis of the alkaloid and of its accompanying acid. The distribution of alkaloids in nature, while not completely random, cannot be described in any simple and unambiguous way. Higher plants are the chief source of alkaloids, yet alkaloids are also known from club mosses (Lycopodium spp.), horsetails (Equisetum spp.), and fungi. Certain nitrogenous compounds occurring in animals are sometimes referred to as animal alkaloids, but many of them are relatively simple bases such as histamine, noradrenaline, and spermine. However, a few compounds with more typical alkaloid structures have been found in animals. Some of these may simply be derived with slight structural modification from plants eaten by the animal, but the possibility that some are synthesized in the animal body from simple precursors cannot be ruled out. The best examples of animal alkaloids are the salamander compounds (see Chapter 13) and the quinazolones excreted as a repellent by certain millepedes [2a]. Among the seed plants a greater variety of alkaloids has been found

Introduction

3

in Dicotyledons than in Monocotyledons or gymnosperms. It has been estimated that 10 to 20% of all plants contain alkaloids, but the accuracy of any such estimate is limited by the sensitivity of the analytical methods used [1]. Three times as many alkaloid-producing plant families are found in the tropics as in temperate zones, but this only reflects the greater number of tropical plants generally; since the ratio of tropical alkaloid families to total tropical families is the same as the ratio for temperate families [3]. Although some alkaloids are taxonomically quite restricted in occurrence, others are found widely in unrelated plants. A statistical analysis of 3,600 alkaloid plants showed caffeine occurring in the largest number of families, lycorine in the largest number of genera, and berberine in the largest number of species [4]. . When the same alkaloid has been found in several different plant species, it is tempting to assume an evolutionary relationship between the plants and therefore to describe the occurrences as "homologous". However, decisions about homology must depend more on knowledge of biosynthetic pathways and mechanisms than on knowledge of end products. Far too little is known to make any generalization that the same alkaloid is always produced in the same way. If different pathways or mechanisms are found in different plants to lead to the same alkaloid, the occurrences must be described as "analogous", and they then can offer no information regarding phylogenetic relationships. MOTHES has stressed the important point that the presence of an alkaloid in a plant shows not only that the biosynthetic pathway is present but also that the plant can tolerate the alkaloid. The absence of an alkaloid does not mean necessarily that the biosynthetic pathway is absent; it may mean that an active degradative pathway is also present [5]. The foregoing considerations show that taxonomy can be based only approximately on accumulated products of metabolism. For the most part classical taxonomy has taken little account of alkaloid distribution. If the same type of alkaloid is observed in two plants already thought to be related, its presence is used as evidence to support the relationship; its occurrence in two plants thought to be unrelated is cited as an example of independent evolution. The appearance of two apparently quite different structural types of alkaloid in supposedly closely related species cannot be taken as grounds for questioning the closeness of relationship, since it is not difficult to visualize a single-gene mutation setting off an entirely different biosynthetic pathway - for instance using many of the type reactions of the original pathway but in a different order or with a different starting material (see Chapter 2). Still, alkaloids of similar structure are often found within plants of a given taxonomic grouping, and the lower the grouping in the taxonomic hierarchy, the greater the similarities in structure. Practically this generalization is useful in locating plant material for the study of alkaloid biochemistry. Theoretically it probably does argue for similar biosynthetic pathways in plants that are otherwise similar. The occurrence of "chemical races" has been frequently observed among alkaloid plants. A chemical race is an intraspecific variety distinguished from other plants of the same species only by its chemical composition. The intensive breeding of medicinal plants for high content of some active constituent has produced some of these races, but others appear to have arisen naturally and to have persisted in geographical isolation. Some examples of chemical races are tabulated as follows: 1*

4

Introduction

Species

Distinguishing Features

Duboisia myoporoides Sedum acre Hordeum vulgare Papaver somniferum Claviceps purpurea

hycoscyamine or hyoscine or norhyoscyamine sedamine and nicotine, or sedridine hordenine or N-methyltyramine narcotine or absence of narcotine ergotamine or ergocristine

In a few cases some genetic analyses of these races have been performed. Differences in ploidy have sometimes resulted in differences in composition. It has been consistently found among Datura and Atropa spp. that tetraploid plants have a higher alkaloid content than diploid plant!! [6]. The formation of alkaloids varies notably from tissue to tissue within the same plant and also changes during the course of ontogeny. This kind of variability as well as the occurrence of chemical races can account for disagreements over the presence or absence of a particular alkaloid in a given plant [7]. In general, alkaloids tend to accumulate in [8]: 1. Very active tissues. 2. Epidermal and hypodermal ｴｩｳｵｾＮ＠ 3. Vascular sheaths. 4. Latex vessels. It must be emphasized that the sites of accumulation are not necessarily the sites of synthesis. Nicotine, for example, is synthesized in the roots but is translocated and accumulates in the leaves. In other cases the complete synthesis may require cooperation of different plant tissues. Thus those species of tobacco that have nornicotine in their leaves make nicotine in the root, translocate it to the leaves and then demethylate it (see Chapter 4). Tropane alkaloids first made in the roots of Datura spp. are extensively modified in the leaves [9]. Although prominent in very actively growing tissues, alkaloids are found not in the youngest cells of these tissues but in somewhat older cells which are becoming vacuolated. The presence of alkaloids in vacuoles rather than the surrounding cytoplasm was amply shown in early studies and has more recently been demonstrated using electron microscopy [10]. As a result of translocation and of different steps taking place in different tissues, it may be quite difficult to determine the real site of alkaloid synthesis. Grafting experiments and culture of isolated plant parts have been the two techniques giving the greatest amount of useful information, but difficulties exist with both approaches. The chief problem is that negative findings may not necessarily mean that the cultured or grafted organ has no synthetic ability, but only that alkaloid synthesis requires contributions from other parts of the plant. Many specific examples of alkaloid localization are cited by JAMES [8] and MOTHES [9]. Tissue cultures have been shown in several cases to produce alkaloids characteristic of the parent plants [11-13]. The use of plant tissue cultures for commercial production of alkaloids has been proposed [14] but is apparently not yet practical. Ontogenetic changes in alkaloid content of plants have been known for many years and are the basis for various empirical rules about the proper time for harvesting plants whose alkaloids have commercial value. The most general principle of ontogenetic change is that alkaloid content increases rapidly at the time of cell enlargement and vacuolization, the increase being followed by a slow decline in concentration

Introduction

5

during senescence. This pattern has been observed in several tissues of different plants, although leaves have been most often studied. The time of maximum alkaloid content will differ according to whether concentration or total amount is stated, with the concentration maximum coming earlier. Several other circumstances may have striking effects on the overall ontogenetic pattern. The initiation of flowering may stop or inhibit alkaloid formation [9]. A young leaf on an old plant may reflect in its alkaloid content the plant's stage of development rather than its own [8]. There is no consistent pattern for the ontogeny of alkaloids in developing seeds and germinating seedlings, although unfertilized ovules of alkaloid plants normally have alkaloids. In some species the alkaloid concentration may decrease after fertilization giving mature seeds with little or no alkaloid (e.g., Nicotiana, Papaver, Hordeum, Datura, and Erythroxylon spp.). Seeds of other species may contain high concentrations of alkaloid (e.g., Lupinus and Pf?ysostigma spp.). During germination alkaloid synthesis may begin within a few days (Hordeum) or only after several weeks (Datura). The alkaloid content of alkaloid-rich seeds may actually decline in the early stages of germination - both in concentration and, more significantly, in total amount per plant. A striking example of ontogenetic change is offered by Catharanthus roseus, which contains virtually no alkaloid in its seed, then develops alkaloids until at 3 weeks after germination they are present throughout the plant. They then disappear almost completely and reappear at 8 weeks [7]. Very rapid changes in alkaloid pattern have been observed to occur in Conium maculatum. Over a 24 h period the developing fruit shows complementary changes in the two alkaloids coniine and coniceine [15]. Several external factors have been found to influence alkaloid content, but direct biochemical explanations for these influences are mostly lacking. Light is, of course, essential for growth of higher plants, so that its beneficial influence on total alkaloid content is expected. For the most part this influence is indirect; however, more specific light effects have also been observed. Etiolation increases both concentration and total amount of ricinine in Ricinus communis. Ultraviolet light, ineffective in photosynthesis, is most effective in stimulating solanine formation in potato tubers [8]. In the light Catharanthus roseus has vindoline as its predominant alkaloid, but in the dark this alkaloid is absent [7]. The effects of photoperiod on alkaloid content are obviously related to the effects of photoperiod on initiation of flowering. Lycopersicon glandulosum, a short-day plant, if grown under long days contains five times as much tomatine as under short days. Species less dependent on day length for flowering show a lesser response of tomatine content to photoperiod [16]. Such findings raise the possibility that tomatine serves specifically as an inhibitor of flowering and acts as a chemical mediator of photoperiodic flower induction. Another interpretation would be that decomposition of tomatine gives rise to other steroids related to pregnenolone which serve as flowering hormones (see Chapter 13). Effects of nutrition and plant growth substances on alkaloid content have received some attention, with the rather commonplace conclusion that factors favoring growth generally favor alkaloid formation. It does appear that nitrogen supplied to plants in the form of ammonium salts is somewhat better than nitrates for increasing alkaloid formation [8]. This result suggests a rather direct use of ammonia in alkaloid synthesis, while nitrate acts more indirectly by increasing overall growth. A potassium-calcium antagonism has also been observed. A high K/Ca ratio favors protein synthesis, but a low ratio favors alkaloid synthesis [8]. Such growth factors as kinetin,

Introduction

6

adenine, and indole-3-acetic acid have been shown to increase the alkaloid content of lupine embryos in sterile culture [17]. Any discussion of the function of alkaloids in plants runs the danger of becoming teleological, but the matter has been of too much interest to too many people to ignore it altogether. Most often alkaloids have been called nitrogenous "waste products" analogous to urea and uric acid in animals. Their occurrence in vacuoles rather than the living parts of protoplasm supports such a view. However, nitrogen is often scarce for plants, and its reutilization is more often the rule in plant metabolism. The metabolic activity of some alkaloids also puts them in a class apart from ordinary waste products. Fluctuations in concentration and frequently rapid conversion to other products have been observed. Knowledge of alkaloid catabolism is still scanty, so that generalizations are not possible; but there is evidence that some of the pyridine alkaloids can serve as precursors of nicotinic acid and thus can be reservoirs of this vitamin (see Chapter 4). A possible role of alkaloids in protecting plants against parasites or herbivores has been frequently proposed [8], and a few examples can be given in support of such an idea, but there are many more examples of plants whose high content of alkaloids confers no protection against their major enemies. Where specific evidence is available for the protective function of an alkaloid, it is reported in the chapter appropriate to that alkaloid and cited in the Index under "Ecology". The role of alkaloids as detoxification products appears plausible in some instances, as in the case of alkaloids that may remove such active molecules as indole-3-acetic acid or nicotinic acid from sites where they could unbalance metabolism. Because of the great diversity among alkaloids it seems likely that theories which give plausible functions to several of them cannot be universally applicable. Further, it is impossible to state what kind of evidence is acceptable for proving the function of an alkaloid. Complete removal of an alkaloid from a plant, where experimental manipulation has made this possible, usually has no effect on the plant; and, of course, 80 to 90% of all plants never contain alkaloids. It is evident that biosynthesis of alkaloid molecules often must require energy and in some instances the presence of highly specific enzymes. The perpetuation of such a low entropy system through the course of evolution seems to call for an explanation in terms of useful function, but no generally adequate explanation has been forthcoming.

Bibliography 1. MOTHES, K., U. A. ROMEIKE: In: Handbuch der PRanzenphysiologie 8, pp. 989-1049. RUHLAND, W., ed. Berlin-Gottingen-Heidelberg: Springer 1958. 1 a. MANSKE, R. H. F., and H. L. HOLMES: The alkaloids, Vols. 1-8. New York: Academic Press 1950-1965. 2. BOIT, H.-G.: Ergebnisse der Alkaloid-Chemie bis 1960. Berlin: Akademie Verlag 1961. 2a. SCHILDKNECHT, H., U. MASCHWITZ und W. F. WENNEIS: Naturwissenschaften 54,

196-197 (1967). 3. 4. 5. 6. 7.

McNAIR, J. B.: Bull. Torrey Botan. Club 62, 219-226 (1935). WILLAMAN, J. J., and H. L. LI: Econ. Bot. 17, 180-185 (1963). MOTHES, K.: Naturwissenschaften 52,571-585 (1965). ROWSON, J. M.: J. pharm. Bdg. 5-6, 195-221 (1954). MOTHES, K., 1. RICHTER, K. STOLLE und D. GROGER: Naturwissenschaften 52, 431

(1965). 8. JAMES, W. 0.: In Ref. 1 1, 15-90.

Bibliography

7

9. MOTHES, K.: In Ref. 1 6, 1--29. 10. CRANMER, M. F.: Ph. D. thesis. Austin, Texas: University of Texas 1966. 11. GRUrZMANN, K.-D.: Pharmazie 21, 340-345 (1966). 11 a. FURUYA, T., H. KOJIMA, and K. SYONO: Chem. & Pharm. Bull. (Tokyo) 14, 1189-1190 (1966). 12. CAREW, D. P.: Nature 207, ｾＹ＠ (1965). 12a. CAREW, D. P.: J. Pharm. Sci. 55,1153-1154 (1966). 13. SUHADOLNIK, R. J.: Lloydia 27, 315-321 (1964). 14. KAUL, B., and E. J. STABA: !icience 150, 1731-1732 (1965). 15. FAIRBAIRN, J. W., and P. N. SUWAL: Phytochem. 1, 38-46 (1961). 16. SANDER, H.: Planta 52, 447--466 (1958). 17. MACIEJEWSKA-POTAPCZYKOWO\, W., and R. NOWACKI: Acta Soc. Botan. Polon. 28, 83-93 (1959).

Chapter 2

General Theories of Alkaloid Biosynthesis o my soul I if I realize you I have satisfaction, Animals and vegetables I if I realize you I have satisfaction, Laws of the earth and air I if I realize you I have satisfaction. Leaves of Grass,

WALT WHITMAN

In spite of the large numbers and great diversity of alkaloid structures, it seems possible now to discern a few general principles that apply to the biosynthesis of many different alkaloids. Some broad theories of alkaloid formation have borne the test of many experimental investigations, some have received no support, some have been refuted, and some newcomers have not yet been tested adequately. In the present chapter brief mention will be made of experimental findings that have general significance. More information on biosynthetic pathways can be found by consulting the appropriate specific chapters or by referring to the Index for names of compounds. Various earlier proposals for the derivation of alkaloid structures from common amino acids were considered and incorporated into the far-reaching proposals presented in 1917 by Sir ROBER'!' ROBINSON [1] and later expanded [2]. These comprehensive proposals were based on analogy with reactions of organic chemistry and on comparisons of structure rather than on biochemical evidence. Nevertheless, biochemical experiments have confirmed the predicted pathways to a great extent. The assumptions of Robinson's scheme of biosynthesis may be summarized as follows: 1. The fundamental skeletons of alkaloids are derived from common amino acids and other small, biological molecules. 2. A few simple types of reaction suffice to form complex structures from these starting materials. For example, the aldol condensation:

OH

" C=O+H-C-X I / I

I I

--+) -C-C-X

I I

the carbinolamine condensation:

I I

I

I I I

I I

I

I I I

N-C-OH+H-C-X - - + N-C-C-X

General Theories of Alkaloid Biosynthesis

9

the aldehyde-amine condensa tion: H ｎＭｈＫｏｾｃ

I

OH

I Ｍｾ＠

I I

-N-C-

I

I

H

as well as simple dehydratior: s, oxidations, and decarboxylations. (X represents an "activating" group, such as carbonyl.) It is important to understand that the ROBINSON proposals were never intended to apply to specific compounds but only to general groups of structurally related compounds. Thus a possible D)rmation of the tropane skeleton from succindialdehyde, methylamine, and acetonedicaboxylic acid could be represented as:

However, the actual reactants in vivo might resemble more closely ornithine, glycine, and citric acid, which by relatively simple reactions could be transformed into the three represented precursors. Summarizing this categorical approach, Fig. 2-1 shows

Q

Omithine ---+

0

Lysine -------+

N

Phenylalanine or T:rrosine

j )C-C-N ｉ

Ｍｾ＠

(HO) Tryp :ophan Ｍｾ＠

ｾ＠

.0

I I Q::J .0

C-C-N

N

Fig. 2-1. Some common structu -al elements of alkaloids and their presumed precursors

some common structural elements of alkaloids and the types of precursors from which they might be derived. A grea1 number of experiments have been done to show that with low concentrations and u 1der very mild conditions of temperature and pH it is possible to bring about reacti:ms of the hypothetical precursors to form complex structures resembling alkaloids_ Biochemical experiments first carried out about 1955 have continued to lend support to the overall ideas of Sir ROBERT ROBINSON, although

General Theories of Alkaloid Biosynthesis

10

certain discrepancies and variations have been found. The biochemical experiments will be considered in the separate chapters on the different groups of alkaloids. Another useful generalization in considering pathways of alkaloid biosynthesis is the probable importance of amine oxidases in the production of alkaloid structures. MANN and SMITHIES [3] and HASSE and MAISACK [4] showed that cyclic compounds were formed as the result of the action of plant diamine oxidase on 1,4-diaminobutane (putrescine) or 1,5-diaminopentane (cadaverine). Initial formation of an imine and ring closure with loss of ammonia, or formation of an aldehyde and ring closure with loss of water were two possible mechanisms suggested. The L11-pyrroline formed in the above reaction or the L1 Cpiperideine formed from cadaverine could then by suitable condensations (since they are very reactive molecules) give rise to various alkaloids containing pyrrolidine or piperidine rings. It was in fact found that reaction of L1cpiperideine with acetoacetic acid resulted not only in condensation but also in spontaneous decarboxylation with formation of the known alkaloid isopelletierine [5]. CH 2-CH2

I

I

CH 2 CH 2 -----+

"-NH2"-NH2

o

o I

+CH 3 CCH 2 COOH ----+ Isopelletierine

More examples of alkaloids believed to be formed through the action of diamine oxidase will be found in Chapters 4 and 6. KACZKOWSKI has reviewed the probable role of diamine oxidase in alkaloid biosynthesis [6]. Some conversions which have been attributed to amine oxidation could just as well have been brought about by transamination, since the action of transaminase also results in formation of a carbonyl group from an amino group. Cases where alkaloid synthesis does not correlate well with the presence of amine oxidase should therefore be investigated to determine the activity of transaminase. JINDRA et al. [6a] have shown the presence of enzymes catalyzing transamination in a number of alkaloid-containing plants, but so far the direct participation of these enzymes in alkaloid biosynthesis has not been established. It has become clear that the O-methyl, methylene dioxy, and N-methyl groups found in the great majority of alkaloids are derived by direct transfer from S-adenosylmethionine. Feeding experiments with 14C-Iabelled methionine have shown this for many different alkaloids. In a few cases this transmethylation has been brought about in vitro by cellfree extracts. Tracer experiments have shown in a few alkaloids the possible origin of methyl groups from formate. Presumably this is first converted to

General Theories of Alkaloid Biosynthesis

11

the methyl group of methionine. The stage at which methylation occurs may well vary from one alkaloid to another. With some (e.g., nicotine) it appears that the methyl group may be introduced at a very early stage, perhaps to the amino acid precursor. Other methyl groups may be added very late - indeed, methylation may preclude any further reaction, as in the case of laudanosine. A feature of many alkaloid structures is the attachment of two aromatic rings to each other. A general explanation for all such structures was put forward by BARTON and COHEN [7] and since then has been applied to many specific alkaloids and thoroughly accepted [8]. Oxidation of a phenol by a one-electron transfer process gives a transient free radical with the odd electron more or less localized at the positions ortho and para to the phenolic hydroxyl group. Coupling of the free radicals with each other then occurs:

0

OH

6 -- Q H

ｾ＠

r H 0

o

H-

6i'O 6Po

-

1

!

1 OH

OH

H0-O-O-0H

E

o

oObo -

QH 0

6-6 ｾ＠

OH Q-O-0H

ｾ＠

The free radical oxidation can be brought about enzymatically by such common plant enzymes as phenol oxidase or peroxidase [9]. The requirement for a free hydroxyl group ortho or para to the coupling position means that O-methylation of precursors can prevent coupling; or when several hydroxyl groups are present in the precursors, methylation of certain ones can control the direction of coupling. A further elaboration of this kind of mechanism is the suggestion of FRANCK [10] that oxidative coupling reactions most likely occur with alkaloids having quaternary nitrogen. With non-quaternary nitrogen alternative reactions are more likely. Since oxidative coupling mechanisms have been applied most extensively to the morphine alkaloids, further consideration is given to these mechanisms in Chapter 8. Many alkaloids are now known to incorporate into their structures units derived from mevalonic acid, the well-known precursor of terpenoids and steroids. With some alkaloids, indeed, the entire carbon skeleton comes from mevalonic acid by typical terpenoid pathways. These terpenoid and steroid alkaloids are considered in Chapter 13. In other alkaloids terpenoid units are more incidental, as for instance the

12

General Theories of Alkaloid Biosynthesis

Cs unit oflysergic acid (Chapter 10) or the C9 or ClO unit which in several variations is quite widespread (see p. 80 et seq.). Several alkaloids having an IX-pyridone structure are known to coexist with corresponding pyridinium salts (e.g., oxysanguinarine and sanguinarine in Sanguinarea canadensis, oxydemethoxyalstonidine and demethoxyalstonidine in Gurouparia gambir). It has been proposed as a general principle that enzymatic oxidation of pyridinium salts to pyridones accounts for the biosynthesis of pyridone alkaloids. Such a reaction has been catalyzed by an enzyme from Ricinlls commllnis [11]. The teleological question of why alkaloids exist in plants has been touched on in Chapter 1. A somewhat more approachable question is, "How did the biochemical pathways of alkaloid formation evolve?" Unfortunately, in no case is the total pathway for synthesis of an alkaloid known. In a few cases it appears that we are close to knowing most of the intermediates, but knowledge of the nature of the mechanisms, enzymatic or otherwise, for bringing about the series of transformations is scarcely approached. Thus speculation regarding evolution of a pathway necessarily remains vague and generalized. JAMES [12, 13] proposed that many of the steps in an alkaloid synthetic pathway are probably not specific to that pathway. Some of them may be catalyzed by enzymes of low specificity which perform other functions in plant metabolism. Some steps may occur spontaneously, without mediation of any enzyme. Thus plants that do not synthesize any alkaloids may nevertheless possess considerable segments of potential biosynthetic pathways, and mutation of a single gene might be enough to start off the entire sequence. Such a single gene mutation might result in appearance of a new compound or it might result in a structural modification within a cell, with the result that cellular constituents kept segregated in the parent cell are able to interact in the mutant. Several observations support this overall viewpoint. Pea plants do not contain alkaloids, but homogenates of pea plants provided with cadaverine synthesize anabasine [14]. Beans, which do not contain alkaloids, if fed certain lupine alkaloids will transform them into other lupine alkaloids [15]. One can argue that peas, beans, and lupines, all members of the Leguminosae, are descended from an alkaloid-containing ancestor and that certain parts of the alkaloid biosynthetic mechanism were lost in the evolution of peas and beans but retained by the lupines. It can as well be argued that the ancestor was alkaloid-free but, like peas and beans, had the potential for synthesizing alkaloids once a mutation had occurred and that such a mutation did occur somewhere during the phylogeny of the lupines. Although a choice cannot be made between these alternatives at the moment, the question is not meaningless. What we need to answer it is detailed knowledge of the pathway and of which steps are missing in which species. An elaboration of the "one special reaction" point of view has been applied by Bu'LoCK and POWELL [16] to account for the formation of secondary products of microorganisms. It might, however, be just as validly applied to higher plants. Using a schematic treatment, these authors have shown that given a single special product outside the general metabolism of a cell and a few enzymes that are not absolutely specific (Le., which can act on several compounds of related structure), a great number of products could be formed. Inclusion of some non-enzymatic reactions would, of course, greatly enlarge the number of possibilities. All of the previously described approaches to the elucidation of alkaloid biosynthetic pathways have been rather hypothetical. They have centered on considera-

Bibliography

13

tions of structures and possible reaction mechanisms for producing them or they have taken note of the co-occurrence of compounds and postulated their interconversions. The more direct approach of feeding isotopically labelled materials to plants, then isolating alkaloids, and determining label incorporation has in recent years provided most of the information we have about synthetic pathways in vivo. Early radiotracer experiments, often with randomly labelled substrates, merely showed that some atoms of the substrate reached the alkaloid. The pathway may have been indirect. Specific labelling of the precursor and degradation of the isolated alkaloid so that the label incorporated into individual atoms could be measured is now a routine approach and gives at least a qualitative idea of how directly the precursor goes to the alkaloid. Finally, double and even triple labelling has been applied in a few cases to establish almost beyond doubt that a precursor has gone directly to an alkaloid (see Chapter 8). If a single molecule labelled with tritium, 14C, and 15N is converted to an alkaloid with the same isotopic ratio (after correction for any loss of atoms), there can be little doubt that it has served as a direct precursor without degradation and reassembly. Still, the tracer technique, however carefully applied, does not usually avoid the problem arising from the likelihood that many non-specific enzymes and reaction pathways exist in plants. A labelled compound could travel through these pathways and arrive at an alkaloid that might normally be made along quite different pathways from a different precursor. The compound fed must be shown to be a normal constituent of the system, and the rate of the proposed steps must be consistent with the normal, overall rate of product formation. A difficult, but powerful way of meeting such objections has been to use the only carbon compound that is a normal substrate for higher plants, carbon dioxide. Studies with labelled carbon dioxide, though still relatively new, have already given some very useful information regarding the biosynthesis of nicotine (Chapter 4) and the opium alkaloids (Chapter 8). In vitro studies of alkaloid biosynthesis using more or less purified plant enzyme systems have so far given few results. Some simple reactions such as methylations and oxidations have been brought about by cell-free preparations, and some knowledge of the enzymes involved has been obtained. Nevertheless, it appears from the literature that such experiments have not been tried very often, or else negative results are not often published [17].

Bibliography

t. ROBINSON, R.: J. Chern. Soc. 111,876-899 (1917). 2. - The structural relations of natural products. Oxford: University Press 1955. 3. MANN, P. J. G., and W. R. SMITHIES: Biochern. J. 61, 89-100 (1955). 4. HASSE, K., U. H. MAISACK: Biochern. Z. 327, 296-304 (1955). 5. CLARKE, A. J., and P. J. G. MANN: Biochern. J. 71, 596-609 (1959). 6. K4,CZKOWSKI, J.: Postepy Biochern. 7,431-443 (1961). Chern. Abstr. 55, 27782. 6a. JINDRA, A., P. KOVACS, H. SMOGROVICOVA, and M. SovovA: Lloydia 30, 158-163 (1967).

7. BARTON, D. H. R., u. T. COHEN: Festschrift Arthur Stoll, pp. 117-143. Basel: Birkhauser Verlag 1957. 8. KUHN, L., U. S. PFEIFER: Pharrnazie 20,659-680 (1965). 9. SCHENK, G., K.-H. FROMMING und H.-G. SCHNELLER: Arch. Pharrn. 298, 855-860 (1965). 10. FRANCK, B.: Angew. Chern. 74, 724 (1962).

14

General Theories of Alkaloid Biosynthesis

ROBINSON, T.: Phytochem. 4, 67-74 (1965). JAMES, W. 0.: J. Pharm. and Pharmacol. 5, 809-822 (1953). - Endeavour 12,76-79 (1953). MOTHES, K., H. R. SCHUTTE, H. SIMON und F. WEYGAND: Z. Naturforsch. 14b, 49-51 (1959). 15. NOWACKI, E.: Bull. acado polon. sci. biol. 6, 11 (1958). 16. Bu'LoCK, J. D., u. A. J. POWELL: Experientia 21,55-56 (1965). 17. BOSE, B. c., S. S. GUPTA, and S. MOHAMED: J. Indian Chern. Soc. 35, 81-82 (1958).

11. 12. 13. 14.

Bibliography of Reviews on Alkaloid Biosynthesis BEZANGER-BEAUQUESNE, L.: Bull. soc. botan. France 105, 267-291 (1958). DAWSON, R. F.: Advances in Enzymol. 8, 203-251 (1948). FRANCK, B.: Naturwissenschaften 47, 169-175 (1960). LEETE, E.: In: Biogenesis of natural products, pp. 739-796. BERNFELD, P., ed. New York: Macmillan 1963. - Science 147, 1000-1005 (1965). - Ann. Rev. Plant Physiol. 18, 179-196 (1967). LUCKNER, M.: Pharmazie 19, 1-14 (1964). MADAN, C. L., and B. MUKERJI: J. Sci. Ind. Res. (India) 17A, 224-234 (1958). MARION, L.: Bull. soc. chim. France, 1958, 109-115. MOTHES, K.: Ann. Rev. Plant Physiol. 6, 393-432 (1955). - Symp. Soc. Exper. Biol. 13,258-282 (1959). - Arch. Pharm. 295, 114-115 (1962). - , u. H. R. SCHUTTE: Angew. Chern. 75, 265-281, 357-374 (1963). POISSON, J.: Annee Biol. 34, 395-427 (1958). RAMSTAD, E., and S. AGURELL: Ann. Rev. Plant Physiol. 15, 143-168 (1964). WENKERT, E.: Experientia 15,165-173 (1959).

Chapter 3

Simple Amino Acid Derivatives and Proto alkaloids With shepherd's purse, and clown's all-heal, The blood I staunch and wound I seal. Only for him no cure is found, Whom Juliana's eyes do wound;

Damon the Mower,

ANDREW MARVELL

A number of naturally occurring nitrogen compounds have basic properties in common with the alkaloids but are usually not classified as alkaloids because their structures are relatively simple. Their nitrogen atoms are not incorporated into heterocyclic skeletons; and they are seen to be derivable from amino acids by a few, simple reactions. Mere decarboxylation of amino acids produces simple amines, which are never classed as alkaloids. Further modification of such simple amines by the introduction of methyl groups or hydroxyl groups, or both, gives rise to a group of ambiguously classified compounds that are sometimes called alkaloids; but it seems useful to distinguish them by the name "protoalkaloids", suggesting both their simple structures and their possible role as precursors of more typical alkaloids (cf. Chapter 1). Simple amines are widely distributed in higher plants, though usually in low concentrations. A survey of 220 species revealed amines in many of them. Since some plants contained only trace amounts, the exact percentage of amine-containing species cannot be stated with precision. Isopentylamine was most widespread, occurring in 75 species [1]. Table 3-1 lists some of the simple amines found in plants with the amino acids from which they are derived by decarboxylation. Radioactive leucine and valine fed to inflorescences of Sorbus aucuparia and Crataegus monogyna gave rise, respectively, to labelled isoamylamine and isobutylamine. Enzyme preparations were also found to carry out these decarboxylation reactions [2]. On the other hand, free ethanolamine, although often detected in leaves, was not found to acquire label when radioactive serine was fed to sugar beet leaves [3]. Possibly the actual decarboxylation occurs in some other tissue. The volatile amines given off by some flowers seem to function as attractants of insect pollinators [4]. Putrescine (1,4-diaminobutane), although obviously derivable by a decarboxylation of ornithine, may be made in a more indirect way from arginine via agmatine (1). Agmatine fed to excised barley leaves was found to be converted to putrescine with N-carbamylputrescine as an intermediate [5]. Studies on the enzymology of this sequence have shown that the enzyme catalyzing the last step has increased activity when plants are grown under potassium deficiency, and therefore such plants are found to be unusually high in putrescine content [6]. Agmatine is also of interest as a component of hordatine A (2),

Simple Amino Acid Derivatives and Protoalkaloids

16

an antifungal compound of barley [7]. The main soluble nitrogen compound of Galega offtcinalis, galegine (3), is also an amidine like agmatine; and its amidine group has been shown by tracer experiments to come from arginine, but the dimethylacrylyl group can not be derived simply from any natural amino acid [8]. Arginine, as a protein amino acid, is much more abundant than ornithine, which is only a transient intermediate of arginine biosynthesis. Thus under conditions of protein Table 3-1. Some simple amines occurring in plants, with their parent amino acids Amine

Amino Acid Precursor

CH3NH2 Methylamine

COOH

I

CH2NH2 Glycine. HOCH 2CH 2NH 2 Ethanolamine

NH2 HOCH 2CHCOOH Serine

ｃｈＳｾ＠

ｃｈＳｾ＠

NH2 /CHCH2NHz

I

/CHCHCOOH

CH3 Isobutylamine

CH 3 Valine

ｃｈＳｾ＠

ｃｈＳｾ＠

NH2 /CHCHzCHzNH2

CH 3 Isopenty lamine

I

/CHCH 2CHCOOH

CH3 Leucine

NH2 HzN (CH2)4NHz Putrescine

I

H2N (CH2)3CHCOOH Ornithine NH2

H 2N(CH 2)sNH 2 Cadaverine

I

H2N (CH2)4CHCOOH Lysine

turnover the formation of putrescine from arginine seems more likely than its formation from ornithine. The possible role of diamines as alkaloid precursors is discussed generally in Chapter 2. Their transformation into heterocyclic rings is presumably mediated by diarnine oxidase, which oxidizes one amino group to an aldehyde that then condenses with the other amino group. A survey of the plant kingdom has shown diamine oxidase to be present in many different dicotyledonous families but absent from monocotyledons and gymnosperms [9]. Some purification and characterization of the diamine oxidase of pea seedlings has also been reported [10].

Simple Amino Acid Derivatives and Protoalkaloids

17

Choline (4), acetylcholine and related compounds are widely distributed in plants both as components of phospholipids and also as simpler derivatives [11]. Phosphorylcholine functions as an important phosphate carrier in plant sap, and the appearance of free choline or other choline derivatives in leaves may signal removal of phosphorus from phosphorylcholine translocated from the roots. During germination of

Galegine (3)

I

H NH

H

I I

0

I I

H2N (CH2)4N--C-NH2 Agmatine (1)

---+ H2N (CH2)4N-C-NH2

N-Carbamylputrescine

1

H2N (CH2)4NH2 Putrescine

O-cx--D-Glucopyranosyl NH

I

I

CNH(CH2)4NHCNH2 Hordatine A (2)

Cicer arietinutlJ, choline content increased to a maximum in 96 h and then decreased. Availability of methyl groups apparently limited choline formation, since supplying methyl donors increased choline content while supplying other acceptors decreased it [12]. In this case it seems likely that de novo choline synthesis was occurring; in other [Cd H 2 NCH2 COOH Ｋ＼ＭｾＩ＠

'4

NH2

I

HOCH2 CHCOOH

HOCH2 CH2 NH 2 ＭｾＩ＠

Glycine

r(4),

1

Ch0 Betaine

Phosphorylcholine

Acetylcholine

Fig. 3-1. Pathways of choline metabolism 2

Robinson, The Biochenustry of Alkaloids

18

Simple Amino Acid Derivatives and Protoalkaloids

cases choline appearance may only indicate breakdown of phospholipids. The biosynthesis of choline has been investigated using leaf discs and homogenates of Beta vUZ[l,aris [13]; the probable pathways are outlined in Fig. 3-1. As expected, the methyl groups are derived from methionine [14]. It is interesting that betaine seems not to be formed by direct methylation of glycine. A compound similar in properties to choline is muscarine (5), which is at least partially responsible for the toxicity of Amanita and Inorybe spp. offungi [15].

Muscarine (5) The presence of the polyamines spermidine and spermine has been demonstrated in ovules and embryos of higher plants, and a possible regulatory role has been proposed for them [15al. H H2N (CH2)4N (CH2)3NH2

H H H2N (CH2)3N (CH2)4N (CH2)3NH2

Spermidine

Spermine

Certain basic substances isolated from plants have been first classed as alkaloids and later found to be more correctly classed as peptides, or slightly modified peptides. The structure of one of these, pandamine from Panda oleosa, is given below [16].

Pandamine Other compounds of this group are julocrotine from Julocroton montevidensis [11], zizyphin from Zi:?')'phus oe!loplia [18], and ceanothine B from Ceanothus americanus [19]. Peptides are also found as an essential part of the lysergic acid group of alkaloids (see Chapter 10) as well as being toxic constituents of Amanita spp. [20] and of Viscum album [21].

Simple Amino Acid Derivatives and Protoalkaloids

19

Protoalkaloids related to the aromatic amino acids tyrosine and dihydroxyphenylalanine are rather common in their own right and are also important as postulated precursors of other groups of alkaloids. While phenylethylamine (6) formed from phenylalanine is of widespread occurrence in plants, derivatives of it are less common than derivatives of tyramine (7); it therefore seems likely that phenylethylamine is metabolized to tyramine before undergoing further reactions. Although dihydroxyphenylalanine is not a common plant amino acid, its decarboxylation product dopamine (8) is well established as a plant constituent and occurs at high concentration in banana peel, where it is the chief substrate for the browning reaction [22]. Successive methylations of the nitrogen in tyramine give rise in sequence to N-methyltyramine (9), hordenine (10), and candicine (11). All four of these compounds have been found to occur together in barley roots [23], and they are found singly in a variety of other plants. Tracer experiments with barley have demonstrated the origin of tyramine and hordenine from phenylalanine [24] or tyrosine [24 to 25a]. Such experiments have also shown the origin of the methyl groups of hordenine from methionine [14]. This methylation is evidently reversible [23]. Some studies have been done on the enzymatic system responsible for methylation of tyramine in Panicum miliaceum [25] and Hordeum vulgare [26]. The enzyme tyramine methylpherase appears in germinating barley roots at the same time as tyramine and hordenine, and its formation in isolated embryos can be stimulated by the addition of amino acids and giberrellin [26]. The conversion ofhordenine-a- 14 C to a radioactive, nitrogen-containing lignin fraction in barley has been reported [27] and is of interest as indicating an active metabolic role for hordenine. Mescaline, an hallucinogenic drug from the peyote cactus (Anhalonium lelJ1inii), has the structure: OCH3

CH'0-o-CH,CH,NH, OCH3 Dopamine is the best precursor of mescaline identified so far, and, as shown in Fig. 3-2, it can be derived either by decarboxylation of DOPA or by hydroxylation of tyramine [28]. Relative percentage recoveries of radioactivity from a- 14 C compounds were phenylalanine 0.025, tyrosine 0.32, tyramine 1.53, DOPA 1.58, and dopamine 1.90 [28]. of the ethylamine side chain of ｾＭｰｨ･ｮｹｬｴ｡ｭｩ＠ Hydroxylation of the ｾＭ｣｡ｲ｢ｯｮ＠ followed by C- and N-methylations gives rise to the useful plant drug ephedrine (12). Tracer feeding experiments have shown formate to be a good precursor for the methyl groups of ephedrine [29]. Similar ｾＭｨｹ､ｲｯｸｬ｡ｴｩｮ＠ of dopamine gives rise to noradrenaline (13), a compound best known as an animal hormone but also occurring in banana fruits (Musa sapientum) and perhaps also in other plants [30]. The enzyme responsible for oxidation of dopamine to noradrenaline has been partially purified from bananas, and molecular oxygen has been shown to be the oxidant [31]. The probable role of noradrenaline as a precursor of the alkaloid berberastine is discussed in Chapter 7. 2*

ｏｃｈＲｾ＠

20

Simple Amino Acid Derivatives and Protoalkaloids

ｾ＠ --

ｎｾ＠

ｈｏＭｯｃＲｾ＠

ｾ＠ - - HOVCH}HCOOH

HO Phenylalanine

Tyrosine

Dihydroxyphenylalanine (DOPA)

I

t

HO-o-CH2CH2NH2 - -

p-Phenylethylamine (6)

1 Ephedrine (12)

Tyramine (7)

Dopamine (8)

1 N-Methyltyramine (9)

Noradrenaline (13)

Hordenine (10)

Candicine (11) Fig. 3-2. Amines and protoalkaloids related to phenylalanine and tyrosine (-)-Stachydrine, the betaine of L-proline:

is known to occur in a large number of unrelated plants. Racemic stachydrine also occurs widely. The two isomeric betaines of hydroxyproline, betonicine and turicine, are known but less common. Homostachydrine, the homologous betaine of pipecolic acid, is known from alfalfa (Medicago salilJa). Biosynthesis of stachydrine and homo-

Simple Amino Acid Derivatives and Protoalkaloids

21

stachydrine has been investigated by tracer feeding experiments using such likelylooking precursors as ornithine, proline, and lysine [32]. After many negative results it was concluded that young alfalfa plants (3 weeks old) do not synthesize stachydrine although they can make proline from ornithine; or if hygric acid is provided, they can methylate it to stachydrine. The block between proline and hygric acid is removed as the plant matures, and mature plants convert proline to stachydrine so rapidly that free proline cannot be detected in them [32]. Evidently, then, two different methylating enzymes function in the following scheme: Ornithine -

ＭＫｾｃｏｈ＠

ｾ＠

H \

COOH - ---+ Stachydrine I

H

\

CH3H

L-Proline

Hygric Acid

The amino acid trytophan is a precursor of the important plant hormones related to indole-3-acetic acid as well as of the indole alkaloids (Chapter 10). A small group of indole derivatives derived from tryptophan by decarboxylation probably includes precursors of the indole alkaloids as well as compounds related in a presently obscure way to the indole hormones [33]. Tryptamine has been reported to occur in many plants of the family Leguminosae. N-methyl and N-dimethyl tryptamine have also been found in several different plants [34]. Serotonin (5-hydroxytryptamine) (14) is also widely distributed, although in small amounts [35]. These compounds are believed to be derived, respectively, from tryptophan and 5-hydroxytryptophan, although experimental evidence on their biosynthesis is not available for higher plants. Various N-methylated derivatives of serotonin are best known as constituents of toad secretions but also occur in certain fungi and higher plants [35a]. Methylated derivatives of 4-hydroxytryptamine are of interest as the hallucinogenic components of certain mushrooms [35a]. Structures of some of the naturally occurring tryptamine derivatives are given in Table 3-2. Tryptamine can act as a plant growth substance since it apparently is converted to indole-3-acetic acid, but the pathway involving tryptamine is not thought to be the only route for the formation of indole hormones from tryptophan [33]. The widely occurring protoalkaloid gramine (3-dimethylaminomethylindole):

Gramine is peculiar in that although it is derived from tryptophan [36], it has a shorter side chain than tryptamine. It is known that its methyl groups come from methionine [37], but there is no other information about its biosynthesis. Although gramine is not present in ungerminated barley seeds, it appears (along with 3-aminomethylindole and 3-methylaminomethylindole) within 4 days after planting and then persists for at least 50 days [38,39]. Tracer experiments have shown that degradation of

Simple Amino Acid Derivatives and Protoalkaloids

22

gramine by barley plants apparently leads to an indole derivative that can be reconverted to tryptophan [40]. Histamine, the decarboxylation product of the amino acid histidine, is, like serotonin, more familiar as an animal hormone than as a plant product. However, it and derivatives of it are firmly established as plant constituents. In the case of nettles (Urtica spp.), the presence of histamine in the stinging hairs may account at least partially for their irritant effects [41]. One of the best plant sources of histamine and Table 3-2. Some derivatives of tryptamine found in higher plants and ftmgi

Dipterin 0-TCH2CH2N (CH 3 )2

ｾｎＩ＠ N, N-Dimethyltryptamine

ｈｏｾｃＲｎ＠ ｾｎＩ＠

Serotonin (14)

Bufotenine

Psilocybin its derivatives is spinach (Spinacea oleracea) [11], and the formation of histamine from labelled histidine has been studied in germinating spinach seeds. While the decarboxylation occurred rapidly in spinach, it was negligible in ten other plant species tested [42]. Phaseolus spp. convert histidine to urocanic acid (imidazoleacrylic acid) [43,441. Histidine is also presumably a precursor of several imidazole alkaloids (Chapter 12).

ｾＭＺｲｃｈ］ｏ＠

__

N H Urocanic Acid

Histidine

Histamine

Bibliography

23

Trigonelline, the betaine of nicotinic acid, may be considered a protoalkaloid; but because of its relationship to other pyridine alkaloids, it is placed in Chapter 4.

Bibliography 1. VON ｋａｾｬｅｎｓｉＬ＠ E. S.: Planta 50, 315-330 (1957). 2. RICHARDSON, M.: Phytochcm. 5, 23-30 (1966). 3. LERCH, B., U. H. STEGEMAN: Z. Naturforsch. 21b, 216--218 (1966). 4. S"lITH, B. N., and B. J. D. MEEUSE: Plant Physiol. 41, 343-347 (1966). 5. S"lITH, T. A., and J. 1.. GARRAWAY: Phytochem. 3, 23-26 (1964). 6. - Phytochem. 4, 599-607 (1965). 7. STOESSL, A.: Tetrahedron Letters 1966, 2287-2292. 8. REUTER, G.: Phytochem. 1,63--65 (1962). 9. WERLE, E., U. A. ZABEL: Biochem. Z. 318, 554-559 (1948). 10. - , 1. TRAUTSCliOLD und D. AURES: Z. Physiol. Chern. 326, 200-211 (1961). 11. ApPEL, W., U. E. WERLE: Arzncimittcl-Forsch. 9, 22-26 (1959). 12. AHMAD, K., and M. A. KARIM: Biochcm. J. 55, 817-820 (1953). 13. DELWICHE, C. C, and H. M. BREGOFF: J. Bio!. Chern. 33, 430-433 (1958). 14. MATCHETT, T . .J., L. MARION, and S. KIRKWOOD: Can. J. Chern. 31, 488-492 (1953). 15. WILKINSON, S.: Quart. Revs. (London) 15, 153-171 (1961). 15a. BAGNI, N., C. 1\1. CALDARERA und G. MORUZZI: I:xperientia 23,139-140 (1967). 16. PAIS, M., F.-X. JARREAU, X. LUSINCHI et R. GOUTAREL: Ann. chim. (Paris) 1, 83-105 (1966). 17. NAKANO, T., c:. DJERASSI, R. A. CORRAL, and O. O. ORAZI: J. Org. Chern. 26, 1184 to 1191 (1961). 18. ZBIRAL, E., E. L. MENARD und J. M. MULLER: Helv. Chim. Acta 48, 404-431 (1965). 19. WARNHOPF, E. W.,]. C N. MA, and P. REYNOLDS-WARNHOFF:]. Am. Chern. Soc. 87, 4198-4199 (1965). 20. WIELAND, T., u. U. GEBERT: Ann. Chern. Liebigs 700,157-173 (1967). 21. SANDBERG, F., and G. SA"II'ELSSON: In: Symposium on phytochemistry, pp. 54-61. ARTHUR, H. R., cd. Hong Kong: Univ. Press 1964. 22. GRIFFITHS, L. A.: Nature 184, 58-59 (1959). 23. RABITZSCH, G.: Planta Med. 7, 268-297 (1959). 24. MASSICOT, .I., and L. J\[ARION: Can. J. Chern. 35,1-4 (1957). 25. BRADY, L. R., and V. E. TYLER jr.: Plant Physio!. 33, 334-338 (1958). 25a. McLAUGHLIN, J. L., and A. G. PAUL: Lloydia 30, 91-99 (1967). 26. MANN, J. D., C. E. STEINHART, and S. H. MUDD: J. Bio!. Chern. 238, 676-681 (1963). 27. FRANK, A. W., and L. MARION: Can. J. Chern. 34,1641-1646 (1956). 28. ROSENBERC, H., ./. L. McLAUGHLIN, and A. G. PAUL: Uoydia 30,100-105 (1967). 1.: Pharm. Bul!. (Tokyo) 5, 594-597 (1957). 29. ｬｾａｓｅｋｉＬ＠ 30. FclY, J. :VI., and]. R. PARRATT: J. Pharm. Pharmacol. 12,360-364 (1960). 31. StlITH, W . .I., and N. KIRSHNER: J. Bio!. Chern. 235, 3589-3591 (1960). 32. ESSERY, J. M., D. J. MCCALDIN, and L. MARION: Phytochem. 1, 209-213 (1962). 33. SHANTZ, E. M.: Ann. Rev. Plant Phvsio!. 17,409-438 (1966). 34. PACHTER, I. J., D. E. ZACHARTlTS, 'and O. RIBEIRO: J. Org. Chern. 24, 1285-1287 (1959). J5. KIRBERGER, E., and L. BRAUN: Biochim. et Biophys. Acta 49, 391-393 (1961). 35 a. BOIT, H.-G.: Ergebnisse der Alkaloid-Chemic bls 1960. Berlin: Akademic Verlag 1961. 36. W[GHnIAN, F., M. D. CHISHOLM, and A. c:. NElSH: Phvtochem. 1, 30-37 (1961). 37. MUDD, S. H.: Biochim. et Biophys. Acta 37, 164-165 (1960). 38. - Nature 189, 489 (1961). 39. TYLER, V. E., jr.: J. Am. Pharm. Ass. 47, 97-98 (1958). 40. DIGENIS, G. A., B. A. FARAJ, and C. 1. ABOU-CHAAR: Bioehem. J. 101, 27c-29c (1966). N., and W. FELDBERG: New Phytologist 48, 143-148 (1949) . 41. ｅｴ｛ｾｉｌｎＬ＠ .f2. VON HAARnIANN, U., G. KAHLSON, and C. STEINHARDT: Life Sciences 5, 1-9 (1966) . .f3. SIVARAMAKRISHNAN, V. M., and P. S. SARtIA: Current Sci. (India) 25, 288-289 (1956). 44. Luu, V.-V., et.J. GRFC;OIRE: Cornpt. rend. soc. bio!. 152, 1260-1262 (1958).

Chapter 4

Pyrrolidine, Piperidine, and Pyridine Alkaloids My heart aches, and a drowsy numbness pains My sense, as though of hemlock I had drunk, Or emptied some dull opiate to the drains One minute past, and Lethe-wards had sunk: Ode to a Nightingale,

JOHN KEATS

Pyrrolidine Alkaloids There are only a few simple pyrrolidine alkaloids found in nature, although pyrrolidine (or pyrrole) rings combined into larger structures are relatively common (e.g., in the indole alkaloids). Methylated derivatives of proline or hydroxyproline could be called alkaloids but are here placed with protoalkaloids in Chapter 2; tJ-methylpyrroline is considered with the terpenoid alkaloids in Chapter 13. The two best-known pyrrolidine alkaloids are hygrine and cuscohygrine, both found in Erythroxylon coca:

Cuscohygrine

( ±)-Hygrine

Both are formed non-enzymatically in mixtures of acetonedicarboxylic acid and Nmethyl-2-hydroxypyrrolidine [1]. They are also produced by exposing a mixture of N-methylputrescine and acetoacetic acid or acetonedicarboxylic acid to the action of diamine oxidase [2]. Presumably in the latter case an amino aldehyde is first formed and condenses: CH 2-CH 2

I

[OJ

------7

?

I

-----+> CH 2 HC-CHCCH 3

1------7 I

Acetoacetic Acid

ｾ＠

1

1

NH OH COOH 1

CH 3

1

25

Piperidine Alkaloids

1 o II

Hygrine + - - - CH 2 CHCHCCH 3

""'/ N

I

COOI-I

Norhygrine is made in a similar system substituting putrescine for its N-methyl derivative [3]. Such in vitro reactions are believed to be analogous to the in vivo biosynthesis. Since the tropane alkaloids closely resemble hygrine and cuscohygrine in structure and often occur together with these pyrrolidine alkaloids [4], experimental results showing derivation of tropane alkaloids in vivo from putrescine plus acetate tend to give support to the idea that a similar pathway exists for the pyrrolidine alkaloids (see Chapter 5). Since putrescine is not normally found as such in plants, it is considered to be only a transient intermediate produced by decarboxylation of ornithine or decarboxylation and deamidination of arginine (see Chapter 3).

Piperidine Alkaloids The piperidine alkaloids, such as isopelletierine and coniine, are usually considered to be made in the same way as hygrine but starting with cadaverine (or lysine) rather than putrescine:

Isopelletierine

Coniine

The same in vitro systems that are found to produce hygrine are found with suitable modification to produce isopelletierine or N-methyl-isopelletierine [1- 3J. However, experiments have shown that in vivo there are probably different pathways for the formation of different piperidine alkaloids. Several in l)ivo studies are available on the biosynthesis of coniine. Feeding uniformly labelled lysine or such related compounds as cadaverine-l,5- 14 C, DL-ex-aminoadipic acid-6- 14 C, LP-piperideine-U-14C, or ex-keto-E-aminocaproic acid-U-14C to ConiulJI lJIaculatulJI plants gave rise to radioactive coniine [5] or y-coniceine [6]. Propionate-2- 14 C was also an effective precursor of y-coniceine, presumably going into the side chain. On the basis of such results CROMWELL and ROBERTS [61 proposed three alternate routes from lysine to coniine, as shown in Fig. 4-1.

26

Pyrrolidine, Piperidine and Pyridine Alkaloids

ｾ＠

Cadaverine

Lysine

1 ｾ＠

ｾｃｏｈ＠

HOOC-C:)

/J1- Piperideine-2Carboxylic Acid

LJI-Piperideine-6Carboxylic Acid

I

t

o

LJl_ ｐｩｰｾｲ､･ｮ｣＠

: d C3]

t t

ｾｃｈＲＳ＠ ;'-Coniceine

Coniine Fig. 4-1. Possible pathways of coniine biosynthesis In disagreement with the proposed derivation of coniine from lysine, LEETh [7J has found acetate to be a much better precursor of coniine and conhydrine than either lysine or cadaverine. Degradation showed label from acetate-J-14C to be evenlv distributed among the even-numbered carbon atoms; and the following pathwav to coniine was suggested:

4 [Acetate I -----+

y-Coniccine

1

Coniine

ｾｃｈＧ＠ 1p-Conhydrine

OH Conhydrine

The source of the nitrogen atom is a problem, since it has been almost axiomatic in the field of alkaloid biosynthesis that nitrogen enters an alkaloid molecule as an organic amino group. The role of y-coniceine as a precursor of coniine is accepted

Piperidine Alkaloids

27

by all workers; and when y-coniceine-I'-14C was fed to C. maculatum plants, 9.9% incorporation into coniine was observed [7a]. Further conversion of coniine to conhydrine was also observed under field conditions; but, strangely, in the greenhouse Vi-conhydrine rather than conhydrine was found [7a]. Mimosine, found in Mimosa pudica and Leucaena glatlca, has been indicated by tracer feeding experiments to have its pyridone ring derived from lysine. Succinate and aspartate were also effective as precursors but are believed to act indirectly, as precursors of lysine [8-9a]. The alanine substituent of mimosine comes from serine [10]. Since L. l!,latlca also contains 5-hydroxypipecolic acid, this is a possible intermediate between lysine and mimosine. In M. pudica hydroxy lysine was found not to be a precursor of mimosine although it did appe:lr to be a precursor of traces of 5-hydroxypipecolic acid [10]:

Lysine -

ｈｏｾｃ＠

HOA --- t.J

H

H

'H

r

N NH2 I I CH 2CHCOOH

. Senne

(-)-S-Hydroxypipecolic Acid

Mimosine

Extracts of L. glauca degrade mimosine to form 3,4-dihydroxypyridine, pyruvate, and ammonia. Further metabolism of the dihydroxypyridine probably occurs. Mimosine acts as a toxic compound toward seedlings of Phaseolus mungo, apparently because degradation stops at the dihydroxypyridine stage, and dihydroxypyridine is a powerful chelator of iron [10al. Sedamine of Sedu"J acre is, like mimosine, derived from lysine rather than from acetate C-6 of lysine going specifically to C-6 of the piperidine ring and C-2 to C-2. The side-chain is derived from phenylalanine and the N-methyl group from methionine. Intermediates oLd Lpiperideine and cinnamic acid have been suggested [1 Ob].

Sedamine The biosynthesis of carpaine in Carica papqya was investigated by feeding labelled acetate, mevalonate, and lysine to excised shoots [11]. In agreement with Leete's proposal for coniine, these experiments showed acetate to be by far the bestincorporated of the three precursors, suggesting the pathway:

o

II

O--C

7 -Acetate- -+

Heb(L,), 3

H

Carpaine

28

Pyrrolidine, Piperidine and P yridine Alkaloids

As in the coniine experiments, the origin of the nitrogen atom remains unknown. The foregoing discussion makes it clear that there are at least two known pathways to the piperidine alkaloids, one from lysine and one from acetate. More experimentation is needed to determine the relative importance of the two pathways for the various piperidine alkaloids. It should also be mentioned that some alkaloids with a piperidine ring are probably derived from terpenoids (see Chapter 13).

Pyridine Alkaloids Although the pyridine alkaloids bear a superficial resemblance to the piperidine alkaloids, there is a distinct pathway leading to the pyridine ring which is different

Bacteria, Higher Plant

Animals, Higher Fungi

ｃｈｏｾ＠

ｾｃｏｈ＠

HOOC

H2

Tryptophan

I

1 H2

ｾ

ｾｃｏｈ＠

-+

ｎＧ＠

.. CHO

H

yc ｾｉ＠

COOH H2

OH

Formylkynurenine

3- HydroxyAnthranilic Acid

Fig. 4-2. Pathways of nicotinic acid biosynthesis

Ricinine

29

from any of the piperidine pathways. Cases of formation of a pyridine alkaloid by dehydrogenation of a piperidine or vice versa are almost nonexistent (but see anabasine, below). The vitamin nicotinic acid is the key compound involved in the biosynthesis of the pyridine alkaloids. Unfortunately details of the synthesis of nicotinic acid itself in higher plants are still unknown. Indeed, more has been learned about the biosynthesis of nicotinic acid in higher plants from the study of alkaloids known to be derived from it than by any direct investigations. The pathway to nicotinic acid from tryptophan via anthranilic acid, which is well elucidated in animals, seems to be used by fungi of the genus Claviceps [12] and by yeast growing aerobically [13]. Anaerobic yeast [131, bacteria [14], and higher plants [151 evidently use a different pathway, in which the pyridine ring is built from a C3 unit closely related to glycerol and another unit closely related to aspartic acid. As a general principle, it has been proposed [16] that those organisms that make lysine from iX-aminoadipic acid make nicotinic acid from tryptophan and those that make lysine from 1,S-diaminopimelic acid use the other route for nicotinic acid. Quinolinic acid is formed via both pathways and in a one step reaction [17] forms nicotinic acid mononucleotide, which may be hydrolyzed to free nicotinic acid or converted to other pyridine nucleotide derivatives. In some cases it may be that pyridine nucleotides rather than free nicotinic acid or nicotinamide are intermediates in pyridine alkaloid biosynthesis [18J. Further discussion of the precursors of nicotinic acid will be deferred until after discussion of ricinine and nicotine. A general view of nicotinic acid metabolism is shown in Fig. 4-2. It should be especially noted that while free nicotinic acid can apparently be converted to its mononucleotide, free nicotinamide is converted to a nucleotide only after loss of its amino group [19]. Consequently, any alkaloids derived from free nicotinamide with retention of the amino group must be made without the involvement of nucleotides.

Trigonelline The simplest pyridine alkaloid is N-methylnicotinic acid, 01 trigonelline. Trigonelline occurs widely distributed in the plant kingdom. An enzyme catalyzing its formation from nicotinic acid and S-adenosylmethionine has been found in the pea plant [20]. Trigonelline can evidently serve as a storage form of nicotinic acid and can also contribute its methyl group to the C1 pool [21,22]. Since free nicotinic acid is rapidly destroyed in pea plants, its storage in a more inert form may be advantageous [22]. There is some indication that trigonelline may have additional physiological significance since it has been found to act similarly to kinetin in inhibiting yellowing of isolated wheat leaves [23J.

Ricinine The most intensively studied of the pyridine alkaloids with a single heterocyclic ring is ricinine (1) of the castor bean plant (Ricinus communis). The advantages of ricinine as an object of study are that the plant is easily grown and that it contains high concentrations of only one alkaloid. Ricinine synthesis is clearly associated with

Pyrrolidine, Piperidine and Pyridine Alkaloids

30

rapid growth both in whole plants [24] and in isolated root cultures [25]. In young seedlings the quantity per plant may increase up to fiftyfold within a week after planting [24]. Derivation of the pyridine ring of ricinine from nicotinic acid or nicotinamide was first shown by LEETE and LEITZ [26] and has since been amply demonstrated by others. One of the most significant findings was that if nicotinamide

ｾ＠

ｯＭ

ｾｃｎｈｺ＠

Ｏｎｩ｣ｯｴｮｲｬｾ＠

llNJ Nicotinamide

CN

oｾ＠

I N'"

r Nicotinamide Nucleotide

ｾ＠ N/.

W ｎｾ＠

CN

oII

/

CNH z

I

CH3 1-Methylnicotinonitrile I

I G) CH 3

1- Methylnicotinamide

- - - - - - - ＿ＭＬｾ＠

, I

ｾ＠

-- - - - - -

-

tJ

108

Terpenoid and Steroid Alkaloids

possible formation of aconitine (8) and related alkaloids from atisine is also suggested in Fig. 13-2 [7]. This whole group of alkaloids is characterized by having an lX-oriented substituent methyl group at the AlB ring juncture. This is in contrast to the more usual p-orientation found in abietic acid and suggests a biosynthetic relationship to phyllocladene [9]. Tracer feeding experiments with mevalonic acid-2-14C have indicated derivation of brownine and lycoctonine (9) in Delphinium brOlvnii by the expected pathway, although incorporation was low [10]. Other feeding experiments, with detached leaves of D. elatum, failed to show any incorporation of mevalonic acid-2-14C into de1pheline (10) [11]. This might be explained by postulating that the roots are the normal site of synthesis [10]. The 0- and N-methyl groups of delpheline could be derived from methionine-methyP4C in the leaf-feeding experiment, showing that the leaf is capable of performing the transmethylation reactions although it does not carry out the complete synthesis [11]. Injection of glycine-2- 14 C and mevalonate-2-14C into D. ajacis has been found to give rise to labelled diterpenoid alkaloids, but degradations have not yet established the location of 14C in any particular alkaloid [12]. The taxine group of alkaloids from yew (Taxus spp.) are esters of various diterpenoids with L-3-dimethylamino-3-phenylpropanoic acid [13]. The nitrogen atom is thus not directly attached to the terpenoid carbon skeleton, and these compounds could reasonably be classified with the simple amino acid derivatives of Chapter 3. Tracer studies have indicated that the phenylpropane moiety originates from phenylalanine, with an evident lX, p-migration of the amino group [13]. NHz OCHztHCOOH Phenylalanine

1

OH

o 3-Dimethylamino-3phenyl propanoic Acid

OR

Taxine II

Steroids Steroid and modified steroid alkaloids are divided into three or four groups according to plant source. The carbon skeletons are also characteristic of each plant group. The Solanum alkaloids present in S. tuberosum and a few related species of the Solanaceae have the CZ7 carbon skeleton of cholesterol changed only by closure of

109

Steroids

additional rings [14]. Modified steroid alkaloids of the genus Veratrum and other Liliaceae genera have the same number of carbon atoms in their skeletons as cholesterol, but modification of rings C and D has occurred as well as additional ring closures [15]. Kurchi alkaloids are found in plants of the Apocynaceae generaHola"hena and Funtumia and have a C21 nucleus similar to pregnane [16]. Buxus alkaloids also

RO

Solanidine

Type

Solasodine

RO

Type

So/anum Alkaloids

RO

Jerveratrum Type

Ceveratrum

RO

Type Veratrum Alkaloids

Funlumia Type

Ho/arrhena Type Kurchi Alkaloids

Buxus Alkaloids Fig. 13-3. Structural types of steroid alkaloids

Terpenoid and Steroid Alkaloids

110

have a C21 pregnane-type nucleus but characteristically have a cyclopropane ring bridging C-9 and C-l0 and usually have two methyl groups at C-4 [17]. These various skeletons and their subgroups are illustrated in Fig. 13-3. Many of them exist naturally as esters or glycosides. Some members of both the Buxus and Funtumia groups have second nitrogen atoms at C-20. Studies on the biosynthesis of the Solanum alkaloids have confirmed their relationship to other steroids derived via the normal mevalonate pathway. Labelled acetate or mevalonate fed to S. aviculare or S. tuberosum were incorporated into solasodine (11), the acetate in C2 units following the pattern found for cholesterol, and mevalonate-214C giving the expected labelling pattern, although complete degradations were not carried out [18, 19]. The presumed transformation of cholesterol to the alkaloid is represented below, with carbon atoms presumably derived from C-2 of mevalonic acid circled.

HO

HO Cholesterol

Solasodine (11)

The conversion of cholesterol to tomatidine has been found to occur in young plants of Lycopersion esculentum [20] or L. pimpinellifolium [21]. There seems to be no information or even hypothesis regarding the reaction by which nitrogen becomes incorporated into the alkaloid. The compound 3-p-hydroxy-S-iX-pregn-16-en-20-one occurs along with tomatidine in L. pimpinellifolium and also received label from added cholesterol-4- 14 C. It could therefore be a precursor of tomatidine. Since it did not receive any label if labeled tomatidine was fed, it cannot be considered a breakdown product of tomatidine [22].

CH3 \

C=O

HO

HO Tomatidine

3p-Hydroxy-SlX-pregn-16-en-20-one

Jerveratrum and ceveratrum alkaloids exist simultaneously in the same plant, the former free or as glucosides, the latter as esters. Rubijervine (12), an alkaloid with a true steroid nucleus, also occurs with them, suggesting a biosynthetic relationship. Several mechanisms have been proposed for the rearrangement of steroid rings C

111

Steroids

HO Rubijervine (12)

and D to form the modified steroid alkaloids [15, 23]. Since the modified steroid nucleus has been found to occur naturally only in these alkaloids, it seems likely that the rearrangement occurs after, and is facilitated by, the introduction of nitrogen. Only one scheme [15] exploits the nitrogen atom as an essential part of the rearrangement mechanism, and this with the unnatural precursor isorubijervine-18-tosylate. Thus no rearrangement mechanism seems entirely satisfactory, and in vivo biosynthetic experiments are completely lacking for the Veratrum alkaloids. The biosynthesis of Holarrhena alkaloids has been investigated by tracer feeding experiments using 4_14C labelled steroids supplied to leaves of H. ftoribunda [24,25]. After feeding of cholesterol-4- 14, the isolated alkaloid fraction contained 14% of the original radioactivity. Pregnenolone (13) and progesterone (14) also served as alkaloid precursors, although none of the steroids that were effective as precursors could be detected in the plant. Progesterone gave rise to five alkaloids of unknown structure, but cholesterol and pregnenolone were precursors of the known compounds holaphyllamine (15), holaphylline (16), and holalnine. Comparison of structures and specific activities suggests the pathway shown in Fig. 13-4. Holamine is enantiomorphic with holophyllalnine at C-3. Those alkaloids with additional nitrogen at C-20 and with a pyrrolidine ring are probably formed by further reaction of a holaphyllamine-type compound, as suggested in Fig. 13-4, but there is no evidence. Holaphyllamine-4- 14 C adlninistered to H. ftoribunda has been found to be converted to radioactive pregnenolone. Thus the pathway between these two compounds is reversible [26]. No experimental work has been done with biosynthesis of the Buxus alkaloids, but from their structures it appears likely that they are made by a pathway silnilar to that for the Holarrhena alkaloids. Their structural peculiarities, though, make plausible an origin from a compound like cycloartenol (17) rather than cholesterol. Some

HO

Cycloartenol (17)

Cyclomicrophyllin A

112

Terpenoid and Steroid Alkaloids

HO Cholesterol

Pregnenolone (13)

Progesterone (14)

j

il

/

Holaphylline (16)

HO I

ｈＧｎｾ＠

Holaphyllamine (15)

CH3 I

H 2C H-C-NH2

Holarrhimine

Unknown Alkaloids

Conarrhimine

Fig. 13-4. Biosynthesis of Holarrhena alkaloids

workers believe that cycloartenol may be a key intermediate in steroid biosynthesis in higher plants taking the role that lanosterol fills in animals and fungi [27,28]. Various functions for the Solanum alkaloids have been suggested from time to time. These include antibacterial activity [29] and activity as insect repellents [30]. A role of tomatine in plant development has also been suggested [31,32]. Tomatine is produced chiefly in shoot meristems in response to photoperiodic control, is accumulated in developing fruits, and then destroyed as the fruits ripen. Of the very few animal products that are called alkaloids, the secretions of salamander skin glands bear the closest resemblance to typical plant alkaloids. Nine of the salamander alkaloids are now known, and it appears that they are probably derived from steroids by opening of ring A and insertion of nitrogen [33]. The structure of samandarine, for example, is:

Bibliography

113

OR

OR

Samandarine Such structures also seem related to the non-nitrogenous toad poisons such as bufotalin.

Bibliography 1. FOWDEN, L.: Ann. Rev. Biochern. 33, 173-204 (1964). 2. AUDA, H., H. R. JUNE]A, E. ]. EISENBRAUN, G. R. WALLER, W. R. KAYS, and H. H. ApPEL: J. Am. Chern. Soc. 89,2476-2482 (1967). 3. YAMAZAKI, M., M. MATSUO, and K. ARAI: Chern. & Pharrn. Bull. (Tokyo) 14, 1058 to 1059 (1966). 4. SCHUrrE, H. R., u. J. LEHFELDT: Arch. Pharrn. 298, 461-465 (1965). 5. MARTIN-SMITH, M., U. T. KHATOON: Fortschr. Arzneirnittelforsch. 6, 279-346 (1963). 6. WENKERT, E.: Chern. & Ind. (London) 1955, 282-284. 7. VALENTA, Z., and K. WIESNER: Chern. & Ind. (London) 1956,354. 8. WHALLEY, W. B.: Tetrahedron 18, 43-54 (1962). 9. D]ERRASSI, c., M. CAIS, and L. A. MITSCHER: J. Am. Chern. Soc. 81, 2386-2398 (1959). 10. BENN, M. H., u. J. MAY: Experientia 20, 252-253 (1964). 11. HERBERT, E. ]., and G. W. KIRBY: Tetrahedron Letters 1963, 1505-1506. 12. FROST, G. M., R. L. HALE, G. R. WALLER, L. H. ZALKOW, and N. N. GIROTRA: Chern. & Ind. (London) 1967, 320-321. 13. LEETE, E., and G. B. BODEM: Tetrahedron Letters 1966,3925-3927. 14. BOlT, H.-G.: Ergebnisse der Alkaloid-Chemie bis 1960. Berlin: Akadernie-Verlag 1961. 15. NARAYANAN, C. R.: Fortschr. Chern. org. Naturstofl'e 20, 298-371 (1962). 16. GOUTAREL, R.: Les alcaloides steroidiques des apocyanacees. Paris: Hermann 1964. 17. NAKANO, T., S. TERAO, and Y. SAEKI: J. Chern. Soc. (c) 1966,1412-1421. 18. GUSEVA, A. R., V. A. PASESHNICHENKO, andM. G. BORIKHINA: Biokhirniya 26, 723-728 (1961). 19. - - Biokhirniya 27,853-858 (1962). 20. TSCHESCHE, R., U. H. HULPKE: Z. Naturforsch. 21b, 893-894 (1966). 21. HEFTMANN, E., E. R. LIEBER, and R. D. BENNETT: Phytochern. 6, 225-229 (1967). 22. BENNETT, R. D., E. R. LIEBER, and E. HEFTMANN: Phytochern. 6, 837-840 (1967). 23. LEETE, E.: In: BERNFELD, P., ed. Biogenesis of natural compounds, pp. 787-796. New York: Macmillan 1963. 24. BENNETT, R. D., and E. HEFTMANN: Arch. Biochem. Biophys. 112,616-620 (1965). 25. - - Phytochem. 4, 873-879 (1965). 26. - - , and S.-T. Ko: Phytochem. 5, 517-521 (1966). 8 Robinson, The Biochemistry of Alkaloids

114

Terpenoid and Steroid Alkaloids

27. GOAD, L. J., and T. W. GOODWIN: Biochern. J. 99, 735-746 (1966). 28. BENVENISTE, P., L. HIRTH, and G. OURISSON: Phytochern. 5, 45-58 (1966). 29. BOLL, P. M., H. A. LILLEVIK, R. Y. GOTTSHALL, and E. H. LUCAS: Antibiotics annual, pp. 255-259. 1955-1956. 30. KUHN, R., U. 1. Low: Chern. Ber. 94, 1088-1095 (1961). 31. SANDER, H.: Planta 47, 374-400 (1956). 32. - Planta 52, 447-466 (1958). 33. HABERMEHL, G.: Naturwissenschaften 53,123-128 (1966).

Chapter 14

Metabolism of Alkaloids by Bacteria and Animals So, naturalists observe, a flea Hath smaller fleas that on him prey; And these have smaller still to bite 'em; And so proceed ad infinitum. On Poetry,

JONATHAN SWIFT

While knowledge about the metabolism of alkaloids in the plants that synthesize them has only recently begun to be developed, pathways of alkaloid degradation by other organisms have been studied for many years, and in a few cases considerable detail is available. The special interest in this area arises, of course, from pharmacological effects of alkaloids, and therefore most studies relate to mammalian metabolism and to alkaloids of widespread use. It is frequently suggested that administered drugs are metabolically converted to the real physiologically active compounds. The metabolism of drugs is a subject of frequent review articles [1-3]; and since many drugs are either natural alkaloids or structurally related compounds, these reviews often include information pertinent to the present discussion. Where not specifically annotated, information in this chapter has been derived from Refs. [1] and [2]. The metabolism of alkaloids by microorganisms has received less attention than their mammalian metabolism, but there is practical reason for such studies, since products of microbial transformation of alkaloids may in some cases themselves be pharmacologically valuable or serve as intermediates for chemical syntheses.

Animals Oxidation, one of the commonest detoxication processes, is often applicable to alkaloid metabolism. It includes dehydrogenation as well as actual introduction of oxygen into the molecule by hydroxylation. A phylogenetic correlation has been suggested to the effect that more advanced organisms tend to detoxify foreign compounds by oxidizing them while primitive organisms have reductive systems instead of the oxidizing systems [4]. A second very general process is conjugation, the conversion of foreign substances to less toxic derivatives by combination with conjugating agents. In mammals phenols are often converted to fJ-glucuronides for excretion, acids are converted to substituted amides by combination with amino acids, some nitrogenous compounds are N-methylated and some are acetylated. Some N-methylated compounds are demethylated. Tertiary amines may be oxidized to N-oxides [5], and preliminary 8*

116

Metabolism of Alkaloids by Bacteria and Animals

formation of an N-oxide has been suggested as part of the mechanism of demethylation. Demethylations are evidently oxidative since 14C-Iabelled N-methyl pyridinium compounds fed to rats have been shown to give rise to labelled CO2 [6]. Combinations are also possible, for instance an aromatic compound may first be hydroxylated and then converted to a glucuronide. Insofar as alkaloids contain the appropriate reactive groups, it may be expected that their metabolism in mammals will follow these general pathways. The avoidance of teleology in considering detoxification mechanisms is difficult, but it is ultimately more difficult to justify specialized enzymes waiting for substrates they may never encounter. The physiological roles of the so-called detoxifying enzymes may lie, rather, in normal metabolism; but, lacking absolute specificity, they are able to act on foreign substances that possess appropriate functional groups. In many cases the products of this action are less toxic than the substrates, but in other cases increased toxicity is the result (e.g., the conversion of codeine to morphine). The site and enzymatic mechanisms of detoxication have been defined to some extent. The light fraction of liver microsomes is active in metabolizing many foreign compounds, and the hydroxylation reactions so characterististic of this metabolism are catalyzed by an enzyme system that incorporates molecular oxygen and requires as cofactors reduced nicotinamide adenine dinucleotide phosphate (NADPH2) and a dihydropteridine [7]. Some of the microsomal enzymes involved in these reactions have been solubilized. Drugs that are hydroxylated by liver micro somes have been found to form a spectrally observable complex with a microsomal cytochrome, and this complex may be the first stage of the hydroxylation reaction [7a]. An N-methylating enzyme purified from rabbit lung has relatively low specificity for methyl acceptor and can catalyze the formation of morphine, codeine, and nicotine from the corresponding nor-derivatives. Oxidation of simple amines is brought about by the widely distributed monoamine and diamine oxidases. The first-formed aldehydes undergo further reaction, often being oxidized to the acids and excreted as such - thus, indole-3-acetic acid is the excreted end-product of tryptamine metabolism. Mescaline, however, is not oxidized by man, and is excreted largely unchanged. Rats and rabbits, on the contrary, do oxidize mescaline to trimethoxyphenylacetic acid and are therefore relatively immune to action of this hallucinogen. This difference between species is probably not due to a difference in enzymatic activities but probably results from a difference in binding of mescaline. Other animals studied degrade mescaline in varying degrees [1]. Although mice metabolize it to a much smaller extent than rats, the oxidation to trimethoxyphenylacetic acid has been shown to occur in mouse brain homogenates, and the enzyme involved seems to be a monoamine oxidase [8]. In addition to being oxidized, mescaline is also acetylated and O-demethylated to some extent by rats, mice, cats, and humans [8a]. Guinea pig liver slices have been shown to split hordenine, probably first to dimethylamine and p-hydroxyphenylacetaldehyde, which then go on to yield further products. The metabolism of histamine is complex. In different animals it is variously oxidized, acetylated, methylated, and converted to a riboside. Ephedrine also has various fates. In several animals the initial reaction is a rapid methylation, but hydroxylation also occurs. The imidazole alkaloid pilocarpine is transformed in rat serum to unknown products that lack both imidazole and lactone rings [9].

117

Animals

The metabolism of heterocyclic nitrogen compounds, including several alkaloids, has been found to follow in general the principles outlined above. Hydroxylation of pyridine rings occurs similarly to the oxidation of benzene rings, although it is possible that different enzymatic mechanisms are responsible. Hydroxylation of pyridine compounds at a fJ-position of the ring seems analogous in its requirements to hydroxylation of a benzene ring. However, oxidation at an 1X- or y-position is evidently catalyzed by a system that contains flavine and has no requirement for reduced pyridine nucleotide [10, 11]. The betel nut (Areca catechu) alkaloid arecoline is hydrolyzed by the action of a liver esterase to arecaidine. Arecaidine appears to account for the physiological properties of betel nut better than arecoline does, and the practice of chewing betel nut with lime probably also favors the ester hydrolysis [11a].

Arecoline

Arecaidine

The metabolism of the tropane alkaloids cocaine and (±)-hyoscyamine has been investigated in a number of animals. Probably the first stage in metabolism of these compounds is hydrolysis of the esters. The further metabolism of the heterocyclic portion may involve hydroxylation and conjugation with glucuronic acid [12]. The metabolism of nicotine (1) by mammals has probably been as extensively studied as that of any alkaloid. In general, only a small percentage of administered nicotine is excreted unchanged, but the actual figures vary from animal to animal in the range of 4 to 12%. Several degradation products have been isolated; and although a complete pathway is not yet available some probable transformations are shown in Fig. 14-1. In dogs approximately one-third of the nicotine administered was found to be converted to compounds in which the pyrrolidine ring was oxidized or opened. The other compounds are relatively minor, but their concentrations depend on the animal and the conditions of nicotine administration [13]. Enzyme preparations from rat liver have been found to convert nicotine to nornicotine (2) and formaldehyde and to oxidize the nornicotine to demethylcotinine (3) [14]. Interestingly, some insects that feed on tobacco handle nicotine in a similar way, excreting the major part of it as cotinine (4) [15]. The metabolism of simple quinoline derivatives has been studied extensively; but the only quinoline alkaloids for which there is any information are the atypical Cinchona compounds. An enzyme from rabbit liver has been known for many years to catalyze the oxidation of quinoline derivatives at the 2-position to yield 1X-pyridones known as carbostyrils. The so-called "quinine oxidase" of rabbit liver has been well characterized and shown to be a soluble, iron-molybdenum-flavoprotein identical to liver aldehyde oxidase [16-19]. The two different types of reaction catalyzed by the same enzyme can be used to support the argument that so-called detoxication reactions are only fortuitous reactions catalyzed by enzymes of low specificity

Metabolism of Alkaloids by Bacteria and Animals

118

normally having other functions. Although the carbostyrils are the major excretion product of cinchonine (5) and cinchonidine (6), some additional oxidation at the £x-position of the quinuclidine ring also occurs. In the case of quinine (7) and quinidine

Nicotine Isomethonium Ion

ｾ＠ ｾＩ＠

N

Ｇｾ＠

CH3

--

-

Nicotyrine

Nicotine

Nornicotine

(1)

(2)

1 5'- Hydroxynicotine

ｾｏｈ＠ ｾｎｊ＠

__

ｾｃｈＳ＠

--

y - (3- Pyridyl) - y-

Cotinine (4)

Methylaminobutyric Acid

+?

(3)

ｾ＠

1

Methylamine

Demethylcotinine

ｾｏ＠ ｾｎＩ＠

ＬｾＮ＠

I(!;)

CH3

CH3 Cotinine Methonium Ion Fig. 14-1. Nicotine metabolism by mammals

(8) the same two positions are oxidized, but oxidation of the quinuclidine ring (at least in man) seems to take precedence over oxidation of the quinoline ring. Among the isoquinoline alkaloids morphine and related compounds seem to be the only ones whose metabolism has received much attention [1]. As with non-

Animals

119

alkaloid phenols, conjugation of the hydroxyl groups to glucuronic acid occurs with morphine and codeine. Morphine, which has two hydroxyl groups, conjugates preferentially at C-3. Codeine, which lacks a hydroxyl at C-3, conjugates at C-6. Ndemethylation of morphine occurs to a very slight extent (3 to 5% in humans). Deuterated morphine is demethylated at a slower rate than ordinary morphine [2]. N-demethylase activity is less in the livers of rats habituated to morphine, but a lower rate is not observed for demethylation in vivo by habituated rats. Codeine, in addition to being conjugated and N-demethylated like morphine, is also partially converted to morphine by an O-demethylation in man and rats, but is not converted in dogs. The N-demethylation reactions can be brought about in vitro by liver microsomes requiring NADPH2 and 02' An N-oxide intermediate has been suggested

ｃｈｏＭＨｾｉ＠ ｒｾ＠

ｾｃｈ］Ｒ＠

ｒｾ＠

UN) R

R=

ｃｈｏＭＨｾｹ＠

H: Cinchonine (5) and Cinchonidine (6) OCH3 : Quinine (7) and Quinidine (8)

ｒｾ＠

2-Hydroxy Compounds

=

ｖｎｾｏ＠

ｃｈｏＭＨｾｉ＠

ｾ＠

VN)

1

t

ｾ＠

Carbostyrils

ｒｾ＠

ｃｈｏＭＨｾｹ＠

ｖｎｾｏ＠

ｾ＠

2,2'-Dihydroxy Derivatives

[20]. Adaptation of rats to morphine involves not simply changes in the enzymes that metabolize it, but also increased ability to excrete unchanged morphine and consequently less accumulation of alkaloid in the tissues [20a]. Strychnine, lysergic acid (in the form of its synthetic derivative the diethylamide), and reserpine are three important indole alkaloids whose metabolism in animals has been investigated. Four metabolites of strychnine produced in rabbit liver have been isolated; but only one of these has been identified, namely 2-hydroxystrychnine, which has only 1 % of the toxicity or strychnine [21]. In lysergic acid hydroxylation at C-2 of the indole nucleus is brought about by the usual liver microsome hydroxylating system; and this is followed by extensive breakdown to unidentified products. It has been shown that reserpine is demethylated and the ester group hydrolyzed, but there are significant species differences in the extent to which these processess occur. It is interesting that the LSD hydroxylating enzyme is inhibited in vitro by reserpine [22]. In vitro experiments have been used to investigate the metabolism of corynantheidine-type alkaloids by liver microsomes. The major reaction with rabbit microsomes was an O-demethylation producing formaldehyde. Ring hydroxylation did not occur [22a].

120

Metabolism of Alkaloids by Bacteria and Animals

The methylated xanthine alkaloids are to some extent oxidized at C-8 to form methylated uric acid derivatives and to some extent demethylated. In man oxidation occurs to a greater extent than demethylation. Methylated uric acids are the chief excretion products of caffeine and theophylline, but theobromine is excreted mostly as methylated xanthines. Animals other than man which have been studied show somewhat different emphases in detail within these general types of processes. For example, while in man and rabbits demethylation occurs preferentially at N-3, in the dog it occurs preferentially at N-1 and N-7.

Microorganisms The metabolism of alkaloids by microorganisms has been studied, but the only case for which a rather complete pathway can be given is the degradation of nicotine (1) by Arthrobacter spp. This pathway, developed primarily through the work of DECKER, RITTENBERG, and their co-workers, interestingly is quite different from the route of nicotine degradation by animals [23-32]. In Arthrobacter spp. the first step in degradation is oxidation to 6-hydroxynicotine (9). The enzyme responsible for this oxidation is induced by growth on nicotine as the sole carbon source and repressed by availability of other carbon-nitrogen sources. Studies with purified enzyme using 180 have shown that the introduced oxygen is derived from water rather than 02' so that the reaction probably involves hydroxylation and dehydrogenation [25]. The second step is an oxidative splitting of the pyrrolidine ring. This second step may also involve two separate reactions - dehydrogenation and hydrolysis [26,21]. A third oxidation to the 2,6-dihydroxypyridine ring is followed in cell-free systems by ring closure to a product that is not metabolized further [28,29], but in intact cells a combination of enzymatic and non-enzymatic reactions leads to further breakdown according to the scheme presented in Fig. 14-2 [30]. The structure of the blue pigment produced is not yet known. It probably belongs to the ill-defined group of azaquinones and has no metabolic function [31]. A suggested structure for a similar blue pigment produced by a pseudomonad from isonicotinic acid is [32]:

Hm CO O N 0 HO ｾ＠ h- OH o

N

oCOOH

For alkaloids other than nicotine there are in the literature some isolated examples of reactions brought about by microorganisms, but no complete metabolic pathways. Ricinine is acted on by a nitrilase of Pseudomonas spp., resulting in conversion of the cyano group to carboxyl: OCH3

--

QCOOH N I

CH3

0

Microorganisms

121

Other (synthetic) 3-cyano-2-pyridones are acted on similarly. It is interesting that although some amide is also produced, it is not an intermediate in formation of the acid [33, 34].

Nicotine (1)

6-Hydroxynicotine (9)

6-Hydroxypseudooxynicotine

6-Hydroxy - Nmethylmyosmine

/

Blue Pigment

/

a

HO >-N

OH

n

o

_

OH

lic

HO "N

II

OH

2,6 - Dihydroxypyridine

T

HO

N

(CH2)3NHCH3

OH

2,6 - Dihydroxypseudooxynicotine

1

f£9tH3

HO "N

ｾ＠

2,6 - Dihydroxy - N methylmyosmine ..-:;CH

HC Y I

ｏｾ＠

C

'COOH ｾ＠

'NH2

Maleic Acid

ｾ＠

Fumaric Acid

Maleamic Acid Fig. 14-2. Metabolism of nicotine by Arthrobacter oxidans [30]

Mimosine has been found to inhibit the growth of Escherichia coli and to have its inhibition reversed by such indole derivatives as tryptophan. It is split by E. coli into serine plus a pyridine derivative of unknown structure (35]. A number of microorganisms metabolize atropine, with the initial reactions being de methylation and ester hydrolysis. Induced enzyme systems are evidently involved in this metabolism since the ability to degrade atropine is not a stable character. Strains of Aspergillus versicolor which utilize atropine as their sole carbon source have been isolated (36].

122

Metabolism of Alkaloids by Bacteria and Animals

Hydroxylation reactions are a common feature of transformations of alkaloids in microbes just as they are in animals. A strain of Pseudomonas lupanii has been found capable of utilizing lupanine as its sole source of both carbon and nitrogen. The first step in lupanine breakdown was hydroxylation at C-17. This is in contrast to the pathway in Lupinus, where hydroxylation occurs at C-13 (see Chapter 6). The hydroxylating enzyme is inducible by lupanine [36a].

c;6D °

17-Hydroxylupanine

Conversion of thebaine to 14-hydroxycodeinone can be brought about by the woodrotting fungus Trametes sanguinea, with the introduced oxygen atom derived from 02 and not from water [37]:

Thebaine

14-H ydroxycodeinone

Microbial hydroxylation of steroids has been an important source of intermediates for steroid hormone syntheses. Not surprisingly, steroid alkaloids can be hydroxylated in the same way as other steroids [38]. For example, Aspergillus ochraceus brings about monohydroxylation at either the 11-£x or 12-fJ positions of funtumine and funtumidine [39]. Hydroxylations of cones sine [40,41], solasodine [42], and tomatidine [43] by other fungi have also been reported. The 7, 9, and 11 positions are the ones usually attacked.

Higher plants There is very little information on the metabolism of foreign alkaloids by normally alkaloid-free higher plants. However, it has been shown that wheat plants can take up from solution such alkaloids as nicotine, berberine, and codeine and decompose them [44]. In Salvia officinalis there was evidently adaptive formation of a nicotinedecomposing system since plants supplied nicotine over a period of time increased in their ability to decompose it [44]. The conversion of thebaine into codeine and morphine by tobacco plants has been mentioned in Chapter 8. In all of these cases, since germ-free plants were not employed, the possibility of alkaloid metabolism by contaminating microorganisms cannot be ruled out.

Bibliography

123

Bibliography WILLIAMS, R. T.: Detoxication mechanisms, 2nd ed. New York: John Wiley 1959. SHUSTER, L.: Ann. Rev. Biochem. 33, 571-596 (1964). BUSH, M. T., and E. SANDERS: Ann. Rev. Pharmacol. 7, 57-76 (1967). ADAMSON, R. H., R. L. DIXON, F. L. FRANCIS, and D. P. RALL: Proc. Nat. Acad. Sci. US 54, 1386-1391 (1965). 5. BAKER, J., and S. CHAYKIN: Biochem. et Biophys. Acta 41, 548-550 (1960). 6. McKENNIS, H., E. R. BOWMAN, A. HORVATH, and J. P. BEDERKA, Jr.: Nature 202, 699-700 (1964). 7. KAUFMANN, S.: J. BioI. Chern. 239, 332-338 (1964). 7a. SCHENKMAN, J. B., H. REMMER, and R. W. ESTABROOK: Mol. Pharmacol. 3, 113-123 (1967). 8. SEILER, N.: Z. physiol. Chern. 341, 105-110 (1965). 8a. MUSACCHIO, ]. M., and M. GOLDSTEIN: Biochem. Pharmacol. 16,963-970 (1967). 9. LAVALLEE, W. F., and H. ROSENKRANTZ: Biochem. Pharmacol. 15, 206-210 (1966). 10. GREENLEE, L., and P. HANDLER: J. BioI. Chern. 239, 1090-1095 (1964). 11. QUINN, G. P., and P. GREENGARD: Arch. Biochem. Biophys. 115, 146-152 (1966). lla. NIESCHULZ, 0., u. P. SCHMERSAHL: Naturwissenschaften 54, 21 (1967). 12. STOLMAN, A., and C. P. STEWART: Prog. Chern. ToxicoI. 2,1-181 (1965). 13. McKENNIS, H. Jr., L. B. TURNBULL, and E. R. BOWMAN: J. BioI. Chern. 238, 719-723 (1963). 14. PAPADOPOULOS, N. M.: Can. J. Biochem. 42, 435-442 (1964). 15. SELF, L. S., F. E. GUTHRIE, and E. HODGSON: Nature 204, 300-301 (1964). 16. KNOX, W. E.: J. BioI. Chern. 163,699-711 (1946). 17. VILLELA, G. G.: Enzymologia 25, 261-268 (1963). 18. RA]AGOPALAN, K. V., and P. HANDLER: J. BioI. Chern. 239,2022-2026 (1964). 19. - - J. BioI. Chern. 239, 2027-2035 (1964). 20. WAY, E. L., and T. K. ADLER: Pharmacol. Revs. 12,383-446 (1960). 20a. DAMBROWSKI, R.: Bull. acado polon. sci., Ser. sci. bioI. 14, 667-669 (1966). 21. TAKAMOTO, H., K. OGURI, T. WATABE, and H. YOSHIMURA: ]. Biochem. (Tokyo) 55, 394-400 (1964). 22. AXELROD, J., R. O. BRADY, B. WITKOP, and E. U. EVARTS: Ann. N. Y. Acad. Sci. 66, 435-444 (1957). 22a. BECKETT, A. H., and D. M. MORTON: Biochem. Pharmacol 16,1609-1615 (1967). 23. DECKER, K., F. A. GRIES und M. BRUHMULLER: Z. physiol. Chern. 323, 249-263 (1961). 24. DECKER, K., H. EBERWEIN, F. A. GRIES und M. BRUHMULLER: Biochem. Z. 334, 227 to 244 (1961). 25. HOCHSTEIN, L. 1., and B. P. DALTON: Biochem. Biophys. Res. Communs. 21, 644-648 (1965). 26. DECKER, K., and H. BLEEG: Biochim. et Biophys. Acta 105,313-324 (1965). 27. HOCHSTEIN, L. 1., and S. C. RITTENBERG: ]. BioI. Chern. 235, 795-799 (1960). 28. RICHARDSON, S. H., and S. C. RITTENBERG: J. BioI. Chern. 236, 959-963 (1961). 29. - - J. BioI. Chern. 236, 964-967 (1961). 30. GHERNA, R. L., S. H. RICHARDSON, and S. C. RITTENBERG: J. BioI. Chern. 240, 3669 to 3674 (1965). 31. ENSIGN, J. c., u. S. C. RITTENBERG: Arch. Mikrobiol. 47, 137-153 (1963). 32. - - Arch. Mikrobiol. 51, 384-392 (1965). 33. ROBINSON, W. G., and R. H. HOOK: ]. BioI. Chern. 239, 4257-4262 (1964). 34. HOOK, R. H., and W. G. ROBINSON: ]. BioI. Chern. 239, 4263-4267 (1964). 35. SUDA, S.: Botan. Mag. (Tokyo) 73, 142-147 (1960). 36. SCHMIDT, G. c., H. W. WALKER, F. R. ROEGNER, and C. G. FISCHER: J. Pharm. Sci. 55, 914-919 (1966). 36a. TOCZKO, M.: Biochim. et Biophys. Acta 128, 570-573 (1966). 37. AIDA, K., K. UCHIDA, K. IIZUKA, S. OKUDA, K. TSUDA, and T. UEMURA: Biochem. Biophys. Res. Communs. 22, 13-16 (1966). 1. 2. 3. 4.

124

Metabolism of Alkaloids by Bacteria and Animals

38. CAPEK, A., o. HANC, and M. TADRA: Microbiological transformations of steroids. The Hague: W. Junk 1966. 39. GREENSPAN, G., R. REES, L. L. SMITH, and H. E. ALBURN: J. Org. Chern. 30, 4215-4219

(1965). 40. PATIERSON, E. L., W. W. ANDRES und R. E. HARTMAN: Experientia 20, 256-257 (1964). 41. MARX, A. F., H. C. BECK, W. F. VAN DER WARD, and J. DE FLINES: Steroids 8, 421-434 (1966). 42. SATO, Y., and S. HAYAKAWA: J. Org. Chern. 28,2739-2742 (1963). 43. - - J. Org. Chern. 29, 198-201 (1964). 44. FRANZ, G.: Z. Pflanzenernaehr., Dueng. u. Bodenk. 96, 218-230 (1962).

Chapter 15

Biochemical Pharmacology of Alkaloids I'll tell thee everything I can: There's little to relate The White Knight's Song,

LEWIS CARROLL

The above quotation may appear preposterously inappropriate in view of the vast literature on pharmacological effects of alkaloids. However, there is indeed little to relate regarding the biochemistry of these effects. Ultimately, gross effects of alkaloids and other foreign molecules on the behavior of animals must have their explanations in terms of specific molecular interactions and chemical reactions. In the course of its development pharmacology has come from observations of total behavior to observations of gross physiological processes, observations on isolated organs and tissues, and elegant and detailed observations at the cellular level. In very few cases has it started to approach observations at the molecular level. At the present, though, it seems important to suggest some of the problems and difficulties that necessarily attend any such molecular approach and to point to a few areas that seem to show some glimmers of success. Essential aspects of pharmacology are doubtless overlooked in this chapter, and it should probably be regarded as a smattering of things that have been of interest to one biochemist rather than a balanced review. Where references to literature are not given, the reader is referred to the general sources [1-7]. The most attractive explanations for the powerful physiological action of small molecules at low concentration concern themselves with the following types of biochemical processes: 1. Mechanisms of DNA replication, RNA transcription, and protein synthesis. Several antibiotics are clearly understood as having their action at this level. Some alkaloids also probably act here (see below). 2. Mechanisms of transport across membranes, both active and passive. The access of substrates to enzymes and the excretion of metabolic products clearly depend on these transport mechanisms. Passive membrane permeability is influenced by many compounds. Active transport is a more specific and complex process known to be influenced by such drugs as the cardiac glycosides as well as several of the Veratrum alkaloids. 3. Mechanisms of enzyme inhibition or activation. These mechanisms are the most convenient to study in vitro but often difficult to relate to gross physiological effects because at a suitable concentration nearly any compound will show some effect on nearly any enzyme. Since active membrane transport is an enzymatic process, it could also be included under these mechanisms (see below).

126

Biochemical Pharmacology of Alkaloids

4. Mechanisms involving structural changes. Changes in macromolecular conformations as the result of adsorption of small molecules or displacement of one small molecule by another may provide the most general kind of explanation for drug effects since all the previous three types of mechanisms can be considered as dependent on particular macromolecular configurations. S. Blocking of receptor sites for chemical transmitters. Before any administered alkaloid can have a pharmacological effect, it must obviously reach its site (or sites) of action. In this simple qualification lurk great difficulties for understanding mechanisms of drug action in complex organisms. Not only are physical barriers and diverse transport systems influential in controlling distribution, but metabolic changes such as those discussed in the previous chapter may intervene. These various and incompletely understood processes cannot be neglected in any complete explanation of the physiological effects of an administered alkaloid, yet they are outside the usual sphere of interest of biochemistry. Biochemistry can begin only with some knowledge of what the active compound is and what its concentration is at the site where it is presumed to act. Considerations of concentration have been a major stumbling block for explanations of pharmacological mechanisms. In whole organisms as well as in simplified systems a given compound at different concentrations may show effects that appear to be qualitatively and not merely quantitatively different. The literature is rife with examples of drugs which at 10-3 M inhibit certain crucial systems in vitro, but at the presumed receptor in vivo the effective concentration may be found to be lower by several orders of magnitude. At least, explanations of gross effects based on such findings should be suspected; probably they should often be regarded as entirely irrelevant. An important adage of pharmacology is that no drug has a single effect even though one effect may predominate or, by adjustment of concentration, one effect may be favored over others. As an example, tranquilizers have been shown to influence respiratory processes, phosphorylation, ion movements, carbohydrate metabolism, lipid metabolism, uptake of amines and amino acids, release of hormones, and activities of several enzymes. For an understanding of their tranquilizing actions some of these effects may be direct and pertinent, others may be pertinent but indirect, and others may be irrelevant. Once knowledge is at hand of a known compound, in a certain concentration range, at a defined site of action, explanations of its action at a molecular level are approachable. Some kind of complementarity between agent and receptor is taken as self-evident. Various types of molecular interaction can be distinguished, and some of these have been shown to have significance in systems of pharmacological interest. The use of gross structural formulae is inadequate for an understanding of such interactions since subtle aspects of shape and electron distribution need to be considered. While quantum biochemistry may have promise of providing deeper understanding in the future [8], at its present stage of development it appears to offer very slight advance over cruder theoretical systems which attempt to relate electronic structures of drugs to their mechanisms of action. In some cases covalent bonding between drug and receptor may occur; but this is probably not as usual as weaker attachments by ionic attractions, hydrogen bonding, metal coordination, and hydrophobic attraction. Considering alkaloids in

Biochemical Pharmacology of Alkaloids

127

particular, this statement of BLOOM and LAUBACH [4] seems especially apt: "The amino group knows no peer in the remarkable variety of binding involvement which can result from subtle changes in its functionality". All trivalent nitrogen atoms can act as electron pair donors to suitable acceptors, either metal ions or electrophilic organic molecules. Primary and secondary amines can participate in hydrogen bond formation either as donors or acceptors. Primary amines can form covalent Schiff bases with carbonyl compounds. All amino compounds on the acidic side of their isoelectric points exist as cations which will be attracted to anionic groups. Changes in structure, by modifying the ionization potential of the amino group, can profoundly alter its reactive capabilities where these depend on ionic form. The quaternary ammonium compounds found among the alkaloids are, of course, cations at all pH values and are often so strongly adsorbed near their site of entry that they reach internal tissues at lower concentrations than do similar but less highly charged molecules. With the foregoing generalizations and qualifications disposed of, a few examples of alkaloid pharmacology at the molecular level can be considered. These mechanisms have been illuminated as much by studies with synthetic compounds as by studies with naturally occurring alkaloids, and it would therefore be impracticable to limit the discussion rigidly to natural compounds. Drugs acting on the autonomic nervous system have received considerable attention in recent years, and some clear cut biochemical findings have resulted. Anatomically, the peripheral autonomic nervous system can be subdivided into four types of fibers; biochemically, these fibers are divided into "adrenergic" types, whose chemical transmitter is noradrenaline, and "cholinergic" types, whose chemical transmitter is acetylcholine. Anatomical details can be found in Ref. [1]. The chemical and anatomical divisions may be summarized as follows: peripheral autonomic system thoracolumbar or sympathetic

craniosacral or parasympathetic

preganglionic fibers

cholinergic

cholinergic

postganglionic fibers

mostly adrenergic; a few cholinergic

cholinergic

The transmitter substances act not only in transmission of the nervous impulse across the synapses between nerve cells but also in the junctions between the nerve cells and the tissues they control. Conduction of the impulse along a nerve fiber is a different kind of process, which will be considered later. Acetylcholine is the most widespread neurohumoral transmitter, functioning not only in the peripheral autonomic system as shown in the table above, but also in motor nerves to skeletal muscle and in certain neurons within the central nervous system.

o II

ED

CH3 COCH2 CH 2 N (CH3)3

Acetylcholine

128

Biochemical Pharmacology of Alkaloids

Arrival of a nerve action potential at an axonal terminal stimulates release of acetylcholine that has been synthesized and stored in synaptic vesicles. The acetylcholine then diffuses across the synaptic cleft and acts on a receptor of the following neuron, muscle, or gland, which then responds appropriately. Removal of the transmitter from the junction must take place before another impulse can be transmitted. This removal occurs through a combination of diffusion and enzymatic destruction hydrolysis mediated by acetylcholinesterase. Based on this rough outline of nerve transmission, several types of mechanisms can be distinguished for drugs acting on cholinergic fibers. Some classes of agents are listed below. 1. Inhibitors of acetylcholine synthesis. 2. Substances that affect the release of acetylcholine. 3. Substances that act on the receptor as mimics of acetylcholine. 4. Substances that occupy the receptor for acetylcholine and thus block its action. 5. Inhibitors of acetylcholine esterase. The mechanisms of these agents cannot in every case be correlated with the kinds of biochemical processes tabulated on p. 125. For instance, inhibitors of acetylcholine synthesis could act either by repressing formation of an enzyme needed for the synthesis or by inhibiting action of an enzyme already present. There do not seem to be any natural alkaloids known at present which owe their effects to inhibition of acetylcholine synthesis. The synthetic drug hemicholinium does act here. The same is true for release of acetylcholine, which is affected by Botulinus toxin but not by any typical alkaloids. Several alkaloids are known to mimic the effect of acetylcholine on its receptors. They may be subdivided according to the type of receptor involved, some acting preferentially on the postsynaptic receptor of a neuron, some at the neuromuscular junction, and others at receptors of secretory glands. These various mimetic agents can all be shown to have some kind of structural resemblance to acetylcholine, but precise structural requirements are quite obscure, and prediction of the site of action of a compound from its structure is difficult. Several cholinomimetic alkaloids are shown in Table 15-1 with their presumed sites of action. Nicotine shows first a mimetic effect but then a blocking action on the receptor similar to that of compounds of the 4th type listed above. With increasing concentrations the blocking action increasingly predominates over the stimulating action. Several other alkaloids act primarily by blocking receptors of acetylcholine; and since they antagonize the stimulatory effect of muscarine, they are known as antimuscarinic agents. Most of these show their greatest action on autonomic effector cells rather than on ganglion cells. There are also differences in the sensitivities of the different types of effector cells, with the general order of some sensitivities being: salivary glands > iris muscles > visceral muscles > gastric secretory cells. The best-known of these blocking agents is atropine [(±)-hyoscyamine]. Other tropane alkaloids act similarly. Cocaine, which is structurally a tropane alkaloid, shares these properties to only a small extent and is more noted for its effects on adrenergic receptors. Studies of the relation of structure to activity indicate that what is required for antimuscarinic action is an ester of tropic acid with a tertiary amino alcohol. This requirement is met by hyoscyamine and hyoscine but not by cocaine. Curare alkaloids (both the bisbenzylisoquinoline and strychnine types) and Erythrina alkaloids are particularly noted for blocking the neuromuscular junction, although they can also block ganglionic transmission, release acetylcholine, and inhibit cholinesterase.

Biochemical Pharmacology of Alkaloids

129

The actions of acetylcholine itself, the cholinomimetic compounds, and the blocking agents all presumably depend on interaction of these compounds with a receptor whose chemical nature remains unknown. It may be inferred that the receptor contains at least an anionic site that is neutralized by ion pair formation with the positively charged nitrogen of acetylcholine. Compounds that mimic acetylcholine, even though not all of them are quaternary, may also attach at this site and activate the receptor. The blocking agents are thought to shield the anionic site of the receptor without activating it. Obviously other sites of the receptor molecule must be inTable 15-1. Some cholinomimetic alkaloids

Site of Stimulating Effect

Alkaloid

ganglion cells

ganglion cells; various autonomic effector cells

Pilocarpine motor end-plate of skeletal muscle; ganglion cells; various automatic effector cells

H0r-----, CH3

Ar-.A CH

°

@

various autonomic effector cells

z N (CH3)3

Muscarine volved because of the complex relationships between structure and activity that have been observed. Although it is most often assumed that the receptor molecule is a protein, proposals that it is a phosphate ester or an acidic nitrogen-containing polysaccharide similar to hyaluronic acid have also been put forward [9a]. It would be expected that a strong cation like tubocurarine would combine with strong anions such as acidic polysaccharides, as has been shown to happen in the electric organ of the electric eel; but this combination probably has no general physiological significance. It has been shown that tubocurarine inhibits the rhythmic movements of chicken embryos too young to have neuromuscular junctions, and an entirely different mechanism such as enzyme inhibition must be invoked in explanation [10]. Atropine has been shown to form spectroscopically observable complexes with the amino groups of proteins. Such complex formation is dependent on the ester group of atropine and is influenced by the presence of acetylcholine [11]. Wehther or not 9

Robinson, The Biochemistry of Alkaloids

130

Biochemical Pharmacology of Alkaloids

such in vitro observations have any pertinence to an understanding of the cholinergic receptor remains questionable. Inhibitors of acetylcholinesterase have become prominent in recent years because of their use as insecticides and their potential use as "nerve gases". These agents cause accumulation of acetylcholine and therefore continuous stimulation of cholinergic receptors. Although the vast majority of cholinesterase inhibitors are synthetic, they do include the alkaloid physostigmine (eserine). There has been intensive study of the mechanism of action of cholinesterase, and there is now quite a clear understanding of the details of its catalysis and inhibition. The enzyme has two spatially separated active sites, an anionic site that binds the quaternary nitrogen of acetylcholine and an esteratic site that binds the carboxyl carbon of the ester. The anionic site is probably a carboxylate group, while the esteratic site contains the hydroxyl group of a serine residue and the basic imidazole group of histidine. The attachment of the substrate may be further strengthened by other forces, such as VAN DER WAALS forces. After formation of the enzyme-substrate complex, splitting of the ester by shift of a proton occurs, leaving an acetylated enzyme that is rapidly hydrolyzed to acetate plus the enzyme. Physostigmine, in contrast, forms a carbamylated enzyme whose hydrolysis is very much slower, so that the enzyme is effectually taken out of action. These reactions are shown diagrammatically in Fig. 15-1. Physostigmine also shows other effects, unrelated to its inhibition of cholinesterase [12]. The synthetic cholinesterase inhibitors form phosphorylated enzymes that are hydrolyzed at a still slower rate, so that their action is considered to be practically irreversible. Simple quaternary ammonium salts also inhibit cholinesterase by binding at the anionic site, but this binding is weak and competitive with that of acetylcholine. Alkaloids and other drugs that act on adrenergic transmission show certain analogies with those that act on the cholinergic system, but the parallels are not strict. The most significant difference is that there is no enzyme analogous to acetylcholinesterase for destruction of noradrenaline, and its removal from the junction occurs by diffusion or active transport. The enzyme monoamine oxidase (MAO) is regarded as a protective device for disposing of nontransmitter amines that might otherwise interfere with adrenergic transmission [3a]. Analogous to the cholinomimetic substances discussed above are the sympathomimetic amines, which mimic the action of noradrenaline at adrenergic receptors. The structure of the adrenergic receptor and its binding of noradrenaline has been depicted diagrammatically as follows [4]:

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p-Site Inhibitory

131

Biochemical Pharmacology of Alkaloids

Binding at the IX-site is by ionic attraction and hydrogen bonding, whereas a divalent metal ion such as Mg++ serves at the jJ-site to link noradrenaline to the receptor. Since noradrenaline and related compounds can cause both excitation and inhibition of

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smooth muscle, the two sites have been proposed, with different types of effector cells having different proportions of the two sites to account for their different responses. Adrenaline with one N-methyl group shows greater jJ-stimulation than 9*

132

Biochemical Pharmacology of Alkaloids

noradrenaline. Substitution on the lX-carbon of a phenylethylamine prolongs whatever action it has by preventing its destruction by monoamine oxidase. Most sympathomimetic compounds do not act by directly mimicking the action of noradrenaline on its receptor. Rather, they are either releasers of noradrenaline, inhibitors of its readsorption, or blockers of either the lX- or fJ-site. Tyramine is primarily a releaser of noradrenaline. The protoalkaloid ephedrine acts as a releaser of noradrenaline and is also a true mimetic substance that stimulates both the lX- and fJ-sites, causing both vasoconstriction and bronchial muscle relaxation. Cocaine potentiates the action of noradrenaline by hindering its reabsorption into the nerve ending. A compound that blocks the lX-site allows responses characteristic of the fJ-site to predominate and thus acts as fJ-site stimulant. Several alkaloids act as lXadrenergic blocking compounds. These include yohimbine and some of the peptide ergot alkaloids. These natural compounds also have other complex effects and are largely replaced in medicine by more specific, synthetic lX-adrenergic blocking agents. fJ-Adrenergic blocking agents are less common and include only synthetic compounds. The RaulJ'olfia alkaloids, especially reserpine, have an antiadrenergic effect in addition to their action on the central nervous system. They act to cause a release of noradrenaline at a rate so slow that it does not act as a stimulus but is dissipated and metabolized. As a result of this depletion of the required transmitter substance, all adrenergic responses are diminished. The analogous release of other amines such as serotonin is suggested as a mechanism for the action of reserpine on the central nervous system. An important group of pharmacological agents are the monoamine oxidase inhibitors [13]. As pointed out above, MAO is not of crucial importance in removal of noradrenaline from junctions but rather acts as a scavenger of other amines that, if present, would simulate the action of noradrenaline by releasing it from stores or mimicking it at its acceptor. Inhibition of MAO therefore has the effect of stimulating adrenergic receptors. The amines that do the actual stimulating may be of endogenous or exogenous origin. For instance, cheese, which contains tyramine, may be quite toxic if consumed concurrently with a MAO inhibitor. Tyramine, dopamine, serotonin, etc., are also normal cell constituents in small amounts. The interpretation of a drug action as due primarily to MAO inhibition is extraordinarily difficult. Most MAO inhibitors show a number of other actions (e.g., inhibition of amine release); and compounds such as cocaine and ephedrine inhibit MAO in vitro, but their familiar pharmacological properties probably have nothing to do with this. The best known MAO inhibitors are synthetic hydrazine derivatives, but non-hydrazine inhibitors are more selective for MAO. The harmala alkaloids are naturally occurring MAO inhibitors, although they may act in other ways, too [14, 14a]. Under certain conditions harmaline counteracts the amine-releasing effect of reserpine. The structural requirements for MAO inhibition are not clearly defined. Axonal conduction of nervous impulses is little affected by most drugs unless they are supplied at high local concentrations as in local anesthesia, but in isolated preparations useful information concerning the mechanism of axonal conduction has been obtained by observation of drug effects. Cocaine and related synthetic compounds such as procaine inhibit conductance by competing with Ca++ for sites on the membrane which are presumed to be the phosphate groups of phospholipids. The presence of bound calcium is essential for proper functioning of the axon, since its concentra-

Biochemical Pharmacology of Alkaloids

133

tion in the membrane regulates the permeability to sodium and potassium. Displacement of Ca++ by cocaine increases the permeability and therefore decreases the ease with which an action potential can be initiated [15, 16]. Caffeine shows a similar calcium-releasing effect on muscle [17] which can actually be prevented but not reversed by cocaine. The Veratrum alkaloids cause repetitive discharges of nerve cells apparently by delaying repolarization. The primary effect may be to increase sodium conductivity of the membrane. The response to Veratrum alkaloids may be prevented by Ca++ or by cocaine. Correlation of the pharmacological effects of Veratrum alkaloids with their surface activity in monolayers is suggestive that their action may be on lipid components of the cell membrane [18]. The Veratrum alkaloids as well as cas saine (a diterpenoid alkaloid of incompletely known structure) have also been shown to inhibit an ATPase of the cell membrane which is activated by K+. The transport of potassium ion across the cell membrane is thus coupled to A TP hydrolysis; and the inhibitors are believed to bind to a phosphorylated enzyme intermediate so as to block access of K+ [19]. So-called "metabolic" effects of several alkaloids have been noted from time to time. These are effects on degradative pathways of metabolism such as glycolysis, the citric acid cycle, or fat oxidation. A few of these effects can now be explained as resulting from more or less specific enzyme inhibitions. The best examples of such effects are connected with the role of cyclic 3',s'-adenosine monophosphate (3',5'AMP). This nucleotide activatives phosphorylase a, thus stimulating glycolysis, and also activates lipase, thus stimulating fat breakdown [20-22]. Its formation from A TP is stimulated by adrenaline or noradrenaline; its destruction by phosphodiesterase is inhibited competitively by theophylline. Thus methylxanthines and catecholamines can act together, but at different sites, to increase blood sugar levels and oxygen consumption, or to stimulate other processes dependent on 3',s/-AMP. With the development of techniques for studying nucleic acid and protein synthesis in vitro it has become possible to study the action of alkaloids on these systems, and there is now evidence that several alkaloids may owe their pharmacological effects to reactions at this level. Colchicine and the Vinca alkaloids vincristine and vinblastine inhibit the incorporation of uridine into RNA by preparations of ascites cells or mouse brain [23,24]. Colchicine also becomes bound specifically to a small protein that is evidently part of the mitotic apparatus of sea urchin eggs [25, 26]. The diaminosteroid alkaloids of Buxlts and Holarrhena spp. interact with the DNA double helix changing its conformation or transforming it into two linked random coils [27]. The observation that ipecacuanha alkaloids have structural resemblances to glutarimide antibiotics such as cycloheximide suggested that certain of these alkaloids might, like cycloheximide, be inhibitors of protein synthesis. This suggestion has been substantiated by the finding that tubulosine hinders the transfer of amino acids from transfer RNA in HeLa cells [28]. A preliminary but promising approach to the mechanism of drug action is the search for specific binding between pharmacologically active compounds and particular cell constituents. Demonstration of complex-formation is of course only a beginning since the binding must be shown to be specific and to have appropriate consequences. The binding of atropine to proteins has been mentioned earlier [11]. Psilocybin has been found to form an insoluble complex with a fraction of rat brain

134

Biochemical Pharmacology of Alkaloids

mitochondria believed to contain particles from nerve endings. Since high concentrations of psilocybin were required, though, this phenomenon may not have any bearing on the hallucinogenic effects of the drug which are produced by much lower concentrations [29]. A ganglioside isolated from human brain has been shown to complex strongly with serotonin. Calcium ion competes with serotonin, and reserpine releases serotonin from the complex quickly and completely. These observations suggest that the natural serotonin receptor may be a ganglioside [30]. The serious social problem of drug addiction has been hardly approached from the biochemical standpoint, but a generalized biochemical theory has been put forward and may be amenable to experimental testing [31,32]. According to this theory, addicting drugs inhibit the action of some enzyme (or enzymes) whose synthesis is repressed by a product (or products) of its action. When the enzyme's action is inhibited, less product is made; and as a result, more enzyme is synthesized in order to preserve the normal metabolic equilibrium. In the presence of an inhibitor normal metabolism can be maintained only by abnormally high concentrations of enzyme - a situation corresponding to drug "tolerance". When the inhibiting agent is removed, drastic metabolic imbalance ensues - a situation corresponding to the "withdrawal reaction". Identification of some of the hypothetical inhibited enzymes would be a great advance in our understanding of the biochemistry of drug addiction. Finally, a few miscellaneous aspects of alkaloid pharmacology which have possible biochemical correlations will be mentioned. Various nitrogen bases have been found to inhibit yeast alcohol dehydrogenase by competing with nicotinamide adenine dinucleotide (NAD) for its binding site [33]. Since NAD-dependent dehydrogenases are of widespread importance in metabolism, this kind of mechanism might be of general significance in explaining some metabolic effects of alkaloids. Several of the pyrrolizidine alkaloids are known to have a powerful hepatotoxic effect, and those that do are all esters of allylic alcohols. Compounds of this structure are known to be alkylating agents, and their various effects are believed to be related to their alkylation of thiol or other nucleophilic groups of cell nuclei [34].

Bibliography 1. GOODMAN, L. S., and A. GILMAN: The pharmacological basis of therapeutics,"3 rd ed. New York: Macmillan 1965. 2. MANSKE, R. H. F.: The alkaloids 5. New York: Academic Press 1955. 3. ARIENS, E. J.: Molecular pharmacology 1. New York: Academic Press 1964. 3a. IVERSEN, L. L.: Nature 214,8-14 (1967). 4. BLOOM, B. M., and G. D. LAUBACH: Ann. Rev. Pharmacol. 2, 67-108 (1962). 5. BURGER, A., and A. P. PARULKAR: Ann. Rev. Pharmacol. 6, 19--48 (1966). 6. TRIGGLE, D. J.: Chemical aspects of the autonomic nervous system. New York: Academic Press 1965. 7. EIDUSON, S., E. GELLER, A. YUWILER, and B. T. EIDUSON: Biochemistry and behavior. New Jersey: D. Van Nostrand, Princeton 1964. 8. PULLMAN, B.: In: PULLMAN, B., ed.: Electronic aspects of biochemistry, pp. 559-577. New York: Academic Press 1954. 9. See Ref. [3J p. 601. 10. SZABO, P.: Naturwissenschaften 53,109-110 (1966). 11. OROSZLAN, S. 1., and G. D. MAENGWYN-DAVIES: Biochem. Pharmacol. 11, 1213-1220 (1962).

Bibliography

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12. BELL, c.: Biochem. Pharmacol. 15, 1085-1092 (1966). 13. PLETSCHER, A.: Pharmacol. Revs.1S, 121-129 (1966). 14. ZIRKLE, C. L., and C. KAISER: In: GORDON, M., ed.: Psychopharmacological agents 1, pp. 445-554. New York: Academic Press 1964. 14a. GORKIN, V. Z., and L. V. TATYANENKO: Life Sciences 6, 791-795 (1967). 15. GOLDMAN, D. E., and M. P. BLAUSTEIN: Ann. N.Y. Acad. Sci. 137,967-981 (1966). 16. BLAUSTEIN, M. P., and D. E. GOLDMAN: Science 153, 429-432 (1966). 17. SANDOW, A., u. M. BRUST: Biochem. Z. 345, 232-247 (1966). 18. GERSHFELD, N. L.: Biochim. et Biophys. Acta 42, 282-289 (1960). 19. PORTIUS, H. ]., u. K. REPKE: Arzneimittel-Forsch. 14, 1073-1077 (1964). 20. TRINER, L., and G. G. NAHAS: Science 150, 1725-1727 (1965). 21. HYNIE, S., G. KRISHNA, and B. B. BRODIE: ]. Pharmacol. Exp!. Therap. 153,90-96 (1966). 22. TRINER, L., and G. G. NAHAS:]' Pharmacol. Exp!. Therap. 153,569-572 (1966). 23. CREASEY, W. A., and M. E. MARKIW: Biochim. et Biophys. Acta 103, 635-645 (1965). 24. AGUSTIN, B. M., and W. A. CREASEY: Nature 215, 965-966 (1967). 25. BORISKY, G. G., and E. W. TAYLOR: J. Cell. BioI. 34, 525-533 (1967). 26. - - J. Cell. Bio!. 34, 535-548 (1967). 27. MAHLER, H. R., and M. B. BAYLOR: Proc. Nat. Acad. Sci. US 58, 256-263 (1967). 28. GROLLMAN, A. P.: Science 157, 84--85 (1967). 29. GILMOUR, L. P., and R. D. O'BRIEN: Science 155, 207-208 (1967). 30. GIELEN, W.: Z. Naturforsch. 21b, 1007-1008 (1966). 31. GOLDSTEIN, D. B., and A. GOLDSTEIN: Biochem. Pharmacal. S, 48 (1961). 32. ATKINSON, D. E.: Science 150, 851-857 (1965). 33. ANDERSON, B. M., M. L. REYNOLDS, and C. D. ANDERSON: Biochem. et Biophys. Acta 113,235-243 (1966). 3-1. CULVENOR, C. c. J., A. T. DANN, and A. T. DICK: Nature 195, 570-573 (1962).

Addendum Annotated bibliography of recent references arranged according to sequence of topics in the main text.

Chapter 1 PANVISAVAS, R., L. R. WORTHEN, and B. A. BOHM: Lloydia 31,63-69 (1968). Evidence that alkaloids are absent in ferns. BRIGGS, D. E.: Phytochem. 7, 539-554 (1968). Basic compounds, including some alkaloids, inhibit formation of (X-amylase in germinating barley.

Chapter 2 JINDRA, A., and E. J. STABA: Phytochem. 7, 79-82 (1968). Several enzymes, possibly involved in alkaloid metabolism, shown to be present in tissue cultures of Datura stramonium. TAYLOR, W. 1., and A. R. BATTERSBY, eds.: Oxidative Coupling of Phenols. New York: Marcel Dekker 1968. Book includes material pertinent to alkaloid biosynthesis. BARTON, D. H. R.: Chemistry in Britain 3, 330-337 (1967). Review on phenol coupling mechanisms in biosynthesis.

Chapter 3 WIELAND, T.: Science 159, 946-952 (1968). A review on poisonous constituents of Amanita spp. including muscarine, isoxazoles, and peptides. AGURELL, S., and J. L. G. NILSSON: Tetrahedron Letters 1968, 1063--1064. Tracer studies with Psilot:ybe cttbensis suggest biosynthetic pathway: tryptophan ......tryptamine -..N-methyltryptamines -..psilocin ......psilocybin. BOCKS, S. M. : Biochem. J. 106, 12P-13P (1968). Laccase catalyzes oxidation of psilocin to a blue pigment. BRECCIA, A., and A. M. CRESPI: Z. Naturforsch. 21b, 832-855 (1966). Tracer feeding experiments with barley showed that tryptophan and pyruvate were good precursors of gramine - the latter via a pathway perhaps not involving tryptophan. - - , and M. A. RAMPI: Z. Naturforsch. 21 b, 1243-1245 (1966). High speed supernatant of a barley seedling homogenate catalyzed formation of gramine from tryptophan, ATP and methionine. DIGENIS, G. A., and B. A. FARAJ: Experientia 23,774-775 (1967). Further report on studies of ref. 40.

Chapter 4 LEETE, E.: J. Am. Chem. Soc. 89, 7081-7084 (1967). Reaction of N-methyl-LP-pyrrolinium-2-14C salt with acetonedicarboxylic acid gave hygrine labelled only at C-2. HOPE, D. B., K. C HORNCASTLE, and R. T. APLIN: Biochem. J. 105, 663--667 (1967). L-amino oxidase of Mytilus edulis converts lysine to LP-piperideine-2-carboxylic acid, which spontaneously dimerizes. DIETRICH, S. M. C, and R. O. MARTIN: J. Am. Chem. Soc. 90, 1921-1923 (1968). Short-term exposure of Conium maculatu1l1 to 14C02 gave y-coniceine with much higher specific activity than other alkaloids.

Addendum

137

O'DONOVAN, D. G., and M. F. KEOGH: Tetrahedron Letters 1968, 265-267. Acetate shown to be precursor only of side chain in N-methylisopelletierine of Pllniea granatllm. Lysine was precursor of ring. GUPTA, R. N., and 1. D. SPENSER: Chern. Commun. 1968,85-86. Double labelling showed that C-6 of lysine went specifically to C-6 of piperidine ring of N-methylisopelletierine in Sedllm sarmentosulJl. DESATY, D., and L. C. VINING: Can. J. Biochem. 45, 1953-1959 (1967). Tryptophan shown to be an efficient precursor of nicotinic acid in Fusarium Ox)'sporum. MACNICOL, P. K.: Biochem. J. 107,473-479 (1968). 6-Hydroxykynurenic acid was identified in leaves of several plants. Kuss, E.: Z. physiol. Chern. 348, 1589-1595, 1602-1608 (1967). Unstable intermediate between 3-hydroxyanthranilic acid and quinolinic acid was identified. GROSS, D., P. BANDITT, A. ZURECK und H. R. SCHUTTE: Z. Naturforsch. 23b, 390-391 (1968). Nicotinic acid of A1yeobaeterium bovis shown to be derived from aspartic acid via quinolinic acid. SCOTT, T. A., E. BELLION, and M. MATTEY: Biochem. J. 107, 23P (1968). N-formylaspartic acid shown to be precursor of nicotinic acid in Clostridium butylicum. SCHUTTE, H. R., u. U. STEPHAN: Z. Naturforsch. 22b, 1355-1357 (1967). (3-pyridyl)-(y-methylaminopropyl)-ketone-14C-carbonyl fed to rooted leaves of Nieotialla mstiea gave rise to nicotine labelled at C-2'. GILBERTSON, T. J., and E. LEETE: J. Am. Chern. Soc. 89, 7085-7088 (1967). t5-N-methyl ornithine was incorporated as a unit into N-methylpyrrolidine moiety of nicotine. ex-N-methyl ornithine was incorporated only 1/10 as well and with randomization. However, normal pathway from ornithine is probably: ornithine --+putrescine-' N-methylputrescine ＭＫＴｭ･ｴｨｹｬ｡ｩｮｯ｢ｵｾｎｰｲ＠ --+nicotine. Results of SCHROTER and NEUMAN and of RAPAPORT et al. are rationalized. SCHUTTE, H. R., W. MAIER, and K. MOTHES: Acta Biochim. Polon. 13,401-404 (1967). N-methylputrescine shown by double labelling to go as an intact unit to N-methylpyrrolidine moiety of nicotine in Nieotiana rustiea. WALLER, G. R., R. RYHAGE, and S. MEYERSON: Anal. Biochem. 18,395 (1967). Erratum to ref. 89.

Chapter 6 SCHUTTE, H. R., u. G. SEELIG: Ann. Chern., Liebigs 711,221-226 (1968). Tracer feeding experiments indicated origin of multiflorine in LuPi1ltts digitatus by pathway: lysine --+cadaverine -+sparteine -->multiflorine. c_15 N of lysine was incorporated as a unit with 14C-2. FERRIS, J. P., C. B. BOYCE, and R. C. BRINER: Tetrahedron Letters 1966, 5129-5131. The quinolizidine alkaloids of Lythraceae are postulated to be derived from units related to isopelletierine, benzaldehyde, and cinnamic acid.

Chapter 7 MULLER, P., U. H. R. SCHUTTE: Z. Naturforsch. 23b, 491-493 (1968). 1-methyl-6-hydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid found in latex of Euphorbia myrsinites was shown to be derived from m-tyrosine, which also occurs in this latex. CHAN, K. c., M. T. A. EVANS, C. H. HASSALL, and A. M. W. SANGSTER: J. Chern. Soc. (C) 1967,2479-2488. Discussion of possible routes for biosynthesis of the bis-benzylisoquinoline alkaloids of Oeotea rodiaei. First oxidative coupling probably forms ether bridge between two B-rings. GEAR, J. R., and A. K. GARG: Tetrahedron Letters 1968, 141-143. Glycine-2-14 C shown to be a specific precursor for the C9 unit of cephaeline. Presumably mevalonic acid was intermediate. BATTERSBY, A. R., and B. GREGORY: Chern. Commun. 1968, 134-135. Geraniol and loganin shown to be specific precursors of the C9 unit of cephaeline.

138

Addendum

Chapter 8 FAIRBAIRN, ]. W., and S. EL-MASRY: Phytochem. 7, 181-187 (1968). Poppy seeds were found to contain bound forms of several alkaloids including codeine. Free alkaloids were released during germination or by pepsin digestion. An enzyme system was found to incorporate morphine into these bound forms. BARTON, D. H. R., D. S. BHAKUNI, G. M. CHAPMAN, and G. W. KIRBY:]. Chem. Soc. (C) 1967,2134--2140. N-methylcoclaurine was better than coclaurine as a precursor of roemerine in Papaver dubium,' N-methylnorcoclaurine was not as good. a-methyl carbon was not retained in methylene dioxy group. BATTERSBY,A.R.,D.M.FoULKES,M.HIRST, G.V.PARRY,and]. STAUNTON:].Chem. Soc.(C) 1968,210-216. Since norlaudanosoline was incorporated into both morphine and papaverine, the two a-methylation steps must occur after isoquinoline formation. Laudanosoline is probable intermediate between norlaudanosoline and reticuline. FRANCK, B., u. L.-F. TIETZE: Angew. Chem., Intern. Ed. 6, 799-800 (1967). Chemical oxidation of laudanosolines to aporphines could be performed without quaternization if o-quinone formation was inhibited by complexing o-diphenolic groups. FROMMING, K.-H.: Arch. Pharm. 300, 977-981 (1967). Tyrosinase, laccase, or peroxidase used to catalyze oxidative coupling of laudanosoline methiodide to aporphine derivative. BARTON, D. H. R., A. ]. KIRBY, and G. W. KIRBY: ]. Chem. Soc. (C) 1968, 929-936. Full report of work cited in ref. 21. HAYNES, L. J., G. E. M. HUSBANDS, and K. L. STUART: J. Chem. Soc. (C) 1968,951-957. Tracer experiments showed conversion of coclaurine, norcoclaurine, or isococlaurine to 8,14-dihydronorsalutaridine in Croton lineari.r. BATTERSBY, A. R., J. A. MARTIN, and E. BROCHMANN-HANSSEN:]. Chem. Soc. (C) 1967, 1785-1788. Double labelling experiments established that conversion of codeinone to codeine is not reversible. BARTON, D. H. R., R. JAMES, G. W. KIRBY, D. W. TURNER, and D. A. WIDDOWSON: J. Chem. Soc. (c) 1968,1529-1536. Structure and biosynthesis of Et:ythrina alkaloids.

Chapter 9 FRANCK, B., U. H. ]. LUBS: Angew. Chem. 80,238-239 (1968). Model experiments showed oxidation of norbelladine derivatives to crinine ring system by ferric chloride. WILDMAN, W. c., and N. E. HEIMER: ]. Am. Chem. Soc. 89, 5265-5269 (1967). ｃ｡ｲｮｩ･ＭＲｰｾｈ＠ fed to Zephyranthe.r candida was converted 7.05% into lycorine. - , and D. T. BAILEY: J. Am. Chem. Soc. 89, 5514-5515 (1967). Tazettine shown to be an artifact derived during isolation by rearrangement of pretazettine. SCHUTTE, H. R., u. U. ORBAN: Naturwissenschaften 54,565 (1967). Phenylalanine deaminase activity in ColchiCflm alltumnale found sufficient to postulate its significance in formation of colchicine ring A.

Chapter 10 SLAYTOR, M., and 1. ]. McFARLANE: Phytochem. 7, 605-611 (1968). Tracer studies indicated that N-acetyltryptamine and harmalan are probable intermediates between tryptamine and harman in Pa.r.riflora eduli.r. BATTERSBY, A. R., R. S. KAPIL, and R. SOUTHGATE: Chem. Commun. 1968, 131-133. Absolute stereochemistry of loganin agrees with stereochemistry of most indole alkaloids apparently derived from it. - - , J. A. MARTIN, and L. Mo: Chem. Commun. 1968, 133-134. Labelled loganin fed to Vinca ro.rea or Rauwolfia .rerpentina gave rise to indole alkaloids all labelled at the expected position.

Addendum

139

INOUYE, H., S. VEDA, and Y. TAKEDA: Tetrahedron Letters 1968, 3453-3458. Sweroside-10-14C fed to Vinca rosea gave labelled vindoline. Sweroside suggested as intermediate between loganin and corynantheine-type skeletons. VOIGT, R., M. BORNSCHEIN und G. RABITZSCH: Pharmazie 22, 326-329 (1967). Tritiated chanoclavine-I fed to unripe sclerotia of Claviceps purpurea was converted to agroclavine, elymoclavine, and peptide alkaloids. GROGER, D., D. ERGE und H. G. FLOSS: Z. Naturforsch. 23b, 177-180 (1968). Tracer feeding studies with Claviceps paspali showed conversion of alanine-2-14C or _IoN to carbinolamide moiety of D-Iysergic acid-1X-hydroxyethylamide. VOIGT, R., u. S. KEIPERT: Pharmazie 22,329-336 (1967). Studies on influence of culture conditions on growth and alkaloid production of CJaviceps purpurea.

Chapter 11 PRAGER, R. H., and G. R. SKURRAY: Australian J. Chern. 21, 1037-1042 (1968). Anthranilic acid-3,5-3H and mevalonic acid-2-14C fed to Acronychia baueri gave rise to labelled acronidine. COBET, M., and M. LUCKNER: Europ. J. Biochem. 4, 76-78 (1968). 2,4-dihydroxyquinoline-3-14C fed to Ruta graveolens gave rise to labelled kokusaginine. The precursor exists in pH-dependent equilibrium with 2-aminobenzoyl acetic acid

Chapter 12 GUPTA, R. N., M. CASTILLO, D. B. MACLEAN,!. D. SPENSER, and J. T. WROBEL: J. Am. Chern. Soc. 90, 1360 (1968). Tracer feeding experiments with Lycopodium flabelliforme showed origin of lycopodine from two lysine units. Lycopodium alkaloids are to be regarded as modified dimers of isopelletierine.

Chapter 13 HEFTMANN, E.: Lloydia 30, 209-230 (1967). Review on biochemistry of steroidal saponins and glycoalkaloids. AUDA, H., G. R. WALLER, and E. J. EISENBRAUN: J. BioI. Chern. 242,4157-4160 (1967). Tracer feeding experiments with Actinidia polygama showed efficient formation of actinidine from terpenoid precursors but not from lysine, aspartic acid, or quinolinic acid. RONSCH, H., K. SCHREIBER und H. STUBBE: Naturwissenschaften 55, 182 (1968). Effects of hybridization on alkaloid composition in varieties of Solanum dulcamara. TSCHESCHE, R., u. H. HULPKE: Z. Naturforsch. 23b, 283-284 (1968). Pregnenolone-4-14 C fed to HoJarrhena antitiysenterica gave rise to labelled alkaloids, including conessine. HABERMEHL, G., u. A. HAAF: Chern. Ber. 101, 198-200 (1968). Skin gland secretion of Salamandra maculosa converted cholesterol-4-14 C to a mixture of labelled alkaloids.

Chapter 14 PARKE, D. V.: The biochemistry of foreign compounds. Oxford: Pergamon Press 1967. MITOMA, c., T. J. SORICH, II, and S. E. NEUBAUER: Life Sciences 7, 145-151 (1968). Caffeine administered orally to rats increased activity of hepatic microsomal oxidizing system, probably by increasing enzyme synthesis. JACCARINI, A., and J. B. JEPSON: Biochim. et Biophys. Acta 156, 347-363 (1968). Studies on hepatic microsomal system catalyzing 6-hydroxylation of lipid-soluble 3-substituted indoles. WERNER, G., u. G. BREHMER: Z. physioI. Chern., Hoppe-Seyler's 348, 1640-1642 (1967). Studies of tropine ester hydrolases in serum of rabbits and other animals. BECKETT, A. H., and E. J. TRIGGS: Nature 216,587 (1967). A higher percentage of administered nicotine was excreted unchanged by non-smokers than by smokers, but percentage excreted as cotinine was the same in both groups.

140

Addendum

MORSELLI, P. L., H. H. ONG, E. R. BOWMAN, and H. McKENNIS, JR.: J. Med. Chern 10, 1033-1036 (1967). Cotinine administered to rats was 90-97% eliminated unchanged. IIZUKA, H., and A. NAITo: Microbial transformation of steroids and alkaloids. Baltimore: University Park Press 1968. DECKER, K., and V. D. DAI: Europ. J. Biochem. 3, 132-138 (1967); - DAI, V. D., K. DECKER, and H. SUND: Europ. J. Biochem. 4, 95-102 (1968). Studies on L-6-hydroxynicotine oxidase of Arthrobacter oxidans. MITSCHER, L. A., W. W. ANDRES, G. O. MORTON, and E. L. PATTERSON: Experientia 24, 133-134 (1968). Cunninghamella echinulata metabolizes 6,14-endo-ethenotetrahydrothebaine alkaloids by N-demethylation and reduction of ketone to secondary alcohol.

Chapter 15 HASSON-VOLOCH, A.: Nature 218, 330-333 (1968). Review on use of curare for identification of acetylcholine receptor. CHANGEUX, ].-P., T. R. PODLESKI, and L. WOFSY: Proc. Nat. Acad. Sci. US 58, 2063-2070 (1967). Studies on acetylcholine receptor protein of electric eel. Receptor shown to be distinct from esteratic site. - Mol. Pharmacol. 2, 369-392 (1966). In vitro studies of interaction of lepto- and pachycurares with acetylcholinesterase. Each type preferentially binds one of two conformations of the enzyme. FAUCHER, A., et R. MONNET: Compt. rend. (D) 264,2247-2249 (1967). Studies of inhibition of horse serum cholinesterase by solanine and tomatine. CLEMENTE, E., and V. DE P. LYNCH: J. Pharm. Sci. 57, 72-78 (1968). From effects on various isolated organs it was concluded that mescaline stimulates .x-adrenergic receptor sites. FRANK, G. B.: Federation Proc. 27, 132-136 (1968). Review of drugs, including veratrine, that modify membrane excitability. GALLAGHER, C. H.: Biochem. Pharmacol. 17, 533-538 (1968). In magnesium-deficient mitochondria oxidative phosphorylation was depressed by tubocurarine, lasiocarpine, or heliotropine in concentrations lower then needed to inhibit NAD-linked dehydrogenations. MALAWISTA, S. E., H. SATO, and K. G. BENSCH: Science 160, 770-772 (1968). Vinblastine at 10-sM caused rapid dissolution of mitotic spindle from Pectinaria gOflldi. Effect was reversible. Vincristine was much less active and resembled colchicine. SAUCIER, J.-M., P. LEFRESNE et C. PAOLETTI: Compt. rend. (D) 266, 731-734 (1968). Irediamine A shown to bind at two sites to thymus DNA. WACKER, A., L. TRAGER, M. MATURO VA und H. BECKMANN: Naturwissenschaften 54, 90 (1967). Reserpine and ergocornine inhibited induction of steroid-metabolizing enzymes in bacteria probably by preventing synthesis of specific mRNA. REDDY, ]., C. HARRIS, and D. SVOBODA: Nature 217,659-661 (1968). Lasiocarpine inhibited in vivo incorporation of 3H-uridine into rat liver RNA. Primary effect may be on nuclear morphology. SHIMADA, K., and Y. TAKAGAI: Biochim. et Biophys. Acta 145, 763-770 (1967). Caffeine inhibited UV-induced breakdown of DNA in Escherichia coli, perhaps by depressing excision of pyrimidine dimers. GIELEN, W.: Z. Naturforsch. 23b, 117-118 (1968); - Naturwissenschaften 55,104-109 (1968). Evidence that a ganglioside is probable receptor for serotonin. MATTOCKS, A. R.: Nature 217,723-728 (1968). A pyrrole-like metabolite isolated from tissues of animals poisoned by pyrrolizidine alkaloids may be the true hepatoxic agent.

Subject Index

Abietic add 108 Accumulation 4 Acetaldehyde 55 Acetate 25-27, 32, 36, 43, 44, 52, 81, 86,93,94,99, 101, 110 Acetoacetate 43,44,99 Acetoacetic add 10,24 Acetonedicarboxylic add 9,24,43 Acetylcholine 17, 127-131 Acethylcholinesterase 130 Actinidine 105, 106 Aconitine 107, 108 Aconitu1ll 106 Acridine 92, 94 Acridone 94 Addiction 134 Adenine 6, 99, 100 Adenocarpine 35, 36 Adenocarpus viscoms 35 Adenosine monophosphate 133 - triphosphate 133 S-Adenosylmethionine 10, 29, 72 Adhota vasica 95 Adrenaline 131, 133 Adsorption 126 Agmatine 15-17 Agroclavine 88, 89 Ajmaline 61, 78, 80, 83, 84, 87 Alanine 27, 36 p-Alanine 36 Aldol condensation 8 Alfalfa 20 Alkaloids, animal 2 - , biosynthesis 8 Allantoic add 100 Allantoin 100 Amanita 18 Amaranthin 103 Amaranthus 103 Amaryllidaceae 72 Amides 115 Amidine 16 Amines 1, 15, 115, 116, 127, 130, 132 Amino adds 8, 15, 19, 34, 49, 81, 99, 103, 133 DL-tX-Aminoadipic add 25 Aminobenzaldehyde 94 tX-Aminobutyric add 49,99 3-Aminomethylindole 21

Ammonia 5, 27 Ammonium salts 5 Anabasine 12, 29, 31, 32, 34, 35, 50 Anagyrine 52 (-)-Anatabine 31,35 Androcymbine 75 Androcymbitl1ll nJethanthiodes 76 Angustifoline 48,51, 52 Anhalamine 55 Anhalonidine 55 Anhalonium lewinii 19, 55 Anhydrocymbine 76 Animals 2, 6, 115 Annotinine 102 Anthranilic add 29, 92-94, 101, 106 Antibiotics 125 -, glutarimide 133 Antimuscarine agents 128 Aphids 49 Apocynaceae 77, 81, 86, 87, 109 Aporphine 62, 63 Areca catechu 117 Arecaidine 117 Arecoline 117, 129 Argemone 70 Arginine 15, 16, 44, 49 Aristolochia 70 Aristolochic add 70 Arthrobacter 120 - oxMans 121 Asdtes 133 Aspartate 27, 32 Aspartic add 29, 36, 37 Aspergillus versicolor 121 - ochraceus 122 Aspidosperma 77,86,87 Aspidospermine 82, 87 Astrocaria phyllantoides 35 Astrocasine 35, 36 Atisine 106---108 Atropa 4,41 - belladonna 44 Atropine 42, 121, 128, 129, 133 Attraction, hydrophobic 126 Azaquinones 120 Bacteria 28, 36, 115 Banana 19 (-)-Baptifoline 48

142

Subject Index

Baptisia 48, 52 Barley 16, 19,21 Beans 12 - , calabar 78 - , castor 29-31, 36 Beet 102 Belladine 72, 73, 74 Benzaldehyde 55 Benzophenanthridine 59 Benzoxazoles 101 Benzoxazolinone 101 Benzylisoquinolines 56, 63 Benzyltetrahydroisoquinoline 54 Berberine 2, 3, 56, 57, 59, 122 Berberastine 19,57, 59 Beta vulgaris 18 Betacyanidins 102 Betacyanins 102 Betaine 17, 18 Betalamic acid 102 Betanidine 103 Betaxanthins 102 Betel nut 117 Betonicine 20 Biochemistry, quantum 126 Biosynthesis, alkaloid 8 Bis-benzylisoquinoline 56 Bonding, hydrogen 126 Boraginaceae 97 Botulinus 128 Brain 116 Brownine 108 Bufotalin 113 Bufotenine 22 Bllxus 109-111, 133

Cadaverine 10, 12, 16, 25, 26, 35, 36, 49, 50-52, 97 Caffeine 3, 99, 100, 120, 133 Calabash-curare 86 Calcium 5,132-134 Calycanthine 79 C alycanthus florUus 79 Candicine 19, 20 Caracurine-VII 85, 86 ｾＭｃ｡ｲ｢ｯｬｩｮ･＠ 78 - derivative 79 Carbon dioxide 33,64,67,69, 73, 100 Carbostyrils 117, 118 Carica papaya 27 Carpaine 27 Casimiroine 93 Cassaine 133 Castor bean 29-31, 36 Catecholamines 133 Catharanthine 82, 87

Catharanthus 77, 87 - roseus 5 Cats 116 Ceanothine B 18 Ceanothus americanus 18 Cephaeline 60 Cephaelis ipececuanha 61 Cell, gastric secretory 128 Cerveratrum 109, 110 Chanoclavine 88-90 Chelerythrine 58 Chelidonine 59 Chelidonium majus 49, 59 "Chemical races" 3 Chinchona 117 Chinchonine 118 Chimonanthine 78, 79 Chinconidine 118 Cholesterol 108, 110-112 Choline 17,18 Cholinesterase 128, 131 Cicer arietinum 17 Cinchona 84 Cinchonamine 84 Cinchona succiruba 86 Cinnamic acid 27,36, 72, 73, 75, 76 Citric acid 9, 43 Claviceps 77, 87, 88, 90, 101 - pllrpurea 4 Clavines 88, 90 Club mosses 2 Cocaine 42-44,46, 117, 128, 132, 133 Coclaurine 55, 56, 64-66 Codeine 67,68,116,119,122 - , methyl ether 67 Codeinone 67,68 Coenzymes 28 Coffea arabica 100 Colchicine 1, 75, 133 Colchicum 75 Compositae 93, 97 Conarrhimine 112 Condensation, aldehyde-amine 9 - , aldol 8 - , carbinolamine 8 Conduction, axonal 132 Conessine 122 Conhydrine 26, 27 1J!-Conhydrine 26, 27 Coniceine 5 y-Coniceine 25, 26 Coniine 1,5,25-27 Conium maculalum 5, 25 Conjugation 115 Convolvulaceae 88 Coordination, metal 126 Corn 100

Subject Jndcx Cornaceae 106 Corynantheidine 119 Corynantheine 61, 80, 81, 83-86 Corynoxeine 85 Cotinine 117, 118 - , methonium ion 118 Coupling, phenol 67, 70, 73 Crataegus monogvna 15 Crinamine 73, 74 Crotolaria 97 Croton linearis 65 Crotonosine 65 Cryptopine 58 Cularines 62 Curare 128 Cuscohygrine 24 (±)-Cuspareine 93 fi-Cyanolanine 36 3-Cyano-2-pyridones 121 Cycloartenol 111, 112 Cyclohexadienone 65, 66 Cycloheximide 133 Cyclomicrophyllin 111 Cyclopropane 110 Cyanide 36 Cytisine 48,51, 52 Cytisus 48 - laburnum 52 Cytochrome 116 Cytokinins 100 Damascenine 92, 93 Datura 4,5,41,44 - metel 43, 45 - meteloides 45 - stramonium 42, 46 Day length 45 Decarboxylation 106 1,2-Dehydroanabasine 35 Dehydrogenation 115, 120 Dehydrolupanine 52 Dehydrosparteine 52 Delphinium 106 - ajacis 108 - brownii 108 - elatttm 108 Delpheline 107, 108 Demecolcine 76 Demethoxyalstonidine 12 Demethylation 34, 116, 119-121 Demethylcotinine 118 Dendrobine 105, 106 Dendrobium nobile 106 Deoxyajmaline 83 Deoxyvasicinone 95 Detoxication 117 Diamines 16

143

Diamine oxidase 24 1,4-Diaminobutane 10, 15 1,5-Diaminopentane 10 1,5-Diaminopimelic acid 29 Dibenzazonine 69 Dibenzocyclooctane 70 Dibenzopyrrocalines 62 Dicentra spectabilis 58 Dicotyledons 3, 16 Dictamnine 93 Dictamnus albus 93 Dihydropteridine 116 1,3-Dihydroxyacridine 94 2,6-Dihydroxy-N-methylmyosime 121 Dihydroxyphenylalanine 19, 20, 57, 102 Dihydroxyphenylacetaldehyde 69 3,4-Dihydroxyphenylethylamine 64 Dihydroxyphenylpyruvic acid 56 2,6-Dihydroxypseudooxynicotine 121 2,6-Dihydroxypyridine 120, 121 3,4-Dihydroxypyridine 27 Dimethylamine 116 3-Dimethylamino-methylindole 21 L-3-Dimethylamino-3-phenylpropanoic acid 108 6,8-Dimethylergoline 87, 88 N,N-Dimethyltryptamine 22 Dipterin 22 Displacement 126 Diterpenoids 106 Dogs 117, 119, 120 DOPA 20 Dopamine 19, 20, 56, 58-60, 64, 69, 132 Drugs 115 Duboisia 41 - myoporoides 4 Echinops 93 Echinorine 93 Echitamine 84 Ecology 6, 15,49, 112 Electric eel 129 Elymoclavine 88, 89 Emetine 60 Enzyme 13, 15, 19, 29, 46, 51, 52, 72, 93, 116,117,119,120,122,125,134 Ephedrine 19,20, 116, 132 Epistephanine 56 Equisetum 2 Ergocristine 4 Ergoline 88 Ergometrine 89,90 Ergot 77,87,132 Ergotamine 4, 89, 90 Erysopine 69, 70 Erythraline 69 Erythratine 69 Erythrina 69, 128

144 Erythrina berteroana 70 Erythrinan 69 Erythroxylon 5,41,42,46 - coca 24 Escherichia coli 121 Eschscboltzia 70 Eschscholtzidine 70 Eserine 130 Ester, Tigloyl 44 Ethanolamine 15-17, 106 Ether, norlaudanosoline dimethyl Euphorbiaceae 31, 81 E::vodia rutaecarpa 95 Evolution 3, 12 Fagara 66 Excretion 117-119

Subject Index

64, 68

Festuclavine 88 Flavine 117 Flowering 5 Formaldehyde 55, 117, 119 Formate 10, 19, 31, 36, 58, 61, 88, 95 Formylkynurenine 28 Free radical 11 Fumaric acid 121 Function 6 Fungi 28, 77, 88, 122 -, ergot 87 FUlltumia 109, 110 Funtumine 122 Funtumidine 122 Furans 93 Furoquinolines 93 Galanthamine 73 Galanthine 74 Galega offtcinalis 16 Galegine 16, 17 Ganglioside 134 Garrya 106 Garryine 107 Gelsemine 83, 85 Genetic analysis 49 Genetics 49 Genista 48 - aetnensis 52 Geraniol 80, 82, 86, 87 Gentianine 105, 106 Giberrellin 19 Gland 128 - , salivary 128 Gleditsia triacanthos 99 Glochidion philippicum 104 Glucuronic acid 117,119 Glucuronide 116 p-Glucuronides 115 Glutamic acid 32, 34, 42, 49, 103

Glyceraldehyde-3-phosphate Glycerol 29, 32, 36 Glycine 9, 16-18, 44, 108 Glycolic acid 34 Glycosides 110, 125 Glyoxylate cycle 36 Glyoxylic acid 55 Grafting 4, 49, 77 Graminae 101 Gramine 21 Grantianic acid 98, 99 Guanine 99, 100 Guinea pig 116 Gymnosperms 3, 16

37

Haemanthamine 74--76 Hallucinogens 19, 21, 90, 116, 134 Harmala 132 Harmaline 132 Herbivores 6 Heliotridine-N-oxide 97 Hemicholinium 128 Heteroxanthine 100 Histamine 2, 22, 103, 104, 116 Histidine 22, 49, 103, 130 Holamine 111 Holaphyllamine 111, 112 Holaphylline 111, 112 Holarrhena 109, 111, 133 - floribunda 110 Holarrhimine 112 Homology 3 Homolycorine 75 Homostachydrine 20 Hordatine A 15, 17 Hordeum 5 - vulgare 4,19,20,116 Hormones 5, 19,21,22, 122 Hyaluronic acid 129 Hybridization 52 Hycoscyamine 4 Hydrastine 57-59 Hydrastis canadensis 57-59 Hydrazine 132 Hydrogen bonding 126 Hydrolysis 117, 120, 121 a-Hydroxyalanine 89 3-Hydroxy-anthranilic acid 28, 92, 93, 101 Hydroxycinnamic acid 72 ,19.10-8-Hydroxyergoline 88 Hydroxylation 19,116,117,122 Hydroxylupanine 51, 52 17-Hydroxylupanine 122 4-Hydroxylysine 49 6-Hydroxy-N-methylmyosime 121 6-Hydroxynicotine 120, 121 p-Hydroxyphenylacetaldehyde 116

Subject Index p-Hydroxyphenylpyruvic 64 5-Hydroxypipecolic acid 27 3-fJ-Hydroxy-5-iX-pregn-16-en-20-one cholesterol 110 Hydroxyproline 20,24,44 6-Hydroxy-pseudooxynicotine 121 2-Hydroxystrychnine 119 4-Hydroxytryptamine 21 5-Hydroxytryptamine 21 Hydroxytryptophan 21, 78 Hygric acid 21 Hygrine 24, 25, 35, 42 Hyoscine 4, 42, 128 Hyoscyamine 41-45, 117,128 Hyosryamus 41,45 Iboga 86,87 Ibogaine 78, 82, 87 Imidazole 103, 130 Imidazoleacrylic acid 22 Imidazoleglycerophosphate 103 Indoles 61,77,78,95,103, 119 - alkaloids 75 Indole-3-acetic acid 6, 21, 78, 88, 90, 116 Indolenine 79 Inhibitors 128 Inorybe

18

90

lriodial 106 Iron 27 Isatinecic acid 99 Isoamylamine 15 Isobutylamine 15, 16 Isoleucine 45, 99 Isomethonium ion 118 Isonicotinic acid 120 Isopelletierine 10, 25, 35 Isopenniclavine 89 Isopentenyl pyrophosphate Isopentylamine 15, 16 Isoquinoline 54 Isosetoclavine 89 Isothebaine 64, 65

93

52

Leucaena glauca

27

Leucine 15, 99 Light 5 Lignin 19 Liliaceae 76, 109 Liver 116, 119 Loganin 80, 82 Loganiaceae 81,86 Lophophorine 55 Lupanine 48,49, 51, 52, 122 Lupines 12, 35, 48 Lupinine 49-52 (-)-Lupinine 48 Lupinus 5,48,49,52,122 - angustifo/ius 51 -luteus 50,51

Lycoctonine 107,108 Lycopersicon glandulosum 5 - esculentum 110 - pimpinellifolium 110

Robinson, The Biochemistry of Alkaloids

94

Magnocurarine 55 Magnoflorine 66 Malate 36 Maleamic acid 121 Maleic acid 121 Malic 36 Malonate 32 Malonic acid 36 Mammals 118 Man 116,119 Mandragora

18

iX-keto-e-aminocaproic acid 25 iX-keto-!5-aminovaleric acid 46 Kinetin 5,29 Kurchi 109 10

Laburnum anagyroides

Lanosterol 112 Laudanidine 65, 68 Laudanosine 11, 55, 68 Laurifoline 66 Leguminosae 12, 21, 48, 52, 69, 97

Mackinlaya

Jaconine 98 Jerveratrum 109, 110 Julocrotine 18 Julocroton montevidensis

Labelling 13

Lycopodine 102 Lycopodium 2, 101 Lycorine 3,73-75 Lysergene 88 Lysergic acid 12,18,78,81,89,119 - aldehyde 88, 89 ' Lysergol 88, 89 Lysine 16,21,25-27, 32, 35, 36,49-52 94,95 - decarboxylase 50

Insect 15, 49 Insecticides 130 Ipecacuanha 60, 62, 133 Ipomoea violacea

145

42

Manganese 44 (-)-Matrine 48,49, 50 Mavacurin 84 Medicago sativa

20

Membranes 125, 133 Menispermaceae 63 Mescaline 1, 19, 116

146

Subject Index

Meteloidine 45 Methionine 10,17-19,21,27,31,33,58, 88,93,95,100,108 6-Methoxybenzoxazolinone 101 Methylamine 9, 16,44, 118 3-Methylaminomethylindole 21 O-Methylandrocymbine 76 Methylation 19,33, 43, 67, 72, 104, 116 N-Methylcadaverine 52 N-Methyl coclaurine 56 l-Methyl-3-cyano-4-pyridone 30 N-Methylcytisine 52 Methylene dioxy 75 Methyl group 11, 17, 19, 27, 29, 30, 88, 93,100 N-Methyl groups 108 N-Methyl-2-hydroxypyrrolidine 24 N-Methyl-isopelletierine 25 N-Methylnicotinic acid 29 l-Methylnicotinamide 28,30 l-Methylnicotininitrile 30 Methylpherase 19 4-Methylproline 106 N-Methylputrescine 24, 43 N-Methyl pyridinium 116 ,B-methylpyrroline 24, 105, 106 N-Methyltryptamines 21,78,95 N-Methyltyramine 4,19,20 Methylxanthines 133 Mevalonate 27,87,93,99,110 Mevalonic acid 11,82,88,105,106,108 Mice 116 - brain 133 Microorganisms 115,120 Microsomes 116 Millepedes 2 Mimetic agents 128 Mimosa pudica 27 Mimosine 27,121 Mitochondria 134 Molluscs 49 Monoamine oxidase 130 Monocotyledons 3,16 Monocrotalic acid 98, 99 Mononucleotide 28 - , nicotinic acid 29, 37 Monoterpenoid 61,82,86 Morphinan 62, 63 Morphine 11, 63, 64, 67-69, 72, 116,119, 122 - , pseudo- 67 Munitagine 70 Musa sapientum 19 Muscarine 128, 129 Muscle 128 - , iris 128 - , visceral 128

Myosmine 31, 34, 35 Narcotine 4, 57-59 N-Carbamylputrescine 15, 17 N-Dimethyl tryptamine 21 Necic acids 97-99 Necines 97 Nerine bowdenii 72 Nerolidol 106, 107 Nerve gases 130 Nerves 127 Nervous system, autonomic 127 Nettles 22 Neurons 127, 128 Nicotiana 5,31,34 - glauca 35, 37 - glutinosa 34 - rustica 32 Nicotinamide 28---31 - adenine dinucleotide (NAD) 52, 134 Nicotine 1, 4, 11, 13,29,31-34,36, 116, 118, 120-122, 128, 129 Nicotinic acid 6, 23, 28---30, 32-34, 36, 37 - - mononucleotide 29,37 Nicotinonitrile 30 Nicotyrine 31, 118 Nigella 92 - damescena 93 Nitidine 60 Nitrates 5, 33 Nitrilase 120 Nitrophenanthrene 70 Noradrenaline 2,19,20,57,59,70,76,127, 130, 132, 133 Norbelladine 72,73-75 Norhyoscyamine 4 Norlaudanosoline 56,57,64,67,68 - demethyl ether 64 Nornicotine 4,31,33--35,117,118 Norpluviine 74 Nuclei 134 Nucleic acid 100, 125, 133 Nucleotide 30,32 - , pyridine 29 Nudifiorine 31 Nuphar Meum 106 Nupharamine 105, 106 Nupharidine 105 Nutrition 5 ,B-Obscurine 102 Ontogeny 4, 5, 45, 49, 52, 67, 90, 100, 112 Opium 13 Opuntia ficus-indica 102 Orientalines 64, 65

Subject Index Ornithine 9, 15, 16, 21, 25, 32, 33, 42-44, 46, 94, 95, 97 Ourouparia gambir 12 Oxidase, aldehyde 117 - , amine 10, 116 - , diamine 10, 16, 24, 35, 50 - , monamine (MAO) 130, 132 - , phenol 11,67 - , quinine 117 Oxidation 115 N-Oxides 98, 115 Oxindole 86 Oxygen 116 Oxymethoxyalstonidine 12 Oxynitidine 60 Oxysanguinarine 12, 60 Panda oleosa 18 Pandamine 18 Panicum miliaceum 19 Papaver 5, 63 - bracteatum 67 Papaver dubium 64 - orientale 64 - somniferum 4, 55, 57, 58, 64, 67 Papaveraceae 59, 62, 63 Papaverine 55 Parasites 6 Pea 12, 16,29,35 Penicillium viridicatum 94 Penniclavine 88, 89 Peptides 18, 89 Perivine 82 Periwinkle 87 Permeability 125 Peroxidase 11,67,88 Peyote 19 Pharmacology 125 Phaseolus 22 - mungo 27 Phenethyl isoquinolines 76 Phenol 11, 115, 119 Phenylacetaldehyde 54, 64 Phenylalanine 9, 20, 27, 46, 54, 61, 72, 73, 75, 76, 89, 108 Phenylethylamine 19, 132 Phenylpropane 94 Phenylpyruvic acid 55 Phloroglucinol 94 Phosphate 17 - , nicotinamide adenine dinucleotide 116 - , pyridoxal 88, 90 Phospholipids 17, 18 Phosphoribosyl pyrophosphate 28 Phosphodiesterase 133 Phosphorylase 133 Phosphorylcholine 17 10*

147

Photoperiod 5, 45, 112 Phthalideisoquinoline 57, 58 Phyllocladene 108 Phylogeny 3, 51, 115 Physostigma 5 Physostigmine 78, 130, 131 Picraline 80, 83, 84 Pilocarpine 103, 116, 129 Pilocarpus 103 Piper nigrum 106 Piperidine 10, 25, 50 ,11-Piperideine 10, 25-27, 35, 50 ,11-Piperideine-2-carboxylic acid 26 ,11-Piperideine-6-carboxylic acid 26, 35 Piperidyllupinine 51 Pisum sativum 35, 95 Plants, tropical 3 Platynecine 98 Ploidy 4 Pluviine 75 Polyamines 18 Polysaccharides 129 Poppy 63,64 Potato 5 Potassium 5, 15, 133 Pregnane 109 Pregnenolone 5, 111, 112 Prephenic acid 61, 81 Procaine 132 Progesterone 111, 112 Proline 20,21, 24, 42, 44, 89, 103 Propionate 25, 32, 36 Protein 5, 16, 34, 125, 129, 133 "Proto alkaloid" 1, 15 Protoberberine 57-60 Protocatechuic aldehyde 72, 73 Protopine 57-60 Protosinomenine 59, 65 Protostephanine 69 Pseudomonad 120 Pseudomonas 120 - lupanii 122 Pseudo-morphine 67 Psilocybin 22, 133 Putrescine 10, 15-17, 25, 32, 33, 43, 44, 97 Pyridine 28, 117 - alkaloids 6 - nucleotide 29 Pyridone 27, 31 (X- Pyridone 12, 117 y-(3-Pyridyl)-y-Methylaminobutyric acid 118 Pyroclavine 88 Pyrophosphate, geranyl 82, 87 - isopentenyl 88, 93 Pyrrolidine 10, 24, 42, 97, 111

148 cx-Pyrroline 43, 44 Lll-Pyrroline 10, 32, 33 Lll-Pyrroline carboxylic acid 46 LI'-Pyrroline-5-carboxylic acid 32 Pyrrolizidine 97, 134 Pyruvate 27 Pyruvic acid 55 Quantum biochemistry 126 Quebrachamine 87 Quinazoline 92, 94 Quinazolinium 95 Quinazolones 2, 94 Quinidine 118 Quinine 77,84-86, 118 Quinoline 79, 92, 93, 117 Quinolinic acid 28-30, 32, 36, 37 Quinolizidine 48, 97 Quinuclidine 118 Rabbits 116, 119, 120 Radical, phenolate free 65 Radiotracer 13 Ranunculaceae 106 Rats 116, 119, 133 Rauwolfia 77, 132 - verticillata 83 Rearrangement, dienol-benzene 66 Receptor 129 - , adrenergic 130 - , cholinergic 129 Relaxation 132 Reserpine 81,83,87,119,132,134 Reticuline 55-59, 64, 66-68 Retronecine 97-99 Ribonucleic acids 100 Ribose 101 Ricinine 1,5,29-31,36,37, 120 Ricininic acid 30 Ricinus communis 5, 12, 29, 31 RNA 100 Roemerine 64, 66 Rubiaceae 81, 84, 86 Rubijervine 110 Rutaceae 66, 93-95 Rutaecarpine 1, 78, 95, 96 Salamander 2, 112 Salutaridine 67, 68 Salutaridinol 67 Salutaridinol-I 68 Salvia officinalis 122 Samandarine 112, 113 Sanguinarine 2, 12, 59, 60 Sanguinarea canadensis 12 Sarothamnus scoparius 49 Sarpagine 84 Schiff-bases 127 Scopolamine 41---45

Subject Index Scopolia 42 Scopoline 42 Scoulerine 58-60 Sea urchin 133 Sedamine 4, 27 Sedum acre 4,27 Sedridine 4 Seeds 5 Selagine 102 Sempervirine 81,84 Senecic acid 98 Senecio 97 Seneciphyllic acid 98, 99 Seneciphylline 99 Serine 15-17, 27, 36, 106, 121, 130 Serotonin 21, 22, 132, 134 Serpentine 78, 82 Setoclavine 88, 89 Shikimic acid 87, 92, 93 Sinapic acid 75 Sinoacutine 67 Sinomenine 66, 67 Sinomenium acutum 66 Skimmianine 93 Skytanthine 105, 106 Skytanthus acutus 106 Sodium 133 Solanaceae 41, 108 Solanidine 109 Solanine 5 Solanum 108-110, 112 - aviculare 110 - tuberoSlim 108, 110 Solasodine 109, 110, 122 Sophora tetraptera 49 Sorbus aucuparia 15 Sparteine 48-52 Sparteine-N-oxide 52 Spermidine 18 Spermine 2, 18 Spinach 22 Spinacea oleracea 22 (-)-Stachydrine 20,21 Stephania Japonica 56, 69 Stephanine 70 Steroids 5, 11, 105, 108, 122 Strychnine 83, 86, 119 Strychnos 86 Stylopine 58-60 Succinate 27, 32,36 Succindialdehyde 9, 43 Sugar beet 15 Sugars 34 Synapses 127

Taxine 108 Taxonomy 3

149

Subject Index Taxus 108 Tazettine 75, 76 Tembetarine 66 Terpenoids 11, 28, 99, 105 Tetrahydroanabasine 35, 50 Tetrahydroanabasinedicarboxylic acid 35 Tetramethyluric acid 100 TigHe acid 45 Tigloyl esters 44 Tissue culture 4 Thebaine 64,65,67,68 Theobromine 100, 120 Theophylline 100, 120, 133 Thermopsine 48, 52 Thiobinupharidine 106 Threonine 49,99 Toad 21 - poisons 113 Tobacco 4,31,32,67,117,122 Tomatidine 110, 122 Tomatine 5 Toxiferine-I 86 Trametes sanguinea 122 Transaminase 46,50 Transamination 10 Translocation 4 Transmethylation 10, 108 Transmitters, chemical 127, 130 Tranquilizers 126 Trewia nudiflora 31 Triacanthine 99, 100 Trigonelline 23, 31 Triketooctanoic acid 101 Trimethoxyphenylacetic acid 116 Tropane 4, 9, 25, 117, 128 Tropic acid 42, 45, 46, 128 Tropine 42, 45 Tropinone 45 Tryptophan 9, 21, 22, 28, 29, 46, 77-79, 81,85-88,92,93,95

Tryptamine 21, 78, 79, 86, 116 Tubocurarine 129 Tubulosine 133 Turicine 20 Tyramine 19,20,54,61,62, 73, 75, 132 Tyrosine 9, 19, 49, 54, 55, 57, 59, 61, 63, 64, 67, 69, 70, 72, 75, 103 Urea 6,100 Uric acid 6, 99, 100, 120 Urocanic acid 22 Urtica 22 Valine 15, 16 Vasicine 94 Vasoconstriction 132 Veratrum 109, 110, 125, 133 Vicia faba 51 Vinblastine 82, 133 Vinca 87, 133 Vincristine 133 Vindoline 5, 78, 81, 82, 87 Viridicatine 94 Viscum album 18 Voacanga 87 V oacangine 82, 87 Wheat 29, 122 Wieland-Gumlich aldehyde 86 Xanthine Yew 108 Yohimbine

99, 100, 120 80,81,83, 84,132

Zapotidine 104 Zea mays 100 Zeatin 100 Zizyphin 18 Zizyphus oenoplia 18

Molecular Biology, Biochemistry and Biophysics Molekularbiologie, Biochemie und Biophysik

Volumes published: Vol. 1

J. H. VANT' HOFF: Imagination in Science.

Translation and Introduction by G. F. SPRINGER. VI, 18 pages 8 vo. With 1 portrait. Soft cover binding DM 6,60/ US $1.65

Vol. 2 K. FREUDENBERG and A. C. NEISH: Constitution and Biosynthesis of Lignin. IX, 129 pages 8 vo. With 10 figures. Bound DM 28,- / US $ 7.Forthcoming volumes: Vol. 4 A. S. SPIRIN and L. P. GAVRILOVA: The Ribosome (in press) Vol. 5 B. JIRGENSONS: Optical Rotatory Dispersion of Proteins and other Macromolecules (in press) Vol. 6 F. EGAMI and K. NAKAMURA: Microbial Ribonucleases. Approx. 106 pages 8 vo. With 5 figures. Bound DM 28,-/US $ 7.Vol. 7 F. HAWKES: Nucleic Acids and Cytology. Approx. 288 pages 8 vo. With approx. 14 figures. Bound DM 58,-/ US $ 14.50 Volumes being planned: ABELES, R. H., and W. S. BECK: The Mechanism of Action of (Cobamide) Vitamin ｂｴｾ＠ Coenzymes AIDA, K6: Amino Acid Fermentation ANDO, T.: Protamines ANKER, H. S.: Studies on the Mechanism and Control of Fatty Acid Biosynthesis ANTONINI, E.: Kinetic Structures and Mechanism of Hemoproteins BEERMANN, W.: Puffs and their Function in Developmental Biology BREUER, H., and K. DAHM: Mode of Action of Estrogens BRILL, A. S.: The Role of Heavy Metals in Biological Oxidation Processes BURNS, R. c., and R. W. F. HARDY: Nitrogen Fixation in Bacteria and Higher Plants CALLAN, H. G.: Lampbrush Chromosomes CHAPMAN, D.: Molecular Biological Aspects of Lipids in Membranes CHICHESTER, C. 0., H. YOKOYAMA and T. O. M. NAKAYAMA: Biosynthesis of Carotenoids DUMONDE, D. C.: Cell and Tissue Antigens Dus, K. M.: Analysis of Proteins ELEY, D. D.: Biological Materials as Semi-Conductors FENDER, D. H.: Problems of Visual Perception GIBSON, 1.: Paramecium Aurelia and Cytoplasmic Factors

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