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Studies related to synthesis and biosynthesis of indole alkaloids Nelson, Verner Robert 1969

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STUDIES RELATED TO SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS by VERNER ROBERT NELSON B.Sc. Honours, University of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Chemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Verner R. Nelson D e p a r t m e n t o f Chemistry  The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a nada Date April 28, 1969 ABSTRACT In Part I of this thesis, an investigation of the reaction of the alkaloid, catharanthine (12), with zinc in glacial acetic acid is presented. Four isomeric carbomethoxydihydrocleavamines (60-63) have been isolated and fully characterized. It is also shown that heating catharanthine in a mixture of acetic acid and sodium borohydride provides a very convenient method for the preparation of the previously unknown 188-carbomethoxy-cleavamine (64). A similar investigation is presented for some related chemistry of the Aspidosperma series. Minovine (72), when heated in a mixture of acetic acid and sodium borohydride, is readily converted to vincaminoreine (71) and vincaminorine. The reversibility of the latter reaction is demonstrated by the transannular cyclization of vincaminoreine to afford minovine. In Part II, Section A, of this thesis, the synthesis of possible precursors of the Aspidosperma, Vinca, and Iboga alkaloids are described. The synthesis of the methyl cyanoacetate adduct (52) and dimethyl ma Ionic ester adduct (53) was accomplished without d i f f i c u l t y . The other synthetic precursor, 96, was prepared by treating the chloroindolenine, 94, of the tetracyclic indole, 93, with methyl cyanoacetate and triethylamine. In Part I I , Section B, of this thesis are also described the biosyn-thetic studies in Vinca rosea L. and Vinca- minor L. plants. The biogenetic importance of the transannular cyclization reaction is described by evaluating appropriate nine-membered ring alkaloids as possible precursors of the Aspidosperma, Vinca, and Iboga alkaloids. To confirm that the transannular cyclization reaction is not important in the plant, a sequential incorporation of DL-tryptophan-3- C into Vinca minor L . plants is presented. The synthetic precursors, 52, 53 and 96, were also evaluated for their biogenetic importance. i i i TABLE OF CONTENTS Page T i t l e Page i Abstract 1 1 Table of Contents * 1 1 1 L i s t of Figures 1 V L i s t of Tables V 1 Acknowledgements • • • • • v 1 1 Part I Introduction 2 Discussion * 9 Experimental 3 4 Bib 1 iography • 4 3 Part II Introduction 4 7 Discussion •• 6 1 Experimental 1 0 8 Bibliography 1 3 4 i v LIST OF FIGURES Page Part I Figure 1. Reaction mechanism proposed for the formation of cleavamine, descarbomethoxycatharanthine and the epimeric dihydrocleavamines 7 2. The transannular cyclization and i t s synthetic application in the Aspidosperma and Iboga series 10 3. Kutney's synthesis of dl-quebrachamine, dl-4a-dihydro-cleavamine, and dl-48-dihydrocleavamine 13 4. Kutney's synthesis of carbomethoxydihydrocleavamine 14 5. Structure determination of 16-methoxy-(-)-tabersonine 16 6. Some properties of akuammicine 17 7. Mass spectrum of carbomethoxydihydrocleavamine (isomer A)(60) 24 8. Nmr spectrum of carbomethoxydihydrocleavamine (isomer A)(60). 24 9. Nmr spectrum of carbomethoxydihydrocleavamine (isomer B)(61). 25 10. Nmr spectrum of carbomethoxydihydrocleavamine (isomer C)(62). 25 11. Nmr spectrum of carbomethoxydihydrocleavamine (isomer D)(63). 26 12. Nmr spectrum of carbomethoxycleavamine (64) 26 13. Formation of carbomethoxydihydrocleavamines and carbo-methoxycleavamine 28 Part II 1. Barger-Hahn-Robinson-Woodward hypothesis 50 2. Prephenic acid hypothesis for the biosynthesis of Corynan-theine alkaloids 50 V Figure Page 3. Wenkert's proposal for the biosynthesis of Aspidosperma and Iboga alkaloids 52 4. Incorporation of geraniol (30) and loganin (31) into Indole alkaloids 54 5. Scheme for the rearrangement of the monoterpene unit 54 6. Summary of the pathway from acetate to Indole alkaloids of tryptamine + Cg_io type 60 7. Preparation of a,g-unsaturated ketone 51 63 8. Mass spectrum of methyl cyanoacetate adducts 52 66 9. Mass spectrum of malonic ester adducts 53 66 10. (a) Partial nmr spectrum of a mixture of malonic ester adducts 53. (b) Partial nmr spectrum of the less polar malonic ester adduct 53. (c) Partial nmr spectrum of the more polar malonic ester adduct 53 67 11. Oxidative rearrangement of indole alkaloids , 73 12. Preparation of tetracyclic ketone 78 74 13. Nmr spectrum of imino ether 87 81 14. Nmr spectrum of 89 81 15. Mass spectrum of imino ether 87 82 16. Mass spectrum of 89 82 17. Mechanisms to account for the loss of the side chain of the piperidine substituent 84 18. Nmr spectrum of 96 89 19. Mass spectrum of 96 90 20. Some later stages of Indole alkaloid biosynthesis 105 v i LIST OF TABLES Page Part II Table I Results of incorporation of nine-membered ring intermediates into V. rosea L. and V. minor L. by various techniques 94 II Results of incorporation of various intermediates into V. rosea L. under i d e n t i c a l conditions 97 II I Results of incorporation of DL-tryptophan-3-1I+C into Vinca minor L. at various time intervals 99 IV Results of incorporation of synthetic intermediates into V. rosea L. and V. minor L. by various techniques 105 ACKNOWLEDGEMENTS I wish to express my thanks to Professor James P. Kutney for his continual encouragement and excellent guidance through-out the course of my research. I am also grateful to the British Columbia Sugar Refining Company Limited and the National Research Council of Canada for scholarships which I received during my study. PART I THE PARTIAL SYNTHESIS OF SOME NINE-MEMBERED RING INDOLE ALKALOIDS AND RELATED DERIVATIVES Introduction Nature has provided a vast array of organic compounds of varying degrees of complexity and i n t e r e s t . A large group of these compounds, known as a l k a l o i d s , have been of considerable i n t e r e s t , even since before the onset of science i n the modern sense of the word. Alkaloids are nitrogenous bases that usually occur i n plants, and are l o c a l i z e d i n the bark, roots, seeds or leaves. The taxonomic d i s t r i b u t i o n of a l k a l o i d s cannot be fixed with any ce r t a i n t y as the chemistry of the f l o r a of only a few regions of the world have been i n t e n s i v e l y studied and the greater majority of plants s t i l l remain to be examined. It has been estimated that 10-20% of a l l plants contain al k a l o i d s Certain a l k a l o i d s have been long known to have c h a r a c t e r i s t i c and profound e f f e c t s on the b i o l o g i c a l systems, and t h i s observation undoubtedly has provided the stimulus to t h e i r examination in ever increasing d e t a i l . These compounds tend to upset the balance of endogenous amines associated with the chemistry of the central nervous 2 system , The range of e f f e c t s produced by alkaloids i s d e t a i l e d i n pharmacological c o l l e c t i o n s ^ and many have been medically u s e f u l . A number of alk a l o i d s have been i s o l a t e d from animals such as the 4 toad poison, bufotenine ( 1 ) . Samandarine (2) occurs with several r e l a t e d a l k a l o i d s as the skin poison of two salamanders (S. maculosa Laurenti and S. atra Laurenti) of European habitat, whereas batracho-t o x i n i n A ^ (3) i s i s o l a t e d from the skin of the Columbian arrow poison 3 frog (Phyllobates aurotaenia). The indole alkaloids constitute a large class of compounds ranging in complexity from simple derivatives such as psilocybine ( 4 ) , the hallucinogenic principle of Mexican mushrooms, to intricate ring systems such as those found in strychnine (5), the well-known poison. Strychnine has also been used as a stimulant for the heart. Over forty alkaloids which bear str iking resemblances to strychnine have been isolated in recent years from the part icularly potent Calabasch curare. Yohimbine (6) was employed as an aphrodisiac in veterinary medicine. Reserpine (7) , isolated from the Indian snakeroot, Rauwolfia serpentina, was widely used in native medicine, usually as a sedative. It is useful in treat-ment of hypertension and of various mental disorders. The alkaloids of Ergot, which is a fungus parasi t ic on cereal grasses, especially rye, have an oxytocic effect useful in chi ldbirth . Of the Ergot alkaloids, the synthetic derivative of lysergic acid (8), in the form of the diethylamide, produces symptons l ike those of schizophrenia when admi-6 7 nistered in extremely small doses . Vinblastine (9), a dimeric alkaloid produced by Vinca rosea Linn, is used c l i n i c a l l y as a potent anti-leukemic agent. g During investigations by the L i l l y group on the dimeric Vinca alkaloids, vinblastine or vincaleukoblastine (VLB), leurosine, and leurocrist ine, they found that each was cleaved by concentrated hydro-chloric acid to an indole compound and a vindoline derivative. In the instances of VLB and leurosine, the latter was desacetylvindoline (10), whereas leurocristine gave des-N-methyl-desacetylvindoline (11). Both VLB and leurocristine afforded the same indole derivative, velbanamine, but the corresponding compound with leurosine was cleavamine. Velbanamine 5 was considered to be a hydroxy-dihydrocleavamine, since i t yielded some COOMe g cleavamine on prolonged heating with acid . When catharanthine was subjected to the same acid treatment, one of the products was found to be cleavamine, which suggested that the dimeric a l k a l o i d s were c o n s t i -tuted of vindol i n e and catharanthine-like moieties. Moreover, the in f r a r e d spectrum of VLB could be approximated by an equimolar mixture of vindoline and catharanthine ^ . Therefore, the L i l l y research group postulated a p a r t i a l structure f o r VLB which consisted of vindoline attached to a hydroxycatharanthine. However, i t was soon r e a l i z e d that VLB must contain a carbomethoxycleavamine rather than a catharanthine u n i t as the indole portion of the molecule. On the basis of t h i s and 9 other evidence, a revised structure (9) was proposed f o r VLB From the structure determination of VLB, considerable knowledge of the chemistry of catharanthine and cleavamine was accumulated. When catharanthine (12) was treated with concentrated hydrochloric acid i n the presence of t i n and stannous c h l o r i d e , two of the products obtained were descarbomethoxycatharanthine (16) and cleavamine (18) i n addition to two 11 12 dihydrocleavamine derivatives ' . The structure (18) of cleavamine had been suggested mainly on the basis of a comparison of the mass spectral cracking patterns of cleavamine and dihydrocleavamine with that of quebrach-13 amine (30), and was f i n a l l y established by an x-ray analysis of *u- A-A 1 4 » 1 5 cleavamine methiodide * The formation of a l l the products can be r a t i o n a l i z e d on the basis of 6 the mechanism shown in Figure 1. The lone pair of electrons on N^-j in 12 can participate in a rearrangement to form an iminium ion, with concurrent ring cleavage and protonation at the 6-position of the indole. The resulting ring-opened intermediate, 13, is stabilized by two factors: (a) the conjugated nature of the iminium ion, and (b) the conjugation of the newly generated double bond between C-17 and C-18 with both the ester and anilino functions. After acid hydrolysis of the ester, decarboxy-lation may occur via 14 in a manner analogous to the mechanism proposed 16 17 for the Iboga alkaloids ' . This ring-opened intermediate renders the molecule more flexible and the decarboxylated intermediate, 15, is obtained. The intermediate, 15, may follow either of two reaction paths. If the original electron flow is reversed, then the C-5/C-18 bond is regenerated and the product is descarbomethoxycatharanthine (16). But i f the a-methylene indoline system in 15 merely rearranges to the indole, the intermediate, 17, from which the ring-opened tetracyclic compounds must ultimately be formed, is obtained. If one assumes that 17 is the actual intermediate, 1,2-reduction of the iminium ion will give cleavamine directly. On the other hand, 1,4-reduction can also occur to afford an enamine, 19, which rearranges in the well-known manner to the iminium compound, 20, with subsequent reduction to provide the two isomeric dihydrocleavamines (21). A mixture of 4a- and 46-dihydrocleavamines was obtained, because the approach of the proton to C-4 in 19 can occur from above or below the plane of the ring with equal facility. The last steps of the above mechanism explain the formation of cleavamine and its dihydro derivatives when catharanthine is reacted with acid in the presence of a reducing agent (stannous chloride, mossy tin). Cleavamine is also formed (although in lower yield) by the action Figure 1. Reaction mechanism proposed for the formation of cleavamine, descarbomethoxycatharanthine and the epimeric dihydrocleavamines. 8 18 of concentrated hydrochloric acid alone . In the absence of an inorganic reducing agent, the reduction step possibly takes place via an intra-molecular redox reaction wherein two molecules of the intermediate, 17, react to y ie ld one molecule of cleavamine and one of a pyridinium compound, 22. The increase in y ie ld of cleavamine in a reducing medium is thus H 22 explicable on the grounds that 17 is reduced direct ly to cleavamine and no pyridinium compound is formed. Additional evidence for the formation of the tetracycl ic intermediate, 13, was furnished from the reaction of pentacyclic alkaloids with glacial acetic acid in the presence of zinc dust. The isolat ion of a carbomethoxy-9 dihydrocleavamine (23) with zinc in acetic acid provided evidence that the reduction of the iminium system can occur before the loss of the ester group. 12 13 23 One of the crucial steps in the mechanism of the acid-catalyzed rearrangement of catharanthine implies a transannular cyclization of intermediate 15 to provide a route to descarbomethoxycatharanthine. The use of such transannular cyclizations involving iminium salt intermediates is particularly interesting, since Kutney and coworkers have demonstrated that such reactions are of synthetic importance and have led to syntheses of Iboga and Aspidosperma-type systems. They were the f i r s t to demonstrate the f e a s i b i l i t y of such cyclizations under laboratory conditions. They 8 11 12 19 converted dihydrocleavamine ' ' ' (21) in two steps to 7-ethyl-5-20 21 desethylaspidospermidine (24). Likewise, they were able to convert 9 12 carbomethoxydihydrocleavamine ' (23) into 7-ethyl-5-desethylvinca-difformine (28) having the Aspidosperma skeleton and the Iboga alkaloids, 22 23 coronaridine (29), and i t s C-4 epimer, dihydrocatharanthine . Oxida-tion of carbomethoxydihydrocleavamine in the N^ -C-19 direction led to the iminium ion, 26, which cyclized in the reaction media to provide 28. Alternately, oxidation of carbomethoxydihydrocleavamine in the N^ -C-5 direction led to an iminium ion, which, by virtue of equilibration with the enamine, 27, resulted in the mixture of C-4 epimeric iminium ions, 25. On this basis, one epimer cyclized to coronaridine, the other, to dihydro-catharanthine. Since new asymmetric centres are generated during the course of these transannular cyclizations, one might anticipate that several diastereoisomers would be formed. However, the reaction proceeded in a stereospecific manner. This result was not unexpected 2^>24^ si n c e i t was apparent (from examination of molecular models) that effectively only one conformation of each iminium ion would permit the reacting centres to be in close proximity and at the same time not be highly strained. Furthermore, the marked ri g i d i t y of the cyclic products limits rather severely the stereochemistry of the various asymmetric centres. 14 24 25 Kutney and coworkers ' and, independently, Schmid and coworkers successfully carried out a conversion in the natural series. They found Figure 2. The transannular c y c l i z a t i o n and i t s synthetic application i n the Aspidosperma and Iboga series. 11 3d on that (-)-quebrachamine (30) was transformed i n t o (+)-aspidospermidine ' (31) under s u i t a b l e conditions. Thus, the c y c l i z a t i o n led to the natural diasterioisomer. The s i t u a t i o n with regard to the absolute configuration of the pseudo-Aspidosperma and Iboga compounds produced v i a the trans-annular c y c l i z a t i o n was not unambiguous because the o p t i c a l a c t i v i t y of the C-2 carbon i n the dihydrocleavamine molecule would conceivably have been destroyed i n the process. However, Kutney and coworkers showed that the asymmetry of the C-2 carbon atom was retained i n the transannular c y c l i z a t i o n r e a c t i o n . 7-Ethyl-5-desethylaspidospermidine obtained from 5 7 X \ 10 N 30 32 33 4B-dihydrocleavamine (32) was established to have the absolute configu-r a t i o n as shown i n 33 by x-ray d i f f r a c t i o n of i t s N a-acetyl-N^-methiodide d e r i v a t i v e ^ . The configuration of the C-2 carbon atom 1 4 i n 4g_ dihydrocleavamine thus remained unaltered during the course of the conversion reaction. The configuration of the C-19, C-12 and C-2 carbon atoms r e l a t i v e to the configuration of the C-5 carbon atom of 7-ethyl-5-desethylaspidospermidine was that expected from conformational consideration. 12 In addition, the configuration of the C-4 carbon atom of 4B-dihydro-cleavamine was unaltered during the course of the reaction. Since the transannular cyclization of 46-dihydrocleavamine to 78-ethyl-5-desethyl-aspidospermidine took place in a stereospecific manner, i t followed that the transannular cyclization of carbomethoxydihydrocleavamine to provide 7-ethyl-5-desethylvincadifformine, dihydrocatharanthine and coronaridine was also a stereospecific process. The transannular cyclization,in addition to i t s value in the biogenetic postulates of Wenkert, had an important application for synthesis of alkaloids. The transannular cyclization step was incorpo-rated into a general total synthesis of a number of alkaloids bearing the Aspidosperma or Iboga skeletons. Since this step was shown to be com-pletely stereospecific with the configuration of each new asymmetric centre being completely determined by the configuration at C-2 in the nine-membered ring intermediates, the total synthesis based on this step was not beset with stereochemical problems during the various stages of the synthetic sequence. Also, parallel total syntheses of the dihydro-cleavamine and quebrachamine systems were feasible. Thus, the total synthesis of alkaloids bearing the Aspidosperma skeleton on the one hand and the Iboga skeleton on the other complemented each other with obvious advantages over synthetic approaches that required a completely different method for each skeletal type. The important features of the synthesis 29 30 of dl-quebrachamine (34) and dl-dihydrocleavamine (35), as carried 30 out by Kutney and coworkers, are shown in Figure 3. The extension of the synthesis from dl-dihydrocleavamine (35) involved introduction of a carbomethoxy function at C-18 to provide a carbomethoxydihydrocleavamine (36) (Figure 4). This latter step also in turn completed the total 13 H 34, R! = Et; R2 = H 35, Ri = H; R2 = Et Figure 3. Kutney's synthesis of dl-quebrachamine, dl-4a-dihydrocleavamine, and dl-4g-dihydrocleavamine. 15 synthesis of dl-coronaridine and dl-dihydrocatharanthine i n view of the 21 previous transannular c y c l i z a t i o n studies . Introduction of a carbo-methoxy function at C-3 i n quebrachamine would provide a carbomethoxy-31 quebrachamine or vincadine , which i s a n a t u r a l l y occurring Vinca a l k a l o i d . A successful transannular c y c l i z a t i o n of a carbomethoxy-quebrachamine i n the manner described previously would provide vinca-difformine, another Vinca a l k a l o i d . S i m i l a r transannular c y c l i z a t i o n s have also been accomplished with the Akuamma class of a l k a l o i d s . Oxidation of the t e t r a c y c l i c indole, 37, with e i t h e r oxygen and platinum or potassium permanganate gives the two iminium intermediates, 38 and 39, which undergo transannular c y c l i z a t i o n 32 to a f f o r d t u b i f o l i n e (40) and condifoline (41), r e s p e c t i v e l y . It was also found that c o n d i f o l i n e was converted into t u b i f o l i n e under a v a r i e t y of conditions (e.g. neat at 117-120°, i n t e t r a l i n at 160-180°, i n t r i -ethylamine, i n ammonia-isopropanol, or i n potassium t-butoxide—t-butanol). Even though the transannular c y c l i z a t i o n i s an important step i n the synthesis of a l k a l o i d s , the reverse of t h i s process has also provided some i n t e r e s t i n g chemistry i n addition to the aspects already discussed with respect to the ring-opening reactions i n the catharanthine system. 33 Stork and coworkers used such an opening reaction when they converted dl-l,2-dehydroaspidospermidine (42) to dl-quebrachamine (44) by bringing about the equilibrium, 42 5^43, i n methanol and s e l e c t i v e l y reducing the very r e a c t i v e iminium function i n the t e t r a c y c l i c cation with potassium borohydride. An analogous opening of the pentacyclic system was an important step i n the structure determination of the n a t u r a l l y occurring a l k a l o i d s , 34 35 (-)-tabersonine (45) and 16-methoxy-(-)-tabersonine (46). Figure 5 16 44 outlines the reaction sequence for 16-methoxy-(-)-tabersonine. Hydro-genation followed by decarboxylation afforded 16-methoxy-l,2-dehydro-(-) • aspidospermidine (47) which, on treatment with sodium borohydride in 10% ethanolic sodium hydroxide, afforded the tetracyclic 16-methoxy-(+)-quebrachamine (48). 48, R = OMe 47, R = OMe Figure 5. Structure determination of 16-methoxy-(-)-tabersonine. The reverse of the transannular cyclization is also postulated to occur when akuammicine is heated with methanol. Akuammicine (49) is remarkable in the ease with which it breaks up into aromatic optically Figure 6. Some properties of akuammicine. 18 i n a c t i v e components (Figure 6). Upon heating i n a sealed tube i n methanol at 80°, the "betaine" (53) i s formed. The formation of 53 from akuammicine i s explained by the interconversion of a C-16-protonated species to 50, followed by proton removal at C-14 to give an intermediate enamine (51) that can cleave by an e s s e n t i a l l y i r r e v e r s i b l e retro-Michael reaction to the ester (52). Subsequent aromatization of the l a t t e r leads to 53. This i n t e r p r e t a t i o n of the decomposition of akuammicine was consistent with the observations that 2,16-dihydro-akuammicine and akuammicine methiodide do not break down under comparable conditions; the former i s simply epimerized at C-16, and the l a t t e r remains unaffected Upon acid h y d r o l y s i s , akuammicine, l i k e a 6-ketoester, suffered decarboxylation. The r e s u l t i n g indolenine (54) exists i n equilibrium with the indole iminium s a l t (55) . In the absence of a proton donor, the indolenine form i s f a v o u r e d — e . g . , reduction with lithium aluminum hydride i n ether gave the indo l i n e (57), whereas, i n methanol (proton 37 donor) reduction with potassium borohydride, i t afforded the indole (56) The transannular c y c l i z a t i o n has also been postulated as an important step f o r the biosynthesis of indole-type a l k a l o i d s . In order to gain a bette r knowledge of t h i s enamine-imine chemistry of indoles, and hopefully to provide intermediates s u i t a b l e for biosynthesis i n t h i s area, the reaction mediated by acid with various indole a l k a l o i d s must be examined i n greater d e t a i l . The purpose of the f i r s t section of t h i s thesis i s to discuss a d e t a i l e d i n v e s t i g a t i o n of the acid-catalyzed reactions i n the catharanthine system, as well as some rela t e d chemistry of the Aspido-sperma s e r i e s . Discussion Cleavamine (18) was i n i t i a l l y obtained by the L i l l y group in their 8 12 investigations on Vinca alkaloids ' , and i t s derivation from the alkaloid, catharanthine, provided an example of a novel acid-catalyzed rearrangement which had l i t t l e or no precedent in indole alkaloid chemistry. A further study by Kutney and coworkers on the reaction of this alkaloid with hydrochloric acid in the presence of stannous chloride allowed the isolation of two dihydrocleavamine derivatives in addition to cleavamine 1 1. None of the products possessing the cleavamine skeleton s t i l l retained the ester function and were, therefore, of l i t t l e interest to our immediate requirement. The reaction of catharanthine with zinc in acetic acid, on the other 9 12 hand, was shown to yield a carbomethoxydihydrocleavamine ' , and i t became of immediate importance to our investigations. It was necessary for us to study this reaction in more detail in the hope that other carbomethoxydihydrocleavamine or carbomethoxycleavamine derivatives could be isolated. In our hands, catharanthine, on reaction with zinc dust in refluxing glacial acetic acid, provided a complex mixture from which four compounds (representing approximately 40% of the crude reaction mixture) could be isolated and characterized. The major compound (isomer C a), mp 172°, a For the sake of c l a r i t y , the compounds are designated A, B, C, D in order of increasing polarity on s i l i c a gel chromatoplates. 20 obtained by column chromatography on alumina, followed by c r y s t a l l i z a t i o n from methanol, was i d e n t i c a l with the previously reported carbomethoxy-9 12 dihydrocleavamine ' . The other three compounds were obtained pure a f t e r preparative t h i n - l a y e r chromatography ( t i c ) on s i l i c a g e l . Isomer A, mp 144-147°, appeared to be another carbomethoxydihydro-cleavamine d e r i v a t i v e when high r e s o l u t i o n mass.spectrometry established the molecular formula, C21H28N2O2. The molecular ion (m/e 340) was accompanied by fragments which were immediately reminiscent of the que-12 brachamine and cleavamine fragmentation process (Figure 7). Thus, the fragment at m/e 215 may a r i s e from cleavage at "a" and "b" as shown i n Figure 7, while loss of the ester group (d) from the l a t t e r would generate the species at m/e 156. The accompanying p i p e r i d i n e fragment a r i s i n g from t h i s cleavage would be seen at m/e 124. Alternate f i s s i o n at "b", "c" and "d" would give r i s e to the fragments at m/e 144, 143 and 138. Conclusive evidence f o r the structure of t h i s compound was obtained when the known carbomethoxydihydrocleavamine (isomer C), on treatment with boron t r i f l u o r i d e , was converted to isomer A. This experiment c l e a r l y established that these compounds were merely isomeric at C-18. A discussion on the stereochemistry i s deferred to a l a t e r section. Isomer B, mp 146-149°, was the next compound obtained i n the t i c p u r i f i c a t i o n . High r e s o l u t i o n mass spectrometry again established that t h i s substance was also a carbomethoxydihydrocleavamine d e r i v a t i v e , and analysis o f the fragmentation pattern confirmed t h i s assignment. Hydro-l y s i s and decarboxylation of B ( d i l u t e hydrochloric acid) provided 46-dihydrocleavamine. With regard to the l a t t e r experiment, reaction of isomer C with d i l u t e acid gave r i s e to a dihydrocleavamine i d e n t i c a l to that previously 21 12 obtained by the L i l l y workers , but not identical to 48-dihydro-cleavamine as mentioned above. Isomer D, mp 226-229°, was the fourth compound isolated from the zinc-acetic acid reaction. Evidence for a carbomethoxydihydrocleavamine formulation was obtained as above. Reaction of D with boron trifluoride provided B, and consequently, the relationship between these isomers was apparent. The above results established the gross structures of the four com-pounds obtained, but clearly, insufficient evidence has been presented to differentiate between them. The data which allows complete structural and stereochemical assignments to isomers A, B, C and D w i l l now be discussed. X-ray evidence on cleavamine methiodide 14.28 e s t a b i j s h e c i the absolute configuration at C-2 in this molecule. On this basis, the stereochemistry at C-2 in the dihydrocleavamine, obtained by catalytic reduction of cleava-12 15 mine , is also defined. Furthermore, as shown by the x-ray method , the cyclization product derived from this compound is 7j$-ethyl-5-desethylasido-spermidine (Aspidosperma numbering). It i s , therefore, established that this dihydrocleavamine isomer can now be termed as 48-dihydrocleavamine b c (58) ' . An extension of this argument allows the assignment, 4a-dihydro-cleavamine (59), to the isomer obtained by removal of the ester function in isomer C. In consideration of the experiments mentioned earlier, i t is clear that A and C now belong to the 4a-dihydrocleavamine series and differ only in stereochemistry at C-18. Similarly, isomers B and D are in the 6 series and merely differ at C-18. b It must be noted that C-7 in the conventional Aspidosperma numbering system is C-4 in the Iboga system. c For the sake of convenience, the "B" orientation is designated to the C-4 ethyl group which is trans to the hydrogen atom at C-2 as indicated in the structures 58 and 59. 22 The remaining question of stereochemistry at C-18 in the two series was settled by nmr spectroscopy. Figures 8, 9, 10, and 11 il l u s t r a t e the nmr spectra for isomers A-D, respectively. Isomers A and B possess multi-plets in the region T 4.5-5.0 for C-18-H, whereas the corresponding proton absorbs at higher f i e l d in compounds C and D ( T 6.0-6.2). This rather dramatic difference in the resonance frequency is readily explicable in terms of the appropriate conformational structures which are possible in these two series (60-63). In 60 and 61, the proton at C-18 is in close proximity to the basic nitrogen atom of the piperidine moiety, and i t would be expected to absorb at a lower frequency. Such a situation does not prevail in 62 and 63, and a more normal resonance frequency for C-18-H would be anticipated. On this basis, and in conjunction with the previous arguments presented above, isomer A is now completely defined as 188-carbomethoxy-4a-dihydrocleavamine (60), while B is the 188 isomer in the 48 series (61). Isomer C, the major component obtained previously in q another laboratory , is the 18a-carbomethoxy compound in the 4a series (62), while D can now be assigned structure 63. It is appropriate to mention here that similar nmr arguments have been employed by Mokry and Kompis in establishing the stereochemistry of the structurally related Vinca alkaloids, vincaminoreine (71) and the epimeric 38 vincaminorine Our f u r t h e r i n t e r e s t i n f i n d i n g a route to the unknown carbomethoxy-cleavamine (64) ser i e s led to a consideration of the hydride reduction of the intermediates derived from the acid-catalyzed r i n g opening of 11 12 catharanthine. It had been previously postulated ' that dihydro-py r i d i n e s a l t s were formed i n the i n i t i a l stages of t h i s reaction, whereas a pyridinium d e r i v a t i v e may well explain the formation of cleavamine or i t s dihydro analogues, p a r t i c u l a r l y i n the absence of any reducing agent. Indeed, e i t h e r one o f these intermediates would be expected to reduce with hydride reagents. When catharanthine hydrochloride dissolved i n hot g l a c i a l a c e tic a c i d was treated with sodium borohydride, a s u r p r i s i n g l y good y i e l d (63%) of a c r y s t a l l i n e compound, mp 121-123°, was obtained. Elemental analysis of t h i s compound was i n accord with the molecular formula, C21H26N2O2, but in p a r t i c u l a r , the nmr spectrum (Figure 12) was immediately i n d i c a t i v e of a cleavamine system. A m u l t i p l e t centered at T 4.76, along with a c l e a r t r i p l e t at T 8.96, the l a t t e r occurring at lower f i e l d than i n the dihydro-cleavamine d e r i v a t i v e s , provided strong evidence for the double bond at the 3,4 p o s i t i o n . The C-18 proton resonating at T 4.89 indicated that the ester group i n t h i s molecule was i n the 6-orientation i n accord with the CO 2 IJ LU > 40 r-< _l ID cr 20 0 uu, 50 lillllli I3S 215 2101 llii lei, .il I ill 254 231 299 100 200 m/e Figure 7. Mass spectrum of carbomethoxydihydrocleavamine (isomer A) (60) 25 Figure 9. Nmr spectrum of carbomethoxydihydrocleavamine (isomer B) (61). Figure 10. Nmr spectrum of carbomethoxydihydrocleavamine (isomer C) (62). Figure 12 . Nmr spectrum of carbomethoxycleavamine ( 6 4 ) 27 previous assignment indicated above. Finally, acid hydrolysis and decar-boxylation of this crystalline compound yielded cleavamine. The structure of this product was,thus, established to be 186-carbomethoxycleavamine 39 (64) ^. The formation of the four carbomethoxydihydrocleavamines and carbo-methoxycleavamine which have been isolated from the reaction of catharan-thine with glacial acetic acid and a reducing agent can be rationalized on the basis of the mechanism shown in Figure 13. The lone pair of electrons on in catharanthine can participate in a rearrangement to form an imi-nium ion, with concurrent ring cleavage and protonation at the 8-position of the indole. The resulting ring-opened intermediate 65 is stabilized by two factors: (a) the conjugated nature of the iminium ion, and (b) the conjugation of the newly generated double bond between C-17 and C-18 with both the ester and the anilino functions. The crucial intermediate, 65, may follow either of two reaction paths. I f the original electron flow is reversed, then the C-5/C-18 bond is regenerated and catharanthine is regenerated. But i f the a-methylene indoline system in 65 merely rearranges to the indole, the intermediate, 66, from which the ring-opened tetracyclic compounds must ultimately be formed is obtained. If one assumes that 66 is the actual intermediate, 1,2-reduction of the iminium ion occurs readily in the presence of sodium borohydride and carbomethoxycleavamine is obtained directly. On the other hand, 1,4-reduction can also occur to afford an enamine, 67, which rearranges in the well-known manner to the iminium compound, 68, with subsequent reduction to provide the four isomeric carbo-methoxydihydrocleavamines (60-63). The cleavamine-type nine-membered ring compounds could now be evaluated in plants as possible precursors in the biosynthesis of Iboga alkaloids. 28 Figure 13. Formation of carbomethoxydihydrocleavamines and carbomethoxycleavamine. 29 Chemically, in the laboratory, these nine-membered ring compounds are readily converted to the Iboga-type systems. Carbomethoxydihydrocleavamine 21 (isomer C) has been shown to be converted into coronaridine and i t s C-4 epimer, dihydrocatharanthine (Figure 2). Also, recently, carbomethoxy-40 cleavamine has been converted to catharanthine using mercuric acetate in acetic acid. In order to continue the study of the transannular cyclization reaction, the study of natural nine-membered ring compounds such as quebrachamine (30) was undertaken. Note, the distinction between the quebrachamine and the cleavamine systems is that quebrachamine has the piperidine ethyl group attached at position 2, whereas cleavamine has the ethyl group attached to the 4 position of the alkaloid (see quebrachamine (30)). Quebrachamine i t s e l f is readily converted into 1,2-dehydroaspidosper-midine having the Aspidosperma skeleton using either mercuric acetate or 24 25 oxygen in the presence of a catalyst ' . Recently, vincadine (69) was 41 also converted to the Aspidosperma alkaloid, vincadifformine (70) , using oxygen in the presence of a catalyst. To complete this series and to confirm that the transannular cyclization is a truly general reaction of nine-membered ring indole alkaloids, a N-methyl derivative must be converted to the corresponding Aspidosperma alkaloid. For this purpose, the N-methyl vincadine derivative or vincaminoreine (71) was chosen. A mixture of vincaminoreine and 5% platinum on powdered charcoal in ethanol was shaken under an atmosphere of oxygen. After one hour, the mixture was filtered and the f i l t r a t e concentrated to dryness. The crude product was purified or, a preparative t i c plate using alumina as the adsorbent to afford a pure sample of minovine (72) 4 1. This substance was identical in every respect (infrared, 42 4 3 tic) with an authentic sample of minovine ' 30 COOMe [0] 69, R = II 70, R H 71s R = Me 72, R Me The reverse of the transannular cyclization reaction, which led to minovine (72) and vincadifformine (70), was the next consideration. In effect, the reverse of this cyclization has been already discussed in the Iboga series, since the conversion of catharanthine to the various cleavamine derivatives invokes a similar process. With regard to the Aspidosperma alkaloids, some precedent in the literature was also available. Stork and 33 coworkers were able to convert dl-l,2-dehydroaspidospermidine to dl-quebrachamine by creating the equilibrium 42^=^43 and selectively reducing the very reative iminium function in the tetracyclic cation with potassium borohydride. It was fe l t that i f the equilibrium 72^=^73 could be brought about, then the reactive iminium function in 73 could be readily reduced with hydride reagent. The expected product, vincaminoreine (71), would be stable under these conditions and, thereby, the equilibrium would shift in the desired direction. An alternative pathway which does not invoke the above equilibrium is equally plausible. If protonation of the ester occurs, the equilibrium 72^=^74 w i l l be brought about and vincaminoreine (71) would be formed. When minovine (72) dissolved in hot glacial acetic acid was treated with an excess of sodium borohydride, two compounds exhibiting 32 t y p i c a l indole u l t r a v i o l e t spectra were i s o l a t e d . P u r i f i c a t i o n on preparative s i l i c a gel t i c plates allowed the i s o l a t i o n of two compounds. The major component (60% y i e l d ) , mp 138-139°, possessed the formula, C 2 2 H 2 8 N 2 ° 2 » a s determined by high r e s o l u t i o n mass spectrometry. This 38 44 compound was found to be i d e n t i c a l with authentic vincaminoreine ' (71) by appropriate comparison ( t i c , melting point, nmr). The other com-ponent (10% y i e l d ) , rap 129-131°, had also the molecular formula, C 2 2 H 2 8 N 2 ° 2 -Thus, t h i s compound i s epimeric at the carbomethoxy-bearing carbon atom and must be vincaminorine 3 8 » 4 5 . The s p e c t r a l data and melting point of t h i s compound corresponded i d e n t i c a l l y with that i n the l i t e r a t u r e . Unfor-tunately, an authentic sample could not be obtained f o r purposes of comparison. Thus, these investigations established the chemical i n t e r - r e l a t i o n s h i p s between the nine-membered r i n g systems o f the quebrachamine and cleavamine f a m i l i e s with t h e i r c y c l i c Aspidosperma and Iboga r e l a t i v e s . The usefulness o f these r e s u l t s i n subsequent biosynthetic studies w i l l be discussed l a t e r . For example, i n connection with a b i o s y n t h e t i c study i n Vinca rosea Linn pl a n t s , a comparison of a nine-membered r i n g compound with the corresponding c y c l i c system was necessary. In order to trace the biogenetic pathway i n these p l a n t s , the Aspidosperma a l k a l o i d , tabersonine (45), had to be compared with the unknown nine-membered r i n g compound, 6,7-dehydrovincadine (75). A small amount o f tabersonine hydrochloride, when dissolved i n hot g l a c i a l a c e t i c acid and treated with sodium borohydride, y i e l d e d a pure compound a f t e r p u r i f i c a t i o n on t h i n - l a y e r chromatoplates. No attempt was made to obtain t h i s compound i n c r y s t a l l i n e form because of the small amounts involved. The high r e s o l u t i o n mass spectrum was i n accord with the mole-cular formula, C 2 i H 2 6 N 2 0 2 - The u l t r a v i o l e t spectrum was that of a t y p i c a l 33 COOMe COOMe 45 75 indole (X w 228, 286, and 293 my), while the in f r a r e d exhibited a normal max es t e r absorption at 1715 cm - 1. The nmr spectrum, although weak, was s i g n i f i c a n t i n that a one-proton s i g n a l was observed at x 4.11 and a two-proton s i g n a l at T 4.72. Thus, the two o l e f i n i c protons and the proton adjacent to the carbomethoxy function are accounted f o r , although the p o s i t i o n of each proton could not be assigned with c e r t a i n t y . Since the C-18 proton resonates i n the region x 4.11-4.72, the ester group i n t h i s molecule must be i n the 6-orientation i n accord with the previous assign-ments described e a r l i e r . On t h i s b a s i s , the reaction product i s indeed the desired 6,7-dehydrovincadine (75). The above in v e s t i g a t i o n s provide ample evidence for the v e r s a t i l i t y o f enamine-imine intermediates i n the area of indole a l k a l o i d s . Whether such intermediates are involved i n a l k a l o i d biosynthesis i s c l e a r l y an i n t e r e s t i n g question. Indeed, the transannular c y c l i z a t i o n reaction has been postulated as being an important step i n the biosynthesis of several fa m i l i e s of indole a l k a l o i d s . However, no experiments had been done to confirm t h i s postulate. Part II of t h i s thesis deals with some experiments designed to evaluate t h i s r eaction i n terms of i t s s i g n i f i c a n c e i n the plant. Experimental Melting points were determined on a Kofler block and are uncorrected. The u l t r a v i o l e t (uv) spectra were recorded i n methanol on a Cary 11 recording spectrometer, and the i n f r a r e d ( i r ) spectra were taken on Perkin-Elmer Model 21 and Model 137 spectrometers. Nuclear magnetic resonance (nmr) spectra were recorded i n deuteriochloroform at 100 megacycles per second (unless otherwise indicated) on a Varian HA-100 instrument and the chemical s h i f t s are given i n the T i e r s T scale with reference to t e t r a -methylsilane as the i n t e r n a l standard. Mass spectra were recorded on an Atlas CH-4 mass spectrometer and high r e s o l u t i o n molecular weight deter-minations were determined on an AE-MS-9 mass spectrometer. Analyses were c a r r i e d out by Mr. P. Borda o f the microanalytical laboratory, the University of B r i t i s h Columbia. Woelm neutral alumina and S i l i c a Gel G (acc. to Stahl) containing 2% by weight of General E l e c t r i c Retma p-1, Type 188-2-7 e l e c t r o n i c phosphor were used f o r a n a l y t i c a l and preparative t h i n - l a y e r chromatography ( t i c ) . Chromatoplates were developed using the spray reagents, carbon tetrachloride-antimony pentachloride 2:1 or 35% s u l f u r i c acid saturated with eerie s u l f a t e . Woelm neutral alumina ( a c t i v i t y III) was used for column chromatography (unless otherwise indicated). Cleavamine (18) A mixture of catharanthine hydrochloride (12, 10 g), stannous chloride (11 g), and mossy t i n (1 g) in concentrated hydrochloric acid (130 ml) was heated under r e f l u x i n a nitrogen atmosphere for 75 min. By the end of t h i s time, an orange-red gum had formed in the reaction mixture. The 35 acidic solution was decanted from the gum and washed with methylene chloride (3 x 25 ml). The washings were combined with the gum, and then methanol (15 ml) and methylene chloride (25 ml) were added so that a clear solution was obtained. This solution was shaken with IN aqueous sodium hydroxide (150 ml), separated, and washed with water (50 ml). The sodium hydroxide solution was washed with ether (2 x 25 ml) and the ethereal extract added to the methylene chloride solution. After drying over sodium sulfate, the organic solution was evaporated to leave a reddish o i l (7 g), which was taken up in benzene and chromatographed on alumina (300 g). Cleavamine was eluted in the i n i t i a l benzene-petroleum ether 30/60 1:1 fractions and recrystallized from methanol to give needles (1.7 g), mp 117-119° (Lit 1 1 , 1 2 mp 117-119°). 4B-Dihydrocleavamine (58) A solution of cleavamine (18, 0.75 g) in ethyl acetate (10 ml) was hydrogenated over Adam's catalyst (0.007 g). Uptake of hydrogen ceased after 68 min, at which time one mole of hydrogen had been absorbed. Filtr a t i o n and evaporation of the f i l t r a t e gave 4B-dihydrocleavamine (58), which was recrystallized from methanol as prisms (0.59 g) ; mp 136-138° (Lit 1 1 mp 136-138°). Zinc-Acetic Acid Reduction of Catharanthine (12) A mixture of catharanthine (4.9 g) and zinc dust (34 g) in glacial acetic acid (125 ml) was heated under reflux, with vigorous s t i r r i n g , in a nitrogen atmosphere for 4 hr. The hot mixture was filtered and the f i l t r a t e was evaporated under reduced pressure until most of the acetic acid was removed. The residue was made basic by addition of dilute aqueous ammonia, and the resulting mixture was extracted thoroughly with ether. The combined extracts were washed with saturated brine and dried over 36 anhydrous sodium sulfate. Removal of the ether afforded a gummy residue which was subjected to chromatography on alumina (150 g). Elution with petroleum ether (bp 30-60°) (1200 ml) afforded 1.13 g of crystalline 18a-carbomethoxy-4a-dihydrocleavamine (62) (isomer C). Recrystallization from methanol gave colorless blocks: mp 169-171°; [o]i! 3 +100° (CHC17) ; X ° r D 3 ' max (log E ) : 227 (4.50), 286 (3.89), 293 (3.86) my; v K B r :3375 (-N-H), 2755 m 3.x (Bohlmann band), 1709 (C00CH3)cm'1; nmr: T 1.00 (singlet, IH, N-H), 2.76 (diffuse, 4H, aromatic), 6.13 (doublet, IH, C-18 proton), 6.37 (singlet, 3H, COOCH3) , 9.33 ( t r i p l e t , 3H, -CH2CH3). This compound was found to be identical (mp and mixture mp, infrared, nmr and t i c R^. value) with the 9 12 46 carbomethoxydihydrocleavamine reported previously ' ' Anal. Calcd. for C 2 1H 2 8N 20 2: M.W. 340.215. Found: 340.214 (mass spectrometry). Further elution in the above chromatography with petroleum ether (bp 30-60°) provided a mixture (1.14 g) of the three remaining isomeric carbo-methoxydihydrocleavamines which were separated by preparative t i c . S i l i c a gel plates (20 x 60 cm, 0.5 mm thickness) were used, with 150 mg of the mixture being applied to each plate. After development with 3:1 chloroform-ethyl acetate, each desired band was scraped off the plate and eluted with warm methanol. Evaporation of the eluants gave the desired crystalline carbomethoxydihydrocleavamines. 186- Carbomethoxy-4a-dihydrocleavamine (60, 0.28 g) (isomer A) was 2 3 recrystallized from methanol, affording prisms: mp 144-147°; [a]p +18° (CHC13); X m a x (log e): 227 (4.54), 286 (4.02), 294 (3.97) my; v^:3340 (-N-H), 2760 (Bohlmann band), 1707 (C00CH3) cm"1; nmr: T 1.40 (singlet, 1H, N-H), 2.81 (diffuse, 4H, aromatic), 4.53 (doublet, IH, C-18 proton), 6.40 (singlet, 3H, COOCH3), 9.09 ( t r i p l e t , 3H, -CH2CH3). 37 Anal. Calcd. for C 2 1H 2 8N 20 2: M.W. 340.215. Found: 340.215 (mass spectrometry). 188-Carbomethoxy-4g-dihydrocleavamine (61, 0.25 g) (isomer B) was 23 obtained as prisms from methanol: mp 146-149°; [a]^ -66° (CMC13); \ v (log e): 227 (4.54), 286 (4.02), 294 (3.98) my; v*" 3300 (-N-H), lUdA JT13.X 2755 (Bohlmann band), 1695 (-CC0CH3) cm"1; nmr: T 1.37 (singlet, III, N-H), 2.83 (diffuse, 4H, aromatic), 4.98 (doublet, IH, C-18 proton), 6.39 (singlet, 3H, -COOCH3), 9.12 ( t r i p l e t , 3H, -CH2CH3). Anal. Calcd. for C 2 1H 2 8N 20 2: M.W. 340.215. Found: 340.215 (mass spectrometry). 18a-Carbomethoxy-4B-dihydrocleavamine (63, 0.22 g) (isomer D) was recrystallized from acetone, giving small blocks: mp 226-229°; X (log e ) : 226 (4.50), 286 (3.92, 294 (3.90) my; v ^ : 3335 (-N-H) , 2760 (Bohlmann band), 1720 (C00CH3) cm"1; nmr: T 1.30 (singlet, IH, N-H), 2.84 (diffuse, 4Hj aromatic), 6.12 (pair of doublets, IH, C-18 proton), 6.40 (singlet, 3H, -COOCH3), 9.45 ( t r i p l e t , 3H, -CH2CH3). Anal. Calcd. for C 2 1H 2 80 2N 2: M.W. 340.215. Found: 340.215 (mass spectrometry). Epimerization of 18q-Carbomethoxy-4q-dihydrocleavamine (62) (Isomer C) To a solution of compound 62 (500 mg) in dry benzene (10 ml) was added boron trifluoride etherate (1 ml) and the resulting solution was refluxed under an atmosphere of nitrogen for 6 hr. After cooling, the solution was poured into saturated aqueous sodium bicarbonate and the resulting mixture was extracted thoroughly with dichloromethane. The combined extracts were dried (anhydrous sodium sulfate) and evaporated under reduced pressure. The residual material was purified by preparative t i c ( s i l i c a gel, chloro-form), affording 200 mg of starting material (62), as shown by mp, mixture 38 mp, infrared and t i c , and 175 mg cf 18B-carbomethoxy-4a-dihydrocleavam.ine (60) (isomer A). The latter was identical (mp and mixture mp, infrared, tic) with compound 60 prepared previously (see above). Epimerization of 18a-Carbomethoxy-4g-dihydrocleavamine (63) (Isomer D) Compound 63 (22 mg) was treated with boron trifluoride etherate in benzene solution under conditions identical with those described above for compound 62. Purification of the crude product by preparative t i c ( s i l i c a get, 3:1 chloroform-ethyl acetate) afforded 7 mg of starting material (63), as shown by mp, infrared and t i c , and 8 mg of 186-carbomethoxy-43-dihydro-cleavamine (61) (isomer B) . The latter was identical (nip and mixture mp, infrared, tic) with an authentic sample obtained previously (see above). Decarbomethoxylation of 18B-Carbomcthoxy-4ot-dihydrocleavamine (60) (Isomer A) A solution of compound 60 (33 mg) in 5N hydrochloric acid (1 ml) was heated, under an atmosphere of nitrogen, at 95° for 8 hr. The solution was cooled in ice and made basic by addition of dilute aqueous ammonia. The resulting mixture was extracted with dichloromethane and the combined extracts were dried over anhydrous sodium sulfate. Removal of solvent afforded amorphous material which was identical, as shown by infrared and t i c ( s i l i c a gel, ethyl acetate), with an authentic sample of 4a-dihydro-cleavamine (59) 1 1. Decarbomethoxylation of 1S6-Carbomethoxy-4B-dihydrocleavamine (61) (Isomer B) Compound 61 (30 mg) was decarbomethoxylated under conditions identical with those described above. The product, which was crystallized from methanol, gave mp 136-138°, and was found to be identical, as shown by mp and mixture mp, infrared, and t i c ( s i l i c a gel, 1:1 chloroform-ethyl acetate), with 46-dihydrocleavamine (58) 39 Decarbomethoxylation of 18a-Carbomethoxy-4g-dihydrocleavamine (62)  (Isomer C) Decarbomethoxylation of compound 62 (500 mg) under conditions i d e n t i c a l with those described above, gave 4a-dihydrocleavamine ( 5 9 ) , i d e n t i c a l ( i n f r a r e d and t i c ) with an authentic sample. 18B-Carbomethoxycleavamine (64) A s o l u t i o n of catharanthine hydrochloride (5.5 g) i n g l a c i a l a c e t i c acid (250 ml) was heated to 90° i n a 1 l i t r e 3-necked flas k equipped with a mechanical s t i r r e r and a r e f l u x condenser. Sodium borohydride was added at i n t e r v a l s to keep the s o l u t i o n gently r e f l u x i n g . A f t e r 1 hr, the reaction mixture was cooled to 10°, poured i n t o aqueous ammonia, and the r e s u l t i n g mixture was extracted thoroughly with dichloromethane. The combined extracts were washed with water, drie d over anhydrous sodium s u l f a t e , and evaporated under reduced pressure. The r e s i d u a l l i g h t brown o i l was d i s -solved i n hot methanol (7 ml) and the product was allowed to c r y s t a l l i z e , g i v i n g 1.9 g of pure 188-carbomethoxycleavamine (64), mp 121-123°. The mother l i q u o r was evaporated and the r e s i d u a l o i l was subjected to column chromatography on Woelm s i l i c a g e l , a c t i v i t y I, (75 g). E l u t i o n with chloroform produced a further 1 g of c r y s t a l l i n e 188-carbomethoxycleavamine (64): mp 121-123°; y i e l d = 2.9 g (63%); X (log e): 225 (4.55), 277(sh) (3.89), 287 (3.95), 294 (3.92) mu; v C H C l 3 : 3415 (N-H), 1708 (C00CH 3) cm"1; nmr: T 1.45 ( s i n g l e t , IH, N-H), 2.47-3.10 ( d i f f u s e , 4H, aromatic), 4.76 (multiplet, IH, o l e f i n i c H), 4.89 (doublet, IH, C-18 proton), 6.42 ( s i n g l e t , 3H, -COOCH3), 7.97 (quartet, 2H, -CH 2CH 3), 8.96 ( t r i p l e t , 3H, -CH 2CH 3). Anal. Calcd. f or C 2iH 26N20 2: C, 74.52; H, 7.74; N, 8.28. Found: C, 74.35; H, 7.80; N, 8.50. 40 Decarbomethoxylation of 18B-Carbomethoxycleavamine (64) A solution of compound 64 (50 mg) in 5N hydrochloric acid (2 ml) was heated at 90° for 3 hr. The solution was cooled, poured into aqueous ammonia and the resulting alkaline mixture was extracted thoroughly with dichloromethane. The combined extracts were washed with water, dried (anhydrous sodium sulfate), and evaporated under reduced pressure, pro-ducing 30 mg of a crystalline material. The latter was shown by mp, mixture mp, and infrared spectra to be identical with an authentic sample of cleavamine (18) 1 1. Hydrogenation of 18B-Carbomethoxycleavamine (64) A small sample of compound 64 was hydrogenated (room temperature and atmospheric pressure) in ethyl acetate over Adam's catalyst. F i l t r a t i o n , followed by evaporation of the f i l t r a t e under reduced pressure, afforded 18B-carbomethoxy-4B-dihydrocleavamine (61), identified by comparison (mp and mixture mp, infrared) with an authentic sample (see above). Transannular Cyclization of Vincaminoreine (71) A mixture of vincaminoreine (71, 0.024 g) and 5% platinum on charcoal (0.050 g) in ethanol (10 ml), under an atmosphere of oxygen, was shaken vigorously on a wrist-action shaker. After 1 hr, the mixture was filtered and the f i l t r a t e concentrated to dryness under reduced pressure. The residue was purified by preparative t i c (alumina, chloroform-benzene 1:3) to yield vincaminoreine (71, 0.003 g) and minovine (72, 0.0018 g): X : 308(sh), 337 mu: A . : 267 mu: v (CHC13): 1660 cm-1. This minovine y J' mm ' max v i J was found to be identical ( i r , uv and various t i c systems) with an , 42,43 authentic sample ' Sodium Borohydride Reduction of Minovine (72) A solution of minovine (72, 0.11 g) in glacial acetic acid (12 m l ) , under an atmosphere of nitrogen, was heated at 90° with an excess of 41 sodium borohydride f o r 30 min. The hot thick s o l u t i o n was poured into i c e - c o l d aqueous ammonia and the r e s u l t i n g mixture was extracted thoroughly with dichloromethane. The combined extracts were dried over anhydrous sodium s u l f a t e , and evaporated under reduced pressure. The res i d u a l o i l (0.119 g) was p u r i f i e d by preparative t i c ( s i l i c a g e l , chloroform) to a f f o r d two pure compounds. The less polar compound was vincaminoreine (71, 4 2 4 4 0.063 g): mp 138-139° ( L i t mp 138-139°); X (log E): 229 (4.54), 288 (3.87), 295 (3.86) my; vK*T: 1722 cm"1 (C00CH 3); nmr: T 2.50-3.08 ( d i f f u s e , 4H, aromatic), 6.17 (pair of doublets, IH, C-18 proton), 6.36 ( s i n g l e t , 3H, -CH 3), 6.53 ( s i n g l e t , 3H, -CH3) . Anal. Calcd. f or C 22H 3 0N 2O 2: M.W. 354.231. Found: 354.231 (mass spectrometry). The more polar compound was vincaminorine (0.010 g): mp 129-131° ( L i t 4 5 mp 130-131°); X (log e): 230 (4.57} 288 (3.89), 295 (3.88)my; ITlaX v^ll: 1715 cm"1 (C00CH 3); nmr: x 2.46-3.03 ( d i f f u s e , 4H, aromatic), 3.81 (pair of doublets, IH, C-18 proton), 6.38 ( s i n g l e t , 3H, -CH 3), 6.41 ( s i n g l e t , 3H, -CH 3). Anal. Calcd. f or C 2 2H 3 0N 2O 2: M.W. 354.231. Found: 354.230 (mass spectrometry). Sodium Borohydride Reduction of Tabersonine (45) A s o l u t i o n of tabersonine hydrochloride (0.008 g) i n g l a c i a l a c e t i c a c i d (10 ml), under an atmosphere of nitrogen, was heated at 90° with an excess of sodium borohydride f or 30 min. The hot thick s o l u t i o n was poured i n t o i c e - c o l d aqueous ammonia and the r e s u l t i n g mixture was extracted thoroughly with dichloromethane. The combined extracts were dried (sodium s u l f a t e ) and evaporated under reduced pressure. The res i d u a l o i l (0.008 g) was p u r i f i e d by preparative t i c ( s i l i c a g e l , chloroform) to afford the ring-opened product (75, 0.005 g) as an o i l : X 228, 286, 293 my; v (CHCI3) 3440 (-NH), 1715 (C00CH 3) cm"1; nmr: T 1.44 (singl IH, -NH), 2.47-2.98 (diffuse, 4H, aromatic), 4.11 (diffuse, IH), 4.72 (diffuse, 2H), 6.39 (singlet, 3H, COOCH3). Anal. Calcd. for C21H26N2O2: M.W. 338.199. Found: 338.199 (mass spectrometry). 43 Bibliography 1. K. Mothes, "The Alkaloids", Vol. VII, R.F. Manske, Ed., Academic Press, New York (1960). 2. J.W. Daly and B. Witkop, Angew. Chem. Internat. Edn., 2_, 421 (1963). 3. "The Merck Index of Chemicals and Drugs", 8th ed., Merck and Co., Inc., Rahway, N.J. (1968). 4. G. Habermehl, Naturwiss., 53_, 123 (1966). 5. T. Tokuyama, J. Daly, B. Witkop, I.L. Karle, and J. Karle, J. Amer. Chem. Soc, 90, 1917 (1968). 6. L.F. Fieser and M. Fieser, "Topics in Organic Chemistry", Chapter 3, Reinhold, New York (1963). 7. J.W. Moncrief and W.N. Lipscomb, J. Amer. Chem. Soc.,' 87, 4963 (1965). 8. N. Neuss, M. Gorman, H.E. Boaz, and N.J. Cone, J. Amer. Chem. Soc, 84, 1509 (1962). 9. N. Neuss, M. Gorman, W. Hargrove, N.J. Cone, K. Biemann, G. Buchi, and R.E. Mannine, J. Amer. Chem. Soc, 86, 1440 (1964). 10. M. Gorman, N. Neuss, and G.H. Svoboda, J. Amer. Chem. Soc, 81, 4745 (1959). 11. J.P. Kutney, R.T. Brown, and E. Piers, Can. J. Chem., 43_, 1545 (1965). 12. M. Gorman, N. Neuss, and N.J. Cone, J. Amer. Chem. Soc, 87, 93 (1965). 13. K. Biemann and G. Spiteller, J. Amer. Chem. Soc, 84_, 4578 (1962). 14. J.P. Kutney, J. Trotter, T. Tabata, A. Kerigan, and N. Camerman, Chem. and Ind., 648 (1963). 15. A. Camerman, N. Camerman, J.P. Kutney, E. Piers, and J. Trotter, Tetrahedron Letters, 637 (1965). 16. M.F. Bartlett, D.F. Dickel, and W.I. Taylor, J. Amer. Chem. Soc, 80, 126 (1958). 44 17. U. Renner, D.A. Piens, and W.G. S t o l l , Helv. Chim. Acta, 42, 1572 (1959). 18. M. Gorman and N. Neuss, Abstracts, 144th American Chemical Society Meeting, Los Angeles, Calif., 1963, p. 38M. 19. J.P. Kutney, R.T. Brown,and E. Piers, Canad. J. Chem., 44, 637 (1966). 20. J.P. Kutney and E. Piers, J. Amer. Chem. Soc, 86, 953 (1964). 21. J.P. Kutney, R.T. Brown, and E. Piers, J. Amer. Chem. Soc, 86_, 2286, 2287 (1964). 22. M. Gorman, N. Neuss, N.J. Cone, and J.A. Deyrup, J. Amer. Chem. Soc, 82, 1142 (1960). 23. N. Neuss and M. Gorman, Tetrahedron Letters, 206 (1961). 24. J.P. Kutney, R.T. Brown, and E. Piers, Lloydia, 27_, 447 (1964). 25. B.W. Bycroft, D. Schumann, M.B. Patel, and H.Schmid, Helv. Chim. Acta, 47, 1147 (1964). 26. D. Schumann, B.W. Bycroft, and H. Schmid, Experientia, 20_, 202 (1964). 27. W. Klyne, R.J. Swan , B.W. Bycroft, D. Schumann, and H. Schmid, Helv. Chim. Acta, 4jB, 443 (1965). 28. N. Camerman and J. Trotter, Acta Cryst., 1_7, 384 (1964). 29. J.P. Kutney, N. Abdurahman, P. Le Quesne, E. Piers, and I. Vlattas, J. Amer. Chem. Soc, 88, 3656 (1966). 30. J.P. Kutney, W.J. Cretney, P. Le Quesne, B. McKague, and E. Piers, J. Amer. Chem. Soc, 88_, 4756 (1966). 31. J. Mokry, I. Kompis, M. Shamma, and R.J. Shine, Chem. Ind. (London), 1988 (1964). 32. D. Schumann and H. Schmid, Helv. Chim. Acta, 46, 1996 (1963). 33. G. Stork and J.E. Dolfini, J. Amer. Chem. Soc, 85, 2872 (1963). 34. M. Plat, J. Le Men, M.M. Janot, J.M. Wilson, H. Budzikiewicz, 45 L.J. Durham, Y. Nagakawa, and C. Djerassi, Tetrahedron Letters, 271 (1962). 35. B. Pyuskyulev, I. Kompis, I. Ognyanov, and G. Spiteller, Coll. Czech. Chem. Comm.., 32_, 1289 (1967). 36. P.N. Edwards and G.F. Smith, J. Chem. Soc, 1458 (1961). 37. G.F. Smith and R.J. Wrobel, J. Chem. Soc, 792 (1960). 38. J. Mokry and I. Kompis, Lloydia, 27, 428 (1964). 39. J.P. Kutney, W.J. Cretney, J.R. Hadfield, E.S. Hall, and V.R. Nelson, submitted for publication. 40. J.P. Kutney and J.R. Hadfield, submitted for publication. 41. J.P. Kutney, K.K. Chan, A. F a i l l i , J.M. Fromson, C. Gletsos, and V.R. Nelson, J. Amer. Chem. Soc, 9£, 3891 (1968). 42. We are very grateful to Dr. I. Kompis, Institute of Chemistry, Bratislava, Czechoslovakia, for providing us with samples of vincadine, vincaminoreine, vincadifformine and minovine. 43. J. Mokry, L. Dubravokova, and P. Sefcovic, Experientia, 18, 564 (1962). 44. J. Trojanek, K. Kavkova, 0. Strouf, and Z. Cekan, Coll. Czech Chem. Comm., 24_, 2045 (1960). 45. J. Trojanek, J. Hoffmannova, 0. Strouf, and Z. Cekan, Coll. Czech. Chem. Comm., 24_, 526 (1959). 46. We are very grateful to Dr. M. Gorman, L i l l y Research Laboratories, for an authentic sample of this compound. PART II STUDIES RELATED TO SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS Introduction The study of the biosynthesis of the indole alkaloids has intrigued and interested workers in the f i e l d for many years. The early studies involving biosynthesis were based on natural compounds whose structures resembled proposed intermediates and the many chemical reactions which were believed to be of biogenetic significance. It was not until radio-active labelled precursors became readily available that the biogenetic hypothesis could be tested. The amino acid, tryptophan (1), has a structural similarity to the indole alkaloids, and has long been fe l t to be the precursor to the indole portion of the molecule. Radioactive labelled tryptophan has been shown by a variety of workers to be incorporated into serotonin (2), ajmaline (3), serpentine (4), reserpine 1 (5) and gramine (6), as well as vindoline 4 2 (7), ibogaine (8), catharanthine (9) and ajmalicine (10). In contrast to the general agreement by different workers with regard to the "tryptophan" portion of the indole alkaloids, the biogenetic origin of the "non-tryptophan", or C g_ 1 0 unit, has been the subject of considerable controversy. With regard to the latter, a number of theories have been proposed over the years. The earliest theory was proposed by several workers and was based 5 6 7 upon structural similarities of indole alkaloids. This Barger -Hahn ' -Robinson -^Woodward 9 h y p o t h e s i s involved dihydroxyphenylacetaldehyde (11) or an equivalent compound and two Cj units in the non-tryptamine MeOOC portion of the alkaloids. The formation of yohimbine (12) and strychnine (13) is illustrated in Figure 1. A number of deficiencies arose with the theory, and in 1959, Wenkert 11 12 and Bringi ' proposed an elegant alternative. They i n i t i a l l y proposed that a hydrated prephenic acid (14) was the crucial intermediate, 13 but later Wenkert modified his hypothesis so that prephenic acid i t s e l f was the direct progenitor of these alkaloids. Various rearrangements and condensations afford a crucial intermediate, the seco-prephenate-formaldehyde (SPF) unit (15), which can be incorporated into yohimbine (12) and corynantheine (16) (Figure 2). Sch l i t t l e r and Taylor in 1960 1 4 and Leete in 1961 1 5 - 1 7 postulated that the non-tryptophan portion of the indole alkaloids was derived via the acetate pathway. The suggestion was that the relevant precursor might be formed by condensation of an open chain six carbon acetate unit, a one carbon unit and a three carbon unit. However, this hypothesis was soon withdrawn. Another hypothesis based on structural relationships was proposed by 11-13 18 Wenkert ~ and Thomas . They suggested that the non-tryptophan moiety of the indole alkaloids was monoterpenoid in origin. Wenkert noted the structural identity of the carbon skeleton of the monoterpenes, verbenalin (17), gentiopicrin (18), bakankasin (19), swertiamarin (20), genipin (21) and aucubin (22) , with the seco-prephenate-formaldehyde unit (15). When Wenkert 1 3 proposed his monoterpenoid origin of the non-tryptamine portion of the complex indole alkaloids, he also postulated a biosynthetic scheme for alkaloids of the Aspidosperma and Iboga families (Figure 3). Condensation of the SPF unit (15) with tryptamine was envisaged as being followed by a retro-Michael reaction providing a cleavage product. If 50 B-condensation plus two Cj units Figure 1. Barger-Hahn-Robinson-Woodward hypothesis. COOH OH Figure 2 . Prephenic acid hypothesis f or the biosynthesis of Corynantheine a l k a l o i d s . 51 II 0 20 21 22 t h i s intermediate undergoes ordinary oxidation-reduction changes, p i p e r i -dines of various states of oxidation are formed whose intramolecular Michael and Mannich reactions lead to the Aspidosperma and Iboga-like 19 skeletons, r e s p e c t i v e l y . Wenkert has also used iminium s a l t i n t e r -mediates i n h i s proposed bio s y n t h e t i c pathway to the Akuamma (e.g., akuammicine (27)), Pleicocarpamine (e.g., pleiocarpamine (28)), and Hunteria (e.g., vincamine (29)) - l i k e bases. The i n i t i a l biosynthetic experiments, using radioactive precursors, 52 Aspidosperma-type Iboga-type alkaloids alkaloids Figure 3. Wenkert's proposal for the biosynthesis of Aspidosperma and Iboga alkaloids. 5 3 disproved rather than proved a l l the hypotheses concerning the biosynthesis of the Cg_io portion of the indole alkaloids^ It was not until 1965 that 2 0 Scott and coworkers were the f i r s t to report a successful incorporation of mevalonate into an alkaloid of the tryptamine + Cg_io type. Subsequent 2 1 - 2 4 publication by several groups of workers established that specifically labelled mevalonic acid was incorporated into indole alkaloids in a manner consistent with the monoterpenoid hypothesis. Not long after, the mono-2 5 - 2 8 terpenoid, geraniol (30),was found to be incorporated " as an intact unit into vindoline ( 7 ) , catharanthine ( 9 ) , and ajmalicine ( 1 0 ) in Vinca rosea L. plants (Figure 4 ) . Each of these alkaloids are representative of one of the three types of tryptamine + Cg_i 0 alkaloids. The f i r s t evidence for cyclopentane intermediates in the pathway was 29 obtained by Battersby and coworkers who showed that loganin ( 3 1 ) was incorporated into ajmalicine, vindoline and catharanthine by Vinca rosea L. plants (Figure 4 ) . Recently, loganin was again successfully incorporated 3 0 - 3 3 into the indole alkaloids of Vinca rosea L. plants and of Rauwolfia 34 serpentina plants. Degradation of the labelled alkaloids has led to the scheme shown in Figure 5. The structures represent the C^Q units of the Corynanthe, Iboga, and Aspidosperma groups of alkaloids which together account for the majority of the indole alkaloids. As an extension of his 35 loganin work, Battersby reported evidence from experiments in vivo which impose s t r i c t requirements on the mechanism of the formation of loganin and i t s conversion into the three classes of indole alkaloids. The results were obtained from the incorporation of various doubly labelled specimens of geraniol and of other substrates into young plants of V. rosea L. He concluded that: (a) the stereospecificity established for the formation 3 6 of the two geraniol double bonds in other biological systems also 54 OGlu MeOOC MeOOC" Figure 4. Incorporation of geraniol (30) and loganin (31) into Indole alkaloids. OH COOH V OH Iboga Corynanthe Aspidosperma Figure 5. Scheme for the rearrangement of the monoterpene unit. 55 holds good i n V. rosea L.; (b) i f saturation of the 2,3-double bond of geraniol i s a p r e r e q u i s i t e f o r the formation of loganin, then both the reduction process and the subsequent removal of the proton from C-2 must occur i n a s t e r e o s p e c i f i c fashion; (c) i n accord with the previous 13 suggestion , the configuration of loganin at C-7 i s determining for the stereochemistry of the corresponding centre i n ajmalicine (10), and by extension for a l l other Corynanthe and Strychnos compounds; and (d) the stereochemical c o r r e l a t i o n of C-2 o f loganin with the corresponding centre o f ajmalicine i s f o r t u i t o u s ; the observed loss of a proton from t h i s p o s i t i o n supports the idea of an enamine intermediate. 37 Battersby had proposed a reasonable pathway from mevalonate to the indole a l k a l o i d s of the tryptamine + Cg_io type. He was care f u l to emphasize "that several s i m i l a r schemes could be written i n which the sequence of operations i s a l t e r e d . For example, though at present i t i s a t t r a c t i v e to consider cleavage of the cyclopentane r i n g before the nitrogenous p o r t i o n of the molecule i s introduced, the evidence i s i n d i r e c t . P l a u s i b l e schemes reversing the order can be written." He also stated that "the conversion o f the corynantheine-strychnine Cg_io unit 37 occurs a f t e r introduction of the nitrogen." Battersby i n the same paper describes an a t t r a c t i v e mechanism for the fragmentation of the cyclopentane r i n g o f loganin. A hydroxyloganin with a phosphate residue as a good leaving group could generate the desired aldehyde (32). Recently, the monoerpenic glucoside, sweroside (33) was incorporated i n t o vindoline (7) i n V. rosea L. p l a n t s . Sweroside bears a s t r i k i n g resemblance to the hypothetical condensing unit (32), having the same stereochemistry at C-2 and C-7. The incorporation of sweroside into vindoline was more e f f i c i e n t than the incorporation of loganin into 56 32, R = Glu vindoline and this would tend to suggest that sweroside is further along the biosynthetic pathway than loganin. In this regard, there was good evidence that loganic acid (34) is a precursor of gentiopicroside (35) in 39 Swertia carboliniensis plants and sweroside is readily incorporated into 38 gentiopicroside in Gentiana scabra plants . A few alkaloids have been isolated which have the hypothetical condensing unit (32) occurring as an intact or nearly intact unit. One of these, ipecoside (36), has the 40 mondterpenic portion derived from loganin. Another alkaloid having a similar monoterpene unit i s the indole alkaloid, cordifoline (37). Recently, the tetrahydro-3-carboline monoterpenoid glycoside, stricto-42 sidine (38) was isolated from Rhazya supp., and was also shown to be present in V. rosea L . plants. The monoterpenoid portion of s t r i c -43 tosidine has been shown to be derived from loganin. Using the reasoning that sweroside (33) and some other monoterpenoid derivatives are masked lactol forms of the desired aldehyde (32), such an aldehyde, secocyclopentanoid monoterpene or secologanin (32), has been 44 synthesized . Menthiafolin (39) was converted in good yield to secologanin (32). Indeed, when secologanin (32) was condensed with 3,4-dihydroxyphenethylamine (40), the major product formed was identical with the natural product, ipecoside (36). The radioactive [0-methyl-3H]-secologanin was subsequently taken up by Vinca rosea L. shoots to yield 58 59 the following radioactive alkaloids: ajmalicine (10), vindoline (7), catharanthine (9), perivine (43) and serpentine (4). The main features are now known of the pathway from mevalonate through geraniol and loganin to secologanin which then serves as precursor of the non-tryptamine units present in the three large classes of indole 37 alkaloids . Attention was then focused on the process whereby the tryptamine unit is introduced for assembly of these families by reactions taking place with or without rearrangement. Secologanin (32) was reacted with tryptamine to generate the B-carbolines, vincoside (strictosidine (38)) 45 46 and isovincoside (42) ' . When vincoside (strictosidine) was fed to V. rosea L. shoots, a good incorporation into alkaloids representing a l l three classes of indole alkaloids was obtained. Also, in connection with this work, dilution analysis in V. rosea L. plants which had previously taken up [5-3H]loganin confirmed that secologanin (32), vincoside (strictosidine (38)) and isovincoside (42) are natural products of this plant. A summary of the pathway from acetate to indole alkaloids of tryptamine + Cg_io type is shown in Figure 6. The relatively high incorporation of a B-carboline system into unrearranged [ajmalicine (10)] and rearranged [vindoline (7) and catharanthine (9)] is of considerable interest. Conversion of vincoside (38) into the Corynanthe group [e.g., ajmalicine (10)] requires straight-forward steps. However, the rearrangement processes leading to the Aspidosperma [e.g., vindoline (7)] and Iboga [e.g., catharanthine (9)] systems must s t i l l be elucidated. 3 acetate mevalonate geraniol (30) ajmalicine (10) catharanthine (9) vindoline (7) Figure 6. Summary of the pathway from acetate to Indole alkaloids of tryptamine + Cg_io type. Discussion The Introduction has revealed that in recent years the biosynthesis of indole alkaloids has stimulated considerable interest in various labora-tories. Almost without exception these investigations have concentrated on the nature of the "non-tryptophan" unit necessary in the biosynthesis, and numerous elegant experiments are now in hand which establish the mono-terpene, loganin, as playing an important role in this regard. Our own interests in this area have been concerned with the later stages of the bio synthetic pathway, i.e., the steps involved after the tryptophan-CiQ "compl has been formed. Such questions as (a) the structure of this "complex(es)" and (b) the pathways which i t follows to elaborate the various families in the indole and dihydroindole series were of prime consideration. In Section A of this discussion, the syntheses of possible precursors the Aspidosperma and Vinca alkaloids are described, and in Section B, the biosynthetic studies are presented. Section A Of the various postulates which were available, the one proposed by 13 Wenkert and outlined in Figure 3 of the Introduction was of particular interest in our i n i t i a l considerations. We have already mentioned in Part I of this thesis that the transannular cyclizations, 23 -+ 24 and 25 -*• 26, as shown in Figure 3 of Part I I , have found some parallel in our own chemical studies. The possible significance of these cyclizations in alkaloid biosynthesis w i l l be discussed in later sections of this thesis. At this time, I would like to present some studies on the synthesis of possible precursors prior to the formation of the nine-membered ring intermediates, 23 and 25. Figure 3 illustrates the possible importance of intermediates such as 44 and 45 in this connection, and we were, therefore, prompted to consider the laboratory synthesis of these types of systems. The synthesis of the carbon skeleton present in 44 was facilitated by 47 the reported synthesis of the a,B-unsaturated ketone (51) . Although this synthesis was reported, the experimental procedures of some of the steps were not completely clear and f i r s t attempts to repeat the synthesis met with certain minor d i f f i c u l t i e s . However, these problems were rapidly eliminated and the experimental procedures followed in the present work are in accord with the sequence as shown in Figure 7. The ketalization of 3-acetylpyridine (48) proceeded to give a good yield (78%) of the ketal (49). Tryptophyl bromide (47) was obtained from tryptophol (46) in 80% yield and was used immediately (owing to ins t a b i l i t y of 47) for reaction with 49 to give the salt , 50, in 86% yield. Sodium borohydride reduction of 50, followed by acid hydrolysis, gave the a,B-unsaturated ketone, 51, in 50% yield after crystallization from methanol. A l l the data obtained for this substance 47 was in agreement with the published information A Michael reaction of this a,B-unsaturated ketone with some suitable OOC CHO Figure 7. Preparation of a,6-unsaturated ketone 51. 64 three carbon unit would then afford the desired carbon skeleton, 44. Since the exact oxidation state of the biological intermediate was not known, an attempt was made to synthesize various compounds with this carbon skeleton but having different oxidation states. An obvious choice for this purpose was methyl cyanoacetate, and therefore, a mixture of this reagent, the ketone, 51, and triethylamine was allowed to s t i r at room temperature for 5 days. After purification, a mixture of epimers possessing the functionality shown in 52 was isolated in good yield. No attempt was made to separate these epimers at this time because this was not necessary for the biosynthetic studies. The infrared spectrum exhibited carbonyl absorption at 1700 cm-1 for the methyl ketone, 1737 cm"1 for the methyl ester and weak n i t r i l e absorption at 2235 and 2195 cm"1. The mole-cular formula was established as C21H25N3O3 by high resolution mass spectro-metry (Found: 367.190; Calcd.: 367.190). The mass spectrum (Figure 8) showed a molecular ion peak at m/e 367, as well as two significant peaks at m/e 130 and 237 which are attributed to the simple fragmentation of the parent molecule to the ions, 54 and 55, respectively. The nmr spectrum established that two epimeric compounds had formed in about equal yield. The two strong signals at T 7.92 and 7.97 arise from the protons of the methyl ketone, whereas the other strong signals at T 6.24 and 6.28 are from the 65 protons of the methyl ester. The remainder o f the nmr spectrum corresponded to the desired s t r u c t u r e . A more d e t a i l e d presentation of the appropriate signals i s given i n the experimental section. The preparation of the malonic ester adduct (53), was f i r s t attempted using the same procedure as f o r 52 but only s t a r t i n g material, 51, was recovered. A s o l u t i o n of the anion of dimethyl malonate was then prepared i n dry tetrahydrofuran and the a,6-unsaturated ketone (51) was added to i t . A f t e r p u r i f i c a t i o n , a mixture of epimers of 53 was obtained i n good y i e l d . The i n f r a r e d spectrum exhibited broad carbonyl absorption <at 1745-1695 cm"1 a t t r i b u t e d to the methyl ketone and the two ester groups. The molecular formula was established as C22H28O5N2 by high r e s o l u t i o n mass spectrometry (Found: 400.198; Calcd.: 400.200). The mass spectrum (Figure 9) was domi-nated by intense peaks at m/e 130 and 270 which a r i s e from the simple fragmentation of the parent molecule to the ions, 54 and 56, res p e c t i v e l y . This fragmentation mode i s analogous to that observed f o r the coupling product, 52, mentioned e a r l i e r . Again, the nmr spectrum (Figure 10a) established that two epimeric compounds had formed i n about equal y i e l d . The methyl ester protons appeared as a serie s of s i n g l e t s i n the region T 6.34, corresponding to s i x protons, and the methyl ketone protons appeared at T 7.84 and 7.96. This epimeric mixture of malonic ester adducts (53) was s i m i l a r i n many respects to the mixture of cyanoacetate adducts (52). Thus, both series of compounds were unstable because of the ease of the retro-Michael reaction with the subsequent regeneration of the a, 6-unsaturated ketone (51) and dimethyl malonate or methyl cyanoacetate. This feature along with the fact that these compounds possessed s i m i l a r values on various t i c systems did not allow complete separation o f the pure components without extensive loss of material. However, a f t e r careful Figure 10. (a) P a r t i a l nmr spectrum of a mixture o f malonic ester adducts 53. (b) P a r t i a l nmr spectrum of the less p olar malonic ester adduct 53. (c) P a r t i a l nmr spectrum of the more polar malonic ester adduct 53. 68 preparative t i c separation using s i l i c a g e l , a p a r t i a l separation of the epimers of the malonic ester adducts, 53, was obtained. Now, a series of nmr si g n a l s could be assigned to each epimer. The methyl ester protons of the less p o l a r epimer appeared as three proton s i n g l e t s at T 6.31 and 6.36 while the methyl, ketone protons absorbed at T 7.96 (Figure 10b). The one proton doublet, J = 10 cps, at T 5.91 can be assigned to the proton on the carbon adjacent to the ester groups. The nmr spectrum (Figure 10c) of the more polar epimer possessed a s i m i l a r s e r i e s of signals with the methyl ester protons absorbing at T 6.31 and 6.34 and the methyl ketone protons at T 7.84. The proton on the carbon adjacent to the ester groups appeared as a doublet, J = 4 cps, centered at T 6.54. Having prepared the two d e r i v a t i v e s , 52 and 53, attention was directed towards the synthesis of yet another member i n t h i s s e r i e s . The aldehydo-est e r adduct, 58, would have the f u n c t i o n a l i t y most c l o s e l y r e l a t e d to the intermediates i n Wenkert's postulate. This compound could be formed from the condensation of the a,6-unsaturated ketone (51) with the anion derived from a-formyl methyl acetate (57). The preparation of t h i s anion (57) has been reported as the product of 48 the Cl a i s e n condensation between methyl formate and methyl acetate . It i s acknowledged that sodium formate and the sodium s a l t of acetoacetic 69 .... 49 •; methyl ester are by-products of t h i s reaction . However, the nmr spectrum ( i n D2O)' of the product obtained when the two esters were added to an ethereal suspension o f sodium methoxide showed three s i n g l e t s at T 1.49, 6.br-and '8.07. This was not consistent with rth'e desired product and was e x p l i c a b l e i n terms o f a mixture of sodium formate (T 1.49) and the salt of methyl acetoacetate (T 6.65 and 8.07), assuming exchange of the methylene protons of the l a t t e r compound. In view of the p o s s i b i l i t y that the above mixture may contain a small proportion o f 57numerous attempts were made to add i t to the a,8-unsaturated ketone, 51, using a large excess of the mixture of sodium s a l t s . The use of tetrahydrofuran, t r i e t h y l a m i n e , o r dimethylsulfoxide as solvents gave rise to no reaction and 51 could be r e - i s o l a t e d i n quan t i t a t i v e y i e l d from a l l reactions. In methanol, however, i t at f i r s t appeared that an adduct had been formed. This tentative conclusion was based on the i n f r a r e d spectrum, which showed several carbonyl absorptions at 1697 and 1665 cm"1. The properties of t h i s reaction mixture, however, were l a t e r e xplicable in terms of a mixture of the s t a r t i n g m a t e r i a l , 51 ( v m a x 1665 cm"1) and the product, 59 (v 1697 cm" 1). The l a t t e r substance i s merely the r e s u l t of the addition of methoxide ion to the s t a r t i n g ketone. The nmr spectrum was also consistent with t h i s observation i n that signals at x 7.40 (C-CH3) and 6.31 0 (OCH3) were evident, the formation of 59. The mass spectrum with a peak at m/e 300 supported These conclusions were confirmed in a reaction of 70 sodium methoxide with 51, under which conditions the same mixture was obtained. In view of the lack of success i n the synthesis of 58 by d i r e c t Michael addition of formyl a c e t i c ester to the a>3-unsaturated ketone, 51, the less d i r e c t route of addition o f cyanoacetate followed by reduction of the n i t r i l e group was considered. The following p i l o t route on model p i p e r i d i n e compounds was investigated. Accordingly, 3-acetylpyridine (48) was converted to the known k e t a l , 49, and t h i s compound was treated with methyl iodide i n ether to give the c r y s t a l l i n e s a l t , 60, i n 95% y i e l d . Sodium borohydride reduction of t h i s s a l t y i e l d e d the l i q u i d k e t a l , 61, which on hydrolysis provided N-methyl-3-acet y l - A 3 - p i p e r i d i n e (62) ... The formation of the a,8-unsaturated ketone i n the l a t t e r was confirmed by the strong carbonyl absorption at 1670 cm" i n the i n f r a r e d . The v i n y l proton i n the nmr'spectrum of t h i s compound occurred as a one proton t r i p l e t at unusually low f i e l d (T 3.10), whereas the three proton s i n g l e t s f o r the N-methyl and methyl ketone occurred in the expected region ( t 7.63-and t7.73). Conversion of 62 to the methyl 71 cyanoacetate adducti 63, was successful, but separation from methyl cyano-acetate proved d i f f i c u l t . The separation was f inal ly accomplished by conversion' to the picrate salt followed by regeneration of the base by f i l t r a t i o n through an Amberlite IRA-400 (HCO~) column 5 0 to afford a mixture of the starting material , 62, and the cyanoacetate adduct, 63. thei presence of the adduct, 63, was indicated in the infrared spectrum with characteristic n i t r i l e , ester and ketone absorptions at 2221, 1747, and 1709 c m - 1 , respectively. The nmr spectrum confirmed that the methyl cyanoacetate adduct and the o,0-unsaturated ketone, 62, were present in about equal amounts. The signals which can be assigned to the adduct were: x 6.17 (C00CH 3), 7.60 (N-CH3), 7.78 (C-CH 3) and 6.21 (NC-CH-COOCH3) in addition to the signals of the a,B-unsatufated ketone, 62. A l l attempts to further purify this mixture by column chromatography and preparative t i c always resulted in the formation of additional amounts of the a,B-unsaturated ketone, 62, with the corres-ponding loss of the methyl cyanoacetate adduct. This latter situation again reveals the fac i le retro-Michael reaction to which reference was already made in the compounds, 52 and 53. Attempted sodium borohydride reduction again resulted in the formation of the a,B-unsaturated ketone, 62. F ina l ly , attempts to form the ketal , 64, met with fai lure and further investigations of this approach were, therefore, abandoned. Since several compounds bearing the skeleton as shown in 44 were now available, we turned our attention towards the synthesis of carbon skeleton 45 having an appropriate three carbon side chain attached to the indole system. It w i l l be noted that this system would represent a different rearrangement of C\Q "non-tryptophan" unit and would be of interest to the biosynthetic postulated as outlined in Figure 3. The crucial step in our synthetic considerations for this series focus on the oxidative rearrangement 72 of a,6-disubstituted indole derivatives to their 6-substituted spiro relatives.'- The particular reaction which was i n i t i a l l y considered involved the rearrangement, 67> 68. The 'resulting imirib ether (68) would be expected to combine with :nucleophiies, and thereby, provide an entry into the desired system. Thus, reaction with a suitable three carbon unit would generate the desired carbon skeleton as represented by 69. The oxidative rearrangement of indoles to'spiro compounds is a well-known reaction and has found use in the alkaloid f i e l d . A number of examples of i t s application to alkaloids of particular interest to this study have appeared in the literature and deserve comment. The rearrangement reaction is normally preceded by the oxidative step using t-butyl hypochlorite. For example, the treatment of the chlbro derivative, 71, arising from t-butyl hypochlorite oxidation of the indole system (Figure 11) with aqueous methanol"at pH 6 resulted in rearrangement to give the spiro oxindole analogue, 73 5 1. However, treatment of the chloro derivative, 71, with methanolic a l k a l i gave the corresponding spiro imino ethers, 72, which, when •; V S2 subsequently refluxed with dilute acid gave the oxindole analogue, 73 * . The "imino ethers are known to react with several nucleophiies. However, their reactivity is s t i l l considerably less than that of the imino chlorides and only strong nucleophiies are expected to undergo smooth reactions with 70 73 aqueous methanol at pH 6 d i l u t e methanolic . a l k a l i acid 72 Figure 11. Oxidative rearrangement of Indole a l k a l o i d s . OMe 53 54 them . Ban has recently reported such a study, i n which he was able to convert the imino ether, 74, to the vinylogous amide, 75, using sodium hydride i n dimethylsulfoxide. •COMe ^ COMe OEt sodium hydride 74 75 The s t a r t i n g material f or the synthetic sequence directed towards the carbon skeleton, 69, was the known t e t r a c y c l i c ketone, 78. The synthesis 19 of t h i s t e t r a c y c l i c ketone presented no problems and i s outlined in 74 Figure 12. Tryptophyl bromide (47) was obtained from tryptophol (46) and used immediately (owing to i n s t a b i l i t y of 47) f o r reaction with 3-acetyl-p y r i d i n e (48) to give the s a l t , 76, i n 94%" y i e l d . C a t a l y t i c hydrogenation of 76 using palladium on charcoal i n ethanol gave the; A 2 - p i p e r i d i n e , 77. Figure 12. Preparation of t e t r a c y c l i c ketone 78. Without p u r i f i c a t i o n , the l a t t e r was exposed to Pictet-Spengler c y c l i z a t i o n . treatment of 77,with acid y i e l d e d two epimeric t e t r a c y c l i c ketones, 78. E q u i l i b r a t i o n using sodium methoxide i n methanol afforded the more stable t e t r a c y c l i c ketone i n 75% y i e l d from the s a l t , 76. Formation of the chloroindolenine (79) was accomplished using molar quantity of t-butyl hypochlorite i n carbon t e t r a c h l o r i d e at -12°. Attempted p u r i f i c a t i o n of t h i s chloro compound always resulted i n decomposition. However, the i n f r a r e d spectrum possessed indolenine absorption at 15S5 cm 1 and a normal ketone absorption at 1700 cm 75 Treatment of the chlofoindolenihe (79) with methanolic al k a l i affbrderl a complex mixture of compounds. The major compound was obtained in 40% yield 81 after a combination of column chromatography and preparative t i c purifica-tions. Crystallization of this product from methanol afforded colorless prisms, mp 2 0 5 - 2 0 8 ° , which analyzed for ^ 7 ^ 0 ^ 0 2 . The compound possessed a typical indolenine absorption in the ultraviolet region at 210, 253 and 280 my. In addition to the absorption band at 3122 cm-1 for a proton on the indolic nitrogen in the infrared spectrum,there appeared sharp bands at 1721 cm 1 for the methyl ketone and another carbonyl absorption at 1695 cm The significant features of the nmr spectrum were a one proton singlet at T 1.00, a four proton multiplet centred at x 2.94 due to the aromatic protons of the benzene ring and a three proton signlet at x 7.30 for the protons of the methyl ketone. Clearly, from the spectral data and analysis, this compound was not the desired imino ether (81). The data suggested that the 7 6 oxindole (80) had formed. The infrared spectrum showed the strong carbonyl absorption at 1695 cm"1, characteristic of oxindole, whereas this spectrum did not have the strong absorbance in the region of 1575 cm"1 which characterizes the imino ether system., The nmr evidence confirmed that an oxindole had formed. The one proton signal at T. 1.00 could be assigned to the proton of the indolic nitrogen and the absence of a strong three proton signal in the region T 6.0-6.5 confirmed that the methyl imino ether had not formed. An attempt was made to convert the oxindole, 80, to an imino ether using Meerweirfs reagent, triethyloxonium tetrafluoroborate. However, examination of the crude reaction product by t i c ( s i l i c a gel) showed that a complex mixture of compounds had formed and as a result purification was not attempted oh this mixture. It was now obvious that the tetracyclic ketone was capable of providing various side products, some of which could be the result of the reaction with the carbonyl group i t s e l f . Thus, the formation of a derivative of the ketone was considered. Conversion of the ketone to the ethylene ketal, 8 2 , was accomplished without d i f f i c u l t y and the spectral data confirmed its formation. The ethylene ketal protons produced a four proton signal in the nmr spectrum at x 6.07 and no carbonyl absorption was present in the infrared. The chloroindolenine, 83, was prepared and immediately reacted with potassium hydroxide in methanol. However, the major compound formed (45% yield) was not the desired imino ether but the oxindole, 84. The infrared spectrum was characteristic of an oxindole system with the NH absorption at 3100 cm-1 and ketone absorption at 1700 cm"1. Again, the nmr did not possess a sharp three proton signal in the region x 6.0-6.5 for the methyl protons of a methyl imino ether system. In the hope that yet another derivative of the ketone group would generate the imino ether system, the conversion to an alcohol was considered. Reduction of the ketone to the alcohol, 85, using sodium borohydride was 19 accomplished without d i f f i c u l t y . In addition to the elemental analysis, the molecular formula was established by high resolution mass spectrometry as C 1 7H 2N 20 (Found: 270.172; Calcd.: 270.173). The significant feature of the nmr spectrum was a three proton doublet (J = 6 cps) at T 8.74 due to the methyl protons on the carbon adjacent to the carbon bearing the alcohol group. In addition to the lack of any carbonyl absorption in the infrared spectrum, there appeared broad bands at 3130 and 3030 cm 1 for NH and Oil functions. The chloroindolenine (86) of the tetracyclic alcohol, 85, was formed as before using t-butyl hypochlorite. The ultraviolet spectrum of the crude product exhibited the expected absorption (X 253 and 290 (sh) m y ) , while r , - • r max no indole absorption could be seen. Without purification, this crude 78 product was immediately reacted with a methanolic a l k a l i s o l u t i o n at re f l u x temperature f o r two hours. The crude product was p u r i f i e d by column chromatography on alumina and f i n a l p u r i f i c a t i o n was accomplished by preparative t i c on s i l i c a gel to a f f o r d the c r y s t a l l i n e imino ether (87), mp 140-143°, i n 33% o v e r a l l y i e l d from the t e t r a c y c l i c alcohol. In addition to high r e s o l u t i o n mass spectrometry, the elemental analysis established the molecular formula, 021^8^02. COOMe 88, R = CHOHCH3 89, R = H The imino ether lent i t s e l f to a straightforward s t r u c t u r a l analysis due to the presence of c e r t a i n very c h a r a c t e r i s t i c spectroscopic features. The compound possessed indolenine absorption i n the u l t r a v i o l e t region (A 213 and 253-258 mp). In addition to the broad absorption band at v max 1 r 3300 cm"1 (OH) i n the i n f r a r e d spectrum, there appeared a strong band at 1575 cm"1 c h a r a c t e r i s t i c of the imino ether function. The nmr spectrum 79 (Figure 13) exhibited a four proton m u l t i p l e t centred at T 2.82 f o r the aromatic protons of the benzene r i n g and the protons of the methyl imino ether appeared as a three proton s i n g l e t at x 5.94. A three proton doublet at T 9.29 (J = 6 cps) was assigned to the methyl protons on the carbon adjacent to the alcohol group. The mass spectrum of the imino ether (Figure 15) provided further s t r u c t u r a l evidence. The molecular ion peak was the base peak at the desired value of m/e 300 and the spectrum was dominated by peaks at m/e 141 m/e 300 m/e 141 m/e 96 and 96. The fragment at m/e 141 arises from cleavage o f the indole system from the p i p e r i d i n e system, while loss o f the side chain from the l a t t e r would generate the species at m/e 96. Two other s i g n i f i c a n t peaks i n the mass spectrum at m/e 282 and 255 are the loss of water and side chain, r e s p e c t i v e l y , from the parent molecule. Now that the synthesis o f the imino ether, 87, was successful, we considered i t s reaction with appropriate nucleophiles. Thus, 87 was treated with methyl cyanoacetate and triethylamine at 60-65° i n a sealed tube for 100 hr. A f t e r removing the excess methyl cyanoacetate by d i s t i l l a t i o n at reduced pressure, examination o f the crude product showed that i t was a complex mixture of compounds. The major compound was i s o l a t e d with the aid of column chromatography on alumina and c r y s t a l l i z a t i o n from hexane afforded 80 c r y s t a l s , mp 185-188°. The i n i t i a l spectroscopic evidence, u l t r a v i o l e t and i n f r a r e d , suggested that the desired adduct, 88, had formed. The u l t r a v i o l e t spectrum was that of a t y p i c a l a-methylene indolenine with maxima at 234 and 334 mu and the minimum at 258 my. In addition to the absorption band at 3190 cm - 1 (NH) i n the i n f r a r e d spectrum, there appeared two strong absorptions at 2190 and 1680 cm"1 for the a,B-unsaturated n i t r i l e and ester groups, r e s p e c t i v e l y . However, the nmr spectrum (Figure 14) i n d i c a t e d t h i s was not the desired adduct, 88, but appeared to be a sub-stance which lacked the side chain on the p i p e r i d i n e r i n g (e.g., 89). Most importantly, there were no si g n a l s which could be a t t r i b u t e d to the protons o f the side chain. However, the remainder of the spectrum confirmed that the three carbon adduct r e s u l t i n g from condensation with methyl cyanoacetate had formed. Thus, the i n d o l i c nitrogen proton absorbed at T -1.03 and the methyl ester protons were observed as a three proton s i n g l e t at t 6.19. The loss of the side chain was indeed confirmed by the mass spectrum (Figure 16); the molecular ion exhibited a s i g n i f i c a n t peak at COOMe m/e 323 m/e 97 m/e 323, whereas the base peak was at m/e 97 f o r the p i p e r i d i n e fragment. This fragmentation pattern arose i n a manner analogous to that already described for the imino ether, 87, the l a t t e r having the corresponding fragments at m/e 141 and 96. The elemental analysis along with a high Figure 14. Nmr spectrum of 89. 83 resolution mass spectrum established the molecular formula, C j g l ^ i ^ C ^ , for the adduct, 89. Two plausible mechanisms can account for the loss of the side chain on the piperidine ring from the desired adduct, 88 (Figure 17). If the loss of this substituent occurs with concurrent opening of the cyclopentane ring, then the indole-enamine intermediate, 90, is generated. This same intermediate, 90, can also arise i f the ring opening of the cyclic system involves the nitrogen atom as shown in 88 -+ 91. The subsequent loss of the side chain i s explicable in terms of a retro-Aldol, i.e., 91 90. The enamine, 90, could readily rearrange to afford the iminium ion, 92, and the latter undergoes cyclization to provide the final product (89). The cyclization step, 92 •+ 89 has precedent from our own work, since a similar mechanism is involved in the transannular cyclization of nine-membered ring alkaloids to the corresponding Aspidosperma system, i.e., conversion of vincaminoreine to minovine (Part I of this thesis). In the hope of isolating the desired adduct, 88, before any loss of the side chain could occur, the sealed tube reaction was conducted in the same manner as before except the time of reaction was reduced. Unfortu-nately, this investigation was complicated by the fact that the reaction between methyl cyanoacetate and triethylamine afforded a thick oily mixture of compounds whose spectral characteristics were similar to those of the desired adduct. For example, when methyl cyanoacetate and triethylamine were allowed to react in a sealed tube at 60° for 50 hr, the infrared spectrum exhibited absorption bands corresponding to unsaturated n i t r i l e and ester functions, while the ultraviolet had absorption maxima at 265 and 335 my. The desired adduct also absorbs at 335 my in the ultraviolet region. In addition, this reaction of imino ether, methyl cyanoacetate and tri e t h y l -amine was extremely d i f f i c u l t to purify as the last traces of methyl 84 17. Mechanisms to account for the loss of the s i piperidine substituent. 85 cyanoacetate were d i f f i c u l t to remove. A number of reactions of different time duration were carried out using sufficient amounts of the imino ether, 87, so that the reaction products could be examined by nmr spectroscopy. However, after purification of the various reaction products, no trace of the desired adduct, 88, could be isolated. The only noticeable feature was the consistently lower yields of the adduct, 89, as the reaction time was reduced. Since the presence of the oxygen function on the piperidine system was causing numerous problems with the synthetic sequence, i t s removal became necessary. A number of different methods were studied in order to obtain the most efficient conversion. In one approach, the tetracyclic alcohol, 85, was converted to i t s tosylate and the latter was subsequently reduced with lithium aluminum hydride. The resulting product from this sequence was 19 the tetracyclic component bearing the ethyl side chain . Another method used was to convert the tetracyclic alcohol to i t s bromide using 48% aqueous 19 hydrogen bromide and subsequent hydrogenation of the bromide . Finally, a modified Wolff-Kishner reduction of the tetracyclic ketone, 78, was accom-plished. This latter procedure was by far the best reaction affording the tetracyclic compound, 93, in 70% yield. This substance corresponded in a l l 19 respects to that recorded in the literature The tetracyclic compound, 93, was treated with a molar amount of t-butyl hypochlorite to give the chloroindolenine, 94. The chloroindolenine was exceptionally stable and could be purified on preparative t i c plate ( s i l i c a gel) to afford a pure sample in 75% yield. When a methylene chloride solution of this chloro derivative was concentrated to dryness, a yellow amorphous solid was obtained, mp 95-100°, whose infrared spectrum exhibited the expected imine absorption at 1588 cm-1 and the ultraviolet spectrum was characteristic of an indolenine (A 225. 266 and 293 my). The nmr spectrum max ' K was significant in that the indolic nitrogen proton was absent while the remainder of the spectrum corresponded to the assigned structure. The mass spectrum of the chloroindolenine had small molecular ion peaks at m/e 288 and 290 in the approximate ratio 3:1 which is in accord with the relative abun-dance of chlorine. The base peak at m/e 253 corresponded to the fragment remaining after the loss of chlorine. As final evidence that the desired chloroindolenine, 94, had formed without any skeletal rearrangements, this compound was treated with lithium aluminum hydride in ether and the tetra-cyclic indole, 93, was obtained in quantitative yield. The chloroindolenine, 94, was treated, as in previous instances, with potassium hydroxide in methanol; however, no imino ether, 95, could be isolated even when the reaction times were extended. The only products obtained were unreacted 94 and the tetracyclic indole, 93. In order to 87 obtain a more drastic medium, a methanolic solution of sodium methoxide was added to a solution of the chloro derivative in methanol. A number of runs were conducted in order to obtain the optimum reflux time for the formation of the imino ether, 95. After 11 hr at reflux temperature, a complex mixture of compounds was obtained from which the desired imino ether, 95, was isolated as a mixture of isomers in 13% yield. These two isomers behaved identically on a l l chromatographic systems and the actual presence of isomers could only be determined by nmr spectroscopy. The characterization of the imino ether, 95, was carried out on this mixture of isomers. The infrared spectrum contained no NH absorption while the imino ether system showed strong absorption at 1568 cm-1. The ultraviolet spectrum was that of an indolenine system with the maxima at 215 and 254-258 mu. The s i g n i f i -cant feature of the nmr spectrum was the presence of a strong three proton signal at T 5.97 with a much smaller signal at T 5.92, both attributable to the methyl protons of the methyl imino ether function. As in the case of the other imino ethers prepared,a similar fragmentation pattern in the mass spectrum was evident here: a strong molecular ion at m/e 284 along with the corresponding piperidine fragments at m/e 125 and 96. The conversion of the imino ether, 95, using methyl cyanoacetate and triethylamine in a sealed tube was accomplished as before. After purification m/e 284 m/e 125 m/e 96 88 by column chromatography followed by preparative t i c on s i l i c a gel, the desired adduct, 96, was crystallized from methanol to afford colorless blocks, mp 165-168°. This adduct lent i t s e l f to a straightforward spectral analysis. Thus, high resolution mass spectrometry established the molecular formula, C 2 1 H 2 5 N 3 O 2 (Found: 351.195; Calcd.: 351.195). The infrared spectrum possessed the NH absorption at 3300 cm 1 along with unsaturated n i t r i l e and ester bands at 2210 and 1666 cm respectively. The ultraviolet spectrum was that of a normal a-methylene indolenine with maxima at 236, 295 and 335 mp. The nmr spectrum (Figure 18) showed a broad one proton singlet at T -1.08, the normal four proton multiplet centred at x 2.72 for the aromatic protons, a three proton singlet at T 6.18 for the methyl ester protons and a three proton tr i p l e t at x 9.53 for the methyl protons of the ethyl group. The mass spectrum (Figure 19) was in accord with the structure of the desired adduct having a molecular ion at m/e 351 and the piperidine fragments at m/e 125 and 96. COOMe m/e 351 m/e 125 m/e 96 Now that the desired adduct, 96, was available, attention was directed towards improving the yield of the last steps of the sequence. It was felt that a one-step conversion of the chloroindolenine to the adduct, 96, might be feasible. Thus, reaction of a mixture of the chloroindolenine, methyl cyanoacetate and triethylamine under sealed tube conditions was attempted and encouraging results were noted. A number of other conditions were employed in order to further investigate this reaction. In the hope that a 89 Figure 18. Nmr spectrum of 96. 06 91 p o l a r solvent would a s s i s t the condensation, dimethylsulfoxide was used with sodium hydride as the base to form the anion of the methyl cyanoacetate. However, no desired material could be detected. Another series of reactions involved the formation o f the sodium s a l t o f methyl cyanoacetate i n methyl cyanoacetate and reacting t h i s with the chloroindolenine. These reactions provided only small amounts of the adduct. From a l l of these experiments, the t e t r a c y c l i c indole, 93, was the major product ( y i e l d 50-80%) with varying small amounts of the chloroindolenine. The maximum y i e l d (8%) of the desired adduct was obtained by heating at 76° a s o l u t i o n of the chloroindolenine i n methyl cyanoacetate and triethylamine i n a sealed tube f o r 144 hr. Heating f o r a shorter period r e s u l t e d i n lower y i e l d , whereas heating f o r longer periods (454 hr) d i d not increase the y i e l d . Although t h i s l a s t step of the synthetic sequence proceeded i n low y i e l d , s u f f i c i e n t q u antities o f the adduct, 96, were prepared so that b i o s y n t h e t i c studies could be performed with i t . Some of the l a t t e r experiments are discussed i n the next section of t h i s t h e s i s . Section B As already mentioned, there are various postulates i n v o l v i n g the bio-13 genesis of Indole a l k a l o i d s , but the one proposed by Wenkert was of p a r t i c u l a r i n t e r e s t i n our i n i t i a l considerations, since i t r e l a t e s d i r e c t l y to previous synthetic work i n t h i s area. The transannular c y c l i z a t i o n r eaction developed by Kutney and coworkers, and mentioned i n Part I of t h i s t h e s i s , provides a general entry i n t o Iboga, Aspidosperma and Vinca a l k a l o i d s The fundamental s i m i l a r i t y of t h i s l a t t e r process to the l a t e r steps i n Wenkert's postulates (Figure 3, Part II of t h i s thesis) provided the stimulus f o r i t s evaluation as a b i o s y n t h e t i c a l l y s i g n i f i c a n t reaction. For t h i s purpose, the appropriate nine-membered rin g intermediates represented by quebrachamine (97) and vincaminoreine (99) were evaluated as 92 p o s s i b l e precursors o f the Aspidosperma and Vinca a l k a l o i d s , while the corresponding carbomethoxydihydrocleavamine (105) and carbomethoxycleavamine (106) d e r i v a t i v e s were studied f o r t h e i r p o s s i b l e r o l e i n the biosynthesis of the Iboga family. Numerous experiments were conducted i n Vinca rosea Linn and Vinca minor Linn plants, and a b r i e f resume of the r e s u l t s i s presented i n Tables I and I I . The preparation o f the t r i t i u m l a b e l l e d r adioactive precursor from the i n a c t i v e a l k a l o i d was accomplished by exchanging the aromatic protons of the indole system with r a d i o a c t i v e t r i t i u m atoms. The method developed i n these laboratories proved to be very successful f o r t h i s purpose. T r i t i u m l a b e l l e d t r i f l u o r o a c e t i c a c i d was used f o r t h i s a c i d catalyzed exchange of the aromatic protons and was prepared by reac t i n g molar qu a n t i t i e s of t r i f l u o r o a c e t i c anhydride and t r i t i u m l a b e l l e d water. A simple vacuum t r a n s f e r system was used to bring the t r i t i u m l a b e l l e d t r i f l u o r o a c e t i c acid into contact with the a l k a l o i d and i t was subsequently removed a f t e r reaction was complete. It was soon r e a l i z e d that t h i s method f o r the formation of rad i o a c t i v e a l k a l o i d s possessed some s i g n i f i c a n t features: (a) the al k a l o i d s were recovered v i r t u a l l y unchanged from the a c i d i c medium, (b) the method appears general to e s s e n t i a l l y a l l indole a l k a l o i d s , (c) since a large excess of acid was used, the d i l u t i o n of the r a d i o a c t i v i t y i n the reaction was very small and the recovered t r i t i u m l a b e l l e d t r i f l u o r o a c e t i c acid was s u i t a b l e f o r reuse, and (d) the experimental procedure was very simple i n i t s operation. The experimental method associated with the incorporation of large molecular weight compounds i n terms of permeability, etc., was appreciated, and the i n i t i a l experiments dealt with an evaluation of various techniques f o r the incorporation of such compounds. Table I i l l u s t r a t e s the r e s u l t s from the various methods of feeding. It soon became apparent that no H 1, R = COOH 41, R = H R' 97, R = R' = H 98, R = H; R' = COOMe 99, R = Me; R' = COOMe 100, R = H; R' = COOMe; 6,7-double bond R' 101, R = R' = H 102, R = H; R' = COOMe; 2,3-double bond 103, R = Me; R' = COOMe; 2,3-double bond 104, R = H; R' = COOMe; 2,3- and 6,7-double bonds 3 107 9, 3,4-double bond Table I . Results of incorporation of nine-membered r i n g intermediates i n t o V. rosea L. and V. minor L. by various techniques. Exp. No. Compound Fed Plant Feeding Method A l k a l o i d Isolated % Incor-poration [ar- 3H]-carbomethoxycleavamine V. rosea L. (106, HC1 s a l t ) [ar- 3H]-carbomethoxycleavamine V. rosea L. (106, HC1 s a l t ) [ar- 3H]-carbomethoxycleavamine V. rosea L. (106, acetate s a l t ) [ar- 3H]-carbomethoxycleavamine V. rosea L. (106, HC1 s a l t ) 188-carbomethoxy(1'*C)-4a- V. rosea L. dihydrocleavamine (105, HC1 s a l t ) 18a-carbomethoxy ( l l*C)-4a- V. rosea L. dihydrocleavamine (105, HC1 s a l t ) [ar- 3H]-quebrachamine (97) ^  V. minor L. [ar- 3H]-quebrachamine (97, V. minor L. HC1 s a l t ) 59 [ar- 3H]-vincaminoreine V. minor L. (99, acetate s a l t ) 59 Cotton wick into stems, 8 days Vacuum i n f i l t r a t i o n of l e a f d i s c s , 42 hours Leaf vein i n j e c t i o n , 6 days Hydroponic, cut stems, 46 hours Hydroponic, cut stems, 7 days Hydroponic, cut stems, 5 days Hydroponic, cut stems, Tween 20 emulsion Hydroponic, cut stems, 20 days Absorption through l e a f sections, 4 days Catharanthine (9) Catharanthine Catharanthine Catharanthine Coronaridine (107) Coronaridine Aspidospermidine (101) Aspidospermidine Vincamine (29) Aspidospermidine Minovine (103) <0.015 (0.04) e <0.008 <0.05 <0.011 <0.015 in a c t i v e 0.48 (3.4) a 0.08 (0.03f <0.001 <0.008 0.7 (0.3)' Table I. Results of incorporation of nine-membered r i n g intermediates into V. rosea L. and V. minor L. by-various techniques (continued). Exp. Compound Plant Feeding A l k a l o i d % Incor-No. Fed Method Isolated poration 10 [ar- 3H]-vincadine (98) 5 9 V. minor L. Absorption through Vincamine 0.08 (0.06) a l e a f sections, 4 days Aspidospermidine <0.003 Minovine i n a c t i v e Values i n parentheses r e f e r to blank experiments which were conducted under s i m i l a r conditions to those involved i n the plant feedings. U 3 96 particular technique showed any obvious advantage over the others. In experiments 1-4, carbomethoxycleavamine (106) was administered to V. rosea L. plants by a number of different methods and one of the major alkaloids, catharanthine (9), was isolated. Experiments 5 and 6 were conducted in order to compare the incorporations of the isomeric carbomethoxydihydrocleavamines into coronaridine (107) by V. rosea plants. Again the incorporations were not significant. The most frustrating aspect of these results was the in a b i l i t y to delineate what might be construed as a "positive" demonstration of the transannular cyclization process from the rather t r i v i a l oxygen-catalyzed conversion of the intermediates to the alkaloids during the period of incorporation. The conversion of these compounds to the appropriate alkaloids by oxygen in the presence of a metal catalyst has already been discussed in Part I of this thesis. It was hoped that a much higher level of incorporation in the plants relative to the blank experiment could be obtained. In an attempt to obtain internally consistent data which may shed more light on the cyclization reaction, our attention was turned to a series of experiments in which identical conditions were maintained throughout the entire series. For this purpose, 6-month old V. rosea L. plants were selected and the incorporation of the appropriate precursor was administered by the cotton wick technique into the stems of the plant. In each instance, the number of plants fed was sufficient to provide the direct isolation of the alkaloids which, without any further dilution with "cold" material could be crystallized to constant activity. Conversion of each of these into the corresponding hydrochloride salts confirmed the level of radioactivity. By this sequence, even very low levels of incorporation could be easily detected. The pertinent results for catharanthine (9), vindoline (7), and ajmalicine (10) are summarized in Table I I , and a brief analysis of these is appropriate. Table I I . Results of incorporation of various intermediates into V. rosea L. under identical conditions. Expt. Compound % Incorporation No. Fed Catharanthine Vindoline Ajmalicine 11 DL-tryptophan- [3-11+C] (1) 0.05 0 .15 0.8 12 [ar-3H]-tryptamine (41) 0.01 0 .003 0.4 13 [ar-3H]-100 <0.001 <0 .001 <0.001 14 [ar-3H]-tabersonine (104) 0.05 0 .03 <0.001 15 [ar-3H]-carbomethoxy-cleavamine (106) 0.03 <0 .001 inactive 16 [ar-3H]-carbomethoxy-cleavamine ( 1 0 6 ) — blank experiment 0.04 Experiments 11 and 12 il l u s t r a t e that (a) the age of the plants selected for this study was suitable for biosynthesis, and (b) the experimental method chosen at least provides positive incorporation of established precursors. Experiments 13 and 14 provide an important comparison between two closely related compounds in their role as potential precursors in the biosynthetic pathway. While the alkaloid, tabersonine (104), is converted into catharanthine and vindoline, the 6,7-dehydrovincadine derivative (100) is not incorporated. The latter compound is the immediate precursor of this alkaloid (104) in the laboratory conversion which ut i l i z e s the trans-annular cyclization reaction. The incorporation of tabersonine into these alkaloids furthermore establishes that the experimental method employed allows the incorporation of higher molecular weight "precursors" into the 98 plant system. The l e v e l of incorporation o f the cleavamine d e r i v a t i v e , 106, i n t o the Iboga system i s also n e g l i g i b l e , as shown i n experiments 15 and 16. A l l of these experiments 6 0 strongly suggest that the transannular c y c l i z a -t i o n reaction i s probably not s i g n i f i c a n t i n e i t h e r Aspidosperma or Iboga biosynthesis, although i t i s c l e a r that negative r e s u l t s must be interpreted with caution. In the hope of obtaining more d i s t i n c t l y p o s i t i v e r e s u l t s , a completely d i f f e r e n t approach to t h i s problem was studied. It i s c l e a r that the trans-annular c y c l i z a t i o n process as i l l u s t r a t e d i n the conversion of the a l k a l o i d , vincadine (98), to vincadifformine (102), a reaction e a s i l y accomplished i n the laboratory 6 1 , i s only one o f a number of a l t e r n a t i v e pathways i n the plant elaboration of Aspidosperma a l k a l o i d s . An equally a t t r a c t i v e and p l a u s i b l e scheme could invoke the reverse process, namely the r i n g opening o f the p e n t a c y c l i c system to y i e l d the nine-membered r i n g a l k a l o i d s ( i . e . , 102 -*• 98). Indeed, such a ring-opening process r e a d i l y occurs i n the 98, R = H 102, R = H 99, R = Me 103, R = Me laboratory as discussed i n Part I of t h i s t h e s i s . This l a t t e r process would imply that Aspidosperma a l k a l o i d s of type 102 are biosynthetic precursors of type 98. In order to obtain information on the r e l a t i o n s h i p , i f any, between these a l t e r n a t i v e s , a study was i n i t i a t e d i n Vinca minor L., 62 a plant which possesses a wonderful array of Aspidosperma alk a l o i d s 99 Table III. Results of incorporation of DL-tryptophan-3-I4C into Vinca minor L. at various time intervals. Total % Incorporation Time Vincadine (98) + vincaminoreine (99) (A) Vincadifformine (102) + minovine (103) (B) B/A 4 hr 0.003 0.057 19 1 day 0.015 0.24 16 2 days 0.010 0.21 21 4 days 0.010 0.22 22 7 days 0.009 0.13 14 14 days 0.003 0.06 20 A detailed investigation involving the incorporation of DL-tryptophan-3-LHC into V. minor L. over different time intervals was undertaken, and some of the results are summarized in Table III. The method involved incorporation of a solution of the amino acid in 0.1 N acetic acid containing a few drops of methanol,and after the appropriate time, the isolation of the alkaloids was carried out by chromatographic techniques. In each time interval reported, the experiment was repeated at least twice. There was surprisingly good agreement between the results obtained in the individual experiments, and Table III gives the average values obtained in these studies. For the purposes of this discussion, the total percent incorporation into the nine-membered ring alkaloids, vincadine (98) and vincaminoreine (99), and their respective cyclic relatives, vincadifformine (102) and minovine (103), is presented. The fourth column in Table III shows the relative ratio of activities between these two groups. A critical analysis of these results reveals several interesting 100 features. These are (a) activity in the alkaloids i s noted even after a short exposure of 4 hr, (b) the activity in the pentacyclic alkaloids (102 and 103) is consistently higher than in the nine-membered ring system, and (c) the relative ratio of activities (B/A) is remarkably similar over the time interval, 4 hr - 2 weeks. This latter finding is certainly the most important in terms of providing information about the later stages of Aspidosperma alkaloid biosynthesis. The lack of any tendency for the ratio B/A to progressively increase or decrease with time speaks strongly against any biosynthetic relationship between the two groups of alkaloids. In other words, the previous suggestion that the transannular cyclization is not biosynthetically important is now strongly supported by the above results. Furthermore, the ring-opening process (102 •+ 98) as mentioned above is similarly unimportant in providing a pathway to the nine-membered ring alkaloids found in V. minor L. It is clear that such an interpretation of the results is valid only i f an equilibrium in the plant between the two alkaloid groups can be excluded. The rather constant B/A could be obtained i f an equilibrium mixture were enriched with respect to the pentacyclic alkaloids, vincadifformine (102) and minovine (103), to the extent of approximately 20:1. An evaluation of this process was, thereby, in order. The incorporation of various nine-membered ring alkaloids into V. minor L. was studied, and some experiments in this direction have been already discussed. An approach from the opposite direction was also studied when the radioactive alkaloid, minovine (103), was incorporated into this plant (Feeding Experiment No. 17), In a typical experiment, during which radio-active 103 was administered over a one-week period, the subsequent inves-tigation of the isolated plant alkaloids showed essentially no activity in the nine-membered ring compounds (B/A >2500). On this basis, the above equilibration process is not significant in V. minor L. In turn, the 101 conclusion must be made that the genesis of the quebrachamine-vincadine family is independent of the pathway leading to the ri g i d pentacyclic 63 Aspidosperma series (102, 103, etc.) The question as to how the present results f i t into the biosynthetic pattern which is rapidly evolving from the combined data of other inves-19 tigations is worthy of comment. Wenkert suggested that " a l l indole alkaloids of the tryptamine + CIQ structure type may be derived from corynantheinoid or closely related progenitors". Based on elegant expe-37 riments in his and other laboratories, Battersby was able to postulate the possible role of structures such as 38 in the biosynthesis of corynan-theinoid as well as Aspidosperma and Iboga bases. From very recent reports 42 43 45 this latter postulate has been supported by the isolation ' ' of such a structure, strictosidine (vincoside) (38), and i t s incorporation into 45 46 the Aspidosperma and Iboga bases * . The possible intermediacy of units similar in structure to the alkaloid, stemmadenine (109), was involved in the 13 19 sequence between the corynantheinoid and Aspidosperma bases ' , and 64 again recent work on germinated Vinca rosea L. seeds supports i t s role in this regard. The conversion of the alkaloid, tabersonine (104), to vindoline and most interestingly to the Iboga alkaloid, catharanthine, has been demonstrated in V. rosea L. plants in our laboratory and also by 64 Scott in germinated seeds. This latter result suggests a possible relationship between the Aspidosperma and Iboga alkaloids. Scott in the same paper studied the technique of short-term germination of V. rosea L. seeds and found that the order of formation of identifiable alkaloids was Corynanthe, Aspidosperma and Iboga. In summation, a l l of these results strongly suggest, but do not prove, that the sequential formation of the indole alkaloids may follow the order: Corynanthe -> Aspidosperma •+ Iboga. 102 The r e s u l t s o f the present i n v e s t i g a t i o n r e l a t e to the possible s t r u c t u r a l units which bear on the Corynanthe -*• Aspidosperma pathway. An a t t r a c t i v e sequence (Figure 20) which i s i n accord with present findings may be postulated ( s t r i c t o s i d i n e (38) -»• 108 -»• 109 •* 110 -*• vincadine (98), vinca-difformine (102), catharanthine (9), e t c . ) . The intermediates and mechanisms 13 19 involved represent an important modification o f Wenkert's o r i g i n a l theory ' p a r t i c u l a r l y with regard to sequence, oxidation l e v e l and mechanism, without, however, d e t r a c t i n g from the e s s e n t i a l correctness of h i s views on the i n t e r r e l a t i o n s h i p s o f the main classes of indole a l k a l o i d s . The rearrangement of the double bond i n stemmadenine (109) and subsequent bond f i s s i o n w i l l provide the intermediate, 110. A s i m i l a r postulate f o r the formation of 110 64 has been advanced by Scott . This l a t t e r intermediate may then elaborate to the Aspidosperma and Iboga bases by the formal bond formations as indicated. The r e l a t i v e order o f the l a t t e r processes r e l a t e s d i r e c t l y to the r e s u l t s presented i n t h i s t h e s i s . From the tryptophan incorporations discussed above, i t i s a t t r a c t i v e to postulate that the independent biosynthetic pathways which lead to the vincadine and vincadifformine groups may i n i t i a t e from a common intermediate such as 110. The eventual elaboration of the a l k a l o i d systems depends merely on the r e l a t i v e order in which the bonds are formed. Thus, the process a -*• b converts 110 to vincadine, etc. The r e l a t i v e i n s i g n i f i c a n c e o f the transannular c y c l i z a t i o n process now suggests that the process c -*• d occurs p r i o r to or simultaneously with a •*• b i n the elaboration of 110 to vincadifformine. In s i m i l a r fashion, the conversion of unit 110 to the a l k a l o i d , catharanthine, i s u n l i k e l y to proceed i n i t i a l l y v i a the process a -»• e, since t h i s would lead to a carbomethoxycleavamine (106) system. Our r e s u l t s have shown that we were unable to demonstrate the conversion o f the l a t t e r to t h i s Iboga a l k a l o i d . Having investigated the transannular c y c l i z a t i o n reaction with respect 103 catharanthine (9), etc. Figure 20. Some later stages of Indole alkaloid biosynthesis. 104 to i t s r o l e i n indole a l k a l o i d biosynthesis, our attention was direc t e d towards studying other aspects o f the l a t e r stages of indole a l k a l o i d biosynthesis using the t o t a l l y synthetic substances discussed i n Section A as p o t e n t i a l precursors. The i n i t i a l studies using the s t r u c t u r a l skeleton represented by the cyanoacetic ester analogue, 52, and malonic ester intermediate, 53, were conducted so that another feature o f the biogenetic postulates of Wenkert (Figure 3) could be tested. Wenkert had postulated that s t r u c t u r a l l y r e l a t e d intermediates could be the precursors of a l l the indole a l k a l o i d s of the tryptamine + Cg_io type. Experiments 18-24 (Table IV) reveal b r i e f l y the r e s u l t s o f the i n v e s t i g a t i o n s which u t i l i z e the synthetic substances, 52 and 53, as p o t e n t i a l precursors. From experiments 23 and 24, 52, R = CN 53, R = COOMe i t can be noted that the cyanoacetic ester analogue, 52, was incorporated at a low l e v e l into ajmalicine, while the malonic ester intermediate, 53, was not u t i l i z e d . C l e a r l y , the former substance cannot be s e r i o u s l y consi-dered as a precursor, although the n i t r i l e function can probably be converted in t o an aldehyde or s i m i l a r grouping i n the plant. The other synthetic substance used as a p o t e n t i a l precursor i s repre-sented by the str u c t u r e , 96. Its p o s s i b l e relevance stems from a consideration of the previous b i o s y n t h e t i c r e s u l t s already discussed. These r e s u l t s showed that the transannular c y c l i z a t i o n process i s not b i o s y n t h e t i c a l l y important R COOMe 96 Table IV. Results of incorporation of synthetic intermediates into V. rosea L. and V. minor L. by various techniques. Exp. Compound No. Fed Plant Feeding Method A l k a l o i d Isolated % Incor-poration 18 [ar- 3H]-52 (HCl s a l t ) V. minor L. 19 [ar- 3H]-53 (HCl s a l t ) 20 [ar- 3H]-53 (HCl s a l t ) 21 [ar- 3H]-53 22 DL-Tryptophan-tS-^C] (1) 23 [ar- 3H]-52 (acetate s a l t ) 24 [ar- 3H]-53 (acetate s a l t ) V. minor L. V. rosea L. V. rosea L. V. rosea L. V. rosea L. V. rosea L. Hydroponic, cut stems, 5 days Hydroponic, cut stems, 7 days Dissolved i n 10% DMSO i n ethanol and painted on leaves, 5 days Dissolved i n 10% DMSO i n ethanol and painted on leaves, 6 days Dissolved i n 10% DMSO i n ethanol and painted on leaves, 9 days Cotton wick into stems, 9 days Vincamine (29) Quebrachamine (97) Vincadine (98) Minovine (103) Vincamine Quebrachamine Vincadine Minovine i n a c t i v e i n a c t i v e i n a c t i v e i n a c t i v e i n a c t i v e i n a c t i v e i n a c t i v e i n a c t i v e Catharanthine (9) i n a c t i v e Catharanthine Ajmalicine (10) Vindoline (7) Catharanthine Ajmalicine Vindoline Vindoline Catharanthine Ajmalicine Cotton wick into stems, Vindoline 9 days Catharanthine Ajmalicine i n a c t i v e i n a c t i v e i n a c t i v e 0.009 0.003 0.005 <0.001 <0.001 0.004 <0.001 <0.001 <0.001 o on Table IV. Results of incorporation of synthetic intermediates into V. rosea L. and V. minor L. by various techniques (continued). Exp. Compound No. Fed Plant Feeding Method Alkaloid Isolated % Incor-poration 25 [ar-3H]-96 (acetate salt) 26 [ar-3H]-96 (acetate salt) V. minor L. Hydroponic, cut stems, Vincamine inactive 2 days Minovine inactive V. rosea L. Cotton wick into stems, Vindoline inactive 9 days Catharanthine inactive Ajmalicine inactive 107 i n e i t h e r Aspidosperma or Iboga biosynthesis. Furthermore, the r i n g -opening process i s s i m i l a r l y unimportant i n providing a pathway to the nine-membered r i n g a l k a l o i d s found i n V. minor L. Thus, the conclusion must be made that the genesis of the quebrachamine-vincadine family i s independent o f the pathway leading to the r i g i d p e n t a c y c l i c Aspidosperma and Iboga s e r i e s . From t h i s r e s u l t , i t can be postulated that these independent pathways may i n i t i a t e from a common intermediate such as 110 (Figure 20). The eventual elaboration o f the a l k a l o i d systems depends merely on the r e l a t i v e order i n which the bonds are formed. The r e l a t i v e i n s i g n i f i c a n c e of the transannular c y c l i z a t i o n process suggests that the process c •*• d occurs p r i o r to or simultaneously with a + b i n the conversion of 110 to the Aspidosperma system. In order to t e s t t h i s theory, a study was i n i t i a t e d i n which t o t a l l y synthetic substances, representing an intermediate having the c d bond formed i n 110, could be evaluated as p o t e n t i a l precursors. Experiments 25 and 26 (Table IV) using V. minor L. and V. rosea L. pla n t s , r e s p e c t i v e l y , reveal b r i e f l y the r e s u l t s o f some of our i n i t i a l i n v e s t i g a t i o n s using the synthetic precursor, 96. This synthetic precursor has the c -*• d bond preformed i n 110 and may be o f i n t e r e s t as a precursor o f the Aspidosperma family of a l k a l o i d s , even though i t s f u n c t i o n a l i t y i s not completely appropriate. Although our i n i t i a l r e s u l t s are negative with respect to incorporation o f t h i s s t r u c t u r a l system, the conclusion cannot be made that intermediates possessing the s k e l e t a l features inherent i n 96 are not on the biosynthetic pathway. C l e a r l y , the struct u r e , 96, can be modified so that a more p l a u s i b l e precursor of the same carbon skeleton can be obtained. Such a modification would be to introduce a degree of unsaturation into the p i p e r i d i n e r i n g and conversion of the n i t r i l e function i n t o an aldehyde or an o l e f i n . Some of these inv e s t i g a t i o n s are now underway i n our laboratory. Experimental Melting points were determined on a Kofler block and are uncorrected. The u l t r a v i o l e t (uv) spectra were recorded i n methanol on a Cary 11 recording spectrometer, and the i n f r a r e d ( i r ) spectra were taken on Perkin-Elmer Model 21 and Model 137 spectrometers. Nuclear magnetic resonance (nmr) spectra were recorded i n deuteriochloroform at 100 megacycles per second (unless otherwise indicated) on a Varian HA-100 instrument and the chemical s h i f t s are given i n the T i e r s T scale with reference to t e t r a -methylsilane as the i n t e r n a l standard. Mass spectra were recorded on an Atlas CH-4 mass spectrometer and high r e s o l u t i o n molecular weight deter-minations were determined on an AE-MS-9 mass spectrometer. Analyses were c a r r i e d out by Mr. P. Borda o f the mi c r o a n a l y t i c a l laboratory, the Uni v e r s i t y o f B r i t i s h Columbia. Woelm neutral alumina and S i l i c a Gel G (acc. to Stahl) containing 2% by weight of General E l e c t r i c Retma p-1, Type 188-2-7 e l e c t r o n i c phosphor were used f o r a n a l y t i c a l and preparative t h i n - l a y e r chromatography ( t i c ) . Chromatoplates were developed using the spray reagents, carbon tetrachloride-antimony pentachloride 2:1 or 35% s u l f u r i c acid saturated with eerie s u l f a t e . Woelm neutral alumina ( a c t i v i t y III) was.used f o r column chromatography (unless otherwise indicated). R a d i o a c t i v i t y was measured with a Nuclear-Chicago Mark 1 Model 6860 Liqu i d S c i n t i a l l a t i o n counter i n counts per minute (cpm). The r a d i o a c t i v i t y of a sample i n d i s i n t e g r a t i o n s per minute (dpm) was calculated using the counting e f f i c i e n c y which was determined f o r each sample by the external 65 standard technique u t i l i z i n g the b u i l t - i n barium-133 gamma source. 109 The r a d i o a c t i v i t y of the samples was determined using a s c i n t i l l a t o r s o l u t i o n made up of the following composition: toluene (1 l i t r e ) , 2,5-diphenyloxazole (4 g) and 1,4-bis[2-(5-phenyloxazolyl)]benzene (0.05 g). In p r a c t i c e , a sample o f an a l k a l o i d as the free base was dissolved i n benzene (1 ml) i n a counting-vial,or i n the case of the s a l t of an a l k a l o i d , the sample was dissolved i n methanol (1 ml) i n a counting-vial,and then i n both cases, the volume was made up to 15 ml with the above s c i n t i l l a t o r s o l u t i o n . For each sample counted, the background was determined f o r the counting-vial to be used by f i l l i n g the v i a l with one o f the above s c i n t i l l a t o r solutions and counting (3 x 100 min) to determine the background cpm. The counting-vial was emptied, r e f i l l e d with the sample to be counted and the s c i n t i l l a t o r s o l u t i o n and counted (3 x 200 min). The dif f e r e n c e i n cpm between the background count and the sample count was used f o r subsequent c a l c u l a t i o n s . Tryptophyl bromide (47) A s o l u t i o n of phosphorus tribromide (0.56 ml) i n ether (10 ml) was added (46, 2.8 g) dropwise to an i c e - c o l d s o l u t i o n o f tryptophol i n ether (140 ml). A f t e r 15 hr, the supernatant was decanted, washed with sodium bicarbonate s o l u t i o n (2 x 100 ml), water (100 ml) and dried over sodium s u l f a t e . Removal of the solvent y i e l d e d the product as white c r y s t a l s (3.1 g), mp 101-102° ( L i t 4 7 mp 90-95°). 3-Acetylpyridine ethylene keta l (49) ^ A s o l u t i o n of 3-acetylpyridine (48, 60 g), ethylene g l y c o l (40.5 g) and p-toluene s u l f o n i c acid hydrate (105 g) i n benzene (250 ml) was heated under r e f l u x f o r 17 hr with a Dean-Stark apparatus to remove water. The mixture was poured into excess aqueous sodium bicarbonate s o l u t i o n , the layers separated and the aqueous phase extracted with benzene (3 x 150 ml). 110 The combined extracts were washed with sodium bicarbonate s o l u t i o n (2 x 150 ml), water (150 ml) drie d (sodium s u l f a t e ) and evaporated. D i s t i l l a t i o n gave the product (63.3 g): bp 165°/88 mm; nmr'. T 2.5 (multiplet, 4H, aromatic), 6.1 (multiplet, 4H, ketal) and 8.35 ( s i n g l e t , 3H, C-CH 3). N-[8-(3-Indolyl)ethyl]-3-acetylpyridinium ethylene ketal bromide (50) Tryptophyl bromide (47, 3.1 g) and 3-acetylpyridine ethylene ketal (49, 6.3 ml) were heated at 80° under nitrogen f o r 8 hr. Addition of ether (20 ml) to the cooled reaction mixture y i e l d e d a p r e c i p i t a t e whose c r y s t a l l i z a t i o n from methanol afforded pure 50 (3.9 g), mp 210-211° ( L i t 4 7 mp 209-210°). N-[B-(3-Indolyl)ethyl]-3-acetyl-A 3-piperideine (51) A suspension of sodium borohydride (10 g) i n methanol (150 ml) was added to a s o l u t i o n of the s a l t (50, 3.3 g) i n methanol (50 ml). Aft e r 2 hr at room temperature, the s o l u t i o n was evaporated almost to dryness and water was added (400 ml). The s o l u t i o n was a c i d i f i e d with hydrochloric acid to pH 2, and s t i r r e d at room temperature for 24 hr. The so l u t i o n was n e u t r a l i z e d with sodium bicarbonate and extracted with methylene chlo r i d e (3 x 100 ml). The combined extracts were washed with water (100 ml), drie d over sodium s u l f a t e , and evaporated to give an orange o i l (2.3 g) which was c r y s t a l l i z e d from methanol to a f f o r d c o l o r l e s s c r y s t a l s of 51 (1.1 g): mp 185-190°; v (CHC13): 3465 (NH), 1662 (C=0) cm - 1; Amax ( l 0 g E ) : 2 2 2 ( 4 ' 4 8 ) » 2 7 5 ( 3- 6°)» a n d 2 8 3 C3-63) M U ; N M R : T 1.9 ( s i n g l e t , IH, NH), 2.8 (multiplet, 6H, aromatic and ol e f i n e ) and 7.72 ( s i n g l e t , 3H, CO-CH3) ( L i t 4 7 mp 185-190°). N-[8-(3-Indolyl)ethyl]-3-acetyl-4-(2-methyl cyanoacetate)-piperidine (52) a ,8-Unsaturated ketone (51, 0.10 g) was added to a s o l u t i o n of methyl I l l cyanoacetate (0.185 g), methanol (0.03 ml) and triethylamine (0.01 ml), and the mixture was s t i r r e d at room temperature, under an atmosphere of nitrogen f o r 5 days. As much as poss i b l e of the solvent was removed under vacuum and the residue chromatographed on alumina (7 g). E l u t i o n with benzene afforded a mixture o f two epimeric methyl cyanoacetate adducts and a small amount of the s t a r t i n g m a t e r i a l . F i n a l p u r i f i c a t i o n by preparative t i c ( s i l i c a g e l , ethyl acetate) afforded an inseparable mixture of two epimeric methyl cyanoacetate adducts, 52 (0.090 g): A 223, 283, 290 mu; v (CHC13): 3455 (N-H), 2235 and 2195 (C-N), 1737 (C00CH 3), 1700 (C=0) cm"1; nmr: T 1.96 ( s i n g l e t , IH, N-H), 2.74 (mul t i p l e t , 5H, aromatic), 5.74 (mu l t i p l e t , IH, NC-CH-COOCH3), 6.24 and 6.28 ( s i n g l e t s , 3H, C00CH 3), 7.92 and 7.97 ( s i n g l e t s , 3H, C0-CH 3); mass spectrum ( i n t e n s i t y ) : m/e 367 (5), 237 (35), 130 (55). Anal. Calcd. f o r C 2 i H 2 5 N 3 0 3 : M.W. 367.190. Found: 367.190 (mass spectrometry). N-[B-(3-Indolyl)ethyl]-3-acetyl-4-(2-dimethyl malonate)-piperidine (53) Sodium was cut i n t o f i n e pieces (0.035 g) and suspended i n dry tetrahydrofuran (10 ml, f r e s h l y d i s t i l l e d from sodium). Dimethyl malonate (0.23 g) was added and the mixture heated at r e f l u x for 2 hr,when a l l the sodium had dissolved g i v i n g a cloudy s o l u t i o n , a,B-Unsaturated ketone (51, 0.055 g) was added with s t i r r i n g under dry nitrogen. A f t e r s t i r r i n g for 3 hr at r e f l u x , the s o l u t i o n was cooled and poured into d i l u t e ammonium hydroxide s o l u t i o n . The aqueous s o l u t i o n was extracted with methylene ch l o r i d e (3 x 25 ml); the combined organic extracts were dried (sodium s u l f a t e ) and evaporated to y i e l d an o i l (0.056 g). P u r i f i c a t i o n by preparative t i c ( s i l i c a g e l , e t h y l acetate) afforded a mixture o f the two epimeric dimethyl malonate adducts, 53 (0.034 g): A 223, 283, 292 mu; Tn 3 . X v (CHCld): 3455 (N-H), 1745-1695 (broad, C=0 and COOCH,) cm"1; mass max 3 . 112 spectrum ( i n t e n s i t y ) : m/e 400 (8), 270 (100), 130 (55). Anal. Calcd. f o r C 2 2 H 2 8 ° 5 N 2 : M - w . 400.200. Found: 400.198 (mass spectrometry). Using the above t i c system, a p a r t i a l separation o f the two epimers was obtained. The nmr o f the less p olar adduct was: T 1.90 ( s i n g l e t , IH, N-H), 2.70 (multiplet, 5H, aromatic), 5.91 (doublet, J = 10 cps, IH, CH3OOC-CH-COOCH3), 6.31 and 6.36 ( s i n g l e t s , 6H, COOCH3), 7.96 (C0-CH3) and the nmr of the more polar adduct was: x 2.03 ( s i n g l e t , IH, N-H), 2.70 (multiplet, 5H, aromatic), 6.31 and 6.34 ( s i n g l e t s , 6H, C00CH 3), 6.54 (doublet, J = 4 cps, IH, CH3OOC-CH-COOCH3), 7.84 ( s i n g l e t , 3H, CO-CH3). Reaction o f a,B-unsaturated ketone (51) with sodium methoxide i n methanol A s o l u t i o n of the a,B-unsaturated ketone (51, 0.023 g) i n methanol (5 ml) was added to a s o l u t i o n o f sodium methoxide (0.400 g) i n methanol (10 ml). A f t e r heating at r e f l u x f o r 2 hr, the cooled s o l u t i o n was poured in t o saturated sodium bicarbonate s o l u t i o n (60 ml) and the basic components extracted with methylene chloride (3 x 10 ml). The combined extracts were washed with water (15 ml), dri e d over sodium s u l f a t e and evaporated to give a white s o l i d (0.024 g): mp 175-178°, v CHC1 3): 1697 and 1665 cm"1; X : 222, 286, 291 my; nmr (DMS0-d6): T 6.31 (0CH 3) and x 7.40 (CO-CH3); fflclX mass spectrum: highest m/e was 300 and the remainder of the spectrum was consistent with a mixture of s t a r t i n g m a t e r i a l , 51, and the methoxy adduct (59). l-Methyl-3-acetylpyridinium iodide ethylene ketal (60) A s o l u t i o n of methyl iodide (20 g) i n ether (50 ml) was added to a s t i r r e d i c e - c o l d s o l u t i o n of 3-acetylpyridine ethylene ketal (49, 20 g) in ether (50 ml). The reaction mixture was l e f t at room temperature overnight and the p r e c i p i t a t e d s a l t , 60, was f i l t e r e d (36.8 g) and p u r i f i e d by r e c r y s t a l l i z a t i o n s from methanol, mp 190°. l-Methyl-3-acetyl-A 3-piperideine ethylene keta l (61) A s o l u t i o n of the s a l t (60, 1.0 g) i n methanol (50 ml) was treated with 113 sodium borohydride (5 g) i n methanol (75 ml). A f t e r 2 hr, the s o l u t i o n was evaporated to dryness and water was added (200 ml). The s o l u t i o n was n e u t r a l i z e d with 2N hydrochloric acid and then b a s i f i e d with sodium carbonate p r i o r to ex t r a c t i o n with chloroform (4 x 50 ml). The combined extracts were washed with water (2 x 50 ml), drie d (sodium sulfate) and evaporated to give the product, 61, as a pale yellow l i q u i d (0.373 g); nmr: T 4.18 (multiplet, IH, v i n y l ) , 6.18 (m u l t i p l e t , 4H, k e t a l ) , 7.68 ( s i n g l e t , 3H, N-CH 3), 8.60 ( s i n g l e t , 3H, C-CH 3). 1-Methy1-3-acetyl-A 3-piperideine (62) The keta l (61, 0.370 g) was disso l v e d i n 2N hydrochloric acid (10 ml) and s t i r r e d at room temperature under nitrogen f o r 18 hr. The reaction mixture was b a s i f i e d with sodium bicarbonate and extracted with chloroform (4 x 10 ml). The combined extracts were washed with water (2 x 10 ml), dr i e d (sodium sulfate) and evaporated to give 62 as a pale yellow l i q u i d (0.230 g): v ( f i l m ) : 1670 (C=0) cm"1; nmr: T 3.10 ( t r i p l e t , IH, C-4-H), IDcLX 7.63 ( s i n g l e t , 3H, N-CH3) and 7.73 ( s i n g l e t , 3H, C-CH 3). N-Methyl-3-acetyl-4-(2-methyl cyanoacetate)-piperidine (63) A s o l u t i o n of l-methyl-3-acetyl-A 3-piperideine (62, 0.20 g), methyl cyanoacetate (0.4 ml), triethylamine (3.0 ml) i n methanol (10 ml) was heated under r e f l u x f o r 5 hr. The reaction mixture was cooled and evapo-rated. Saturated sodium bicarbonate s o l u t i o n (10 ml) was added and extracted with chloroform (4 x 10 ml). The combined extracts were washed with water (15 ml), drie d over sodium s u l f a t e , and evaporated to give an orange l i q u i d . This was dissolved i n methanol (1 ml) and a saturated s o l u t i o n of p i c r i c a c i d i n methanol (2.5 ml) was added. A f t e r an orange o i l separated out, the supernatant l i q u i d was decanted o f f . The o i l was washed with an a d d i t i o n a l volume of methanol (0.5 ml), and dissolved i n acetone-water 114 9:1 (30 ml). An Amberlite IRA-400 HC0 3 " column (1 x 15 cm) was prepared by washing through with acetone-water 9:1 (30 ml) and the above s o l u t i o n was then f i l t e r e d through the column, which was subsequently washed with a further 30 ml portion o f acetone-water 9:1. The combined eluate and washings were evaporated, saturated sodium bicarbonate s o l u t i o n (10 ml) was added and the mixture extracted with chloroform (3 x 10 ml). The combined extracts were washed with water (10 ml), dried over sodium s u l f a t e and evaporated to give a mixture o f s t a r t i n g material (62) and the desired methyl cyanoacetate adduct (63) as a yellow o i l (0.090 g): v (CHCI3): 2221 (CN), 1747 (COOCH3), 1709 (saturated ketone), 1664 (unsaturated ketone) cm - 1; nmr: (methyl cyanoacetate adduct 63) T 6.17 (COOCH3), 6.21 (CH-CN), 7.60 (N-CH 3), 7.78 (C-CH 3) and ( s t a r t i n g material 62) T 3.10 ( d e f i n e proton), 7.60 (N-CH 3), 7.73 (C-CH 3). N-[8-(3-Indolyl)ethyl]-3-acetylpyridinium bromide (76) Tryptophyl bromide (47, 3.98 g) and 3-acetylpyridine (48, 6.5 ml) was heated at 70° f o r 12 hr and then at 100° f o r 4 hr under a nitrogen atmosphere. The c r y s t a l l i n e mass was t r i t u r a t e d with an ethanol-ether 2:1 s o l u t i o n (10 ml) and the crude product r e c r y s t a l l i z e d from 60% aqueous methanol, giving N-[8-3-indolyl)ethyl]-3-acetylpyridinium bromide (76, 5.8 g), mp 214-217° dec ( L i t 1 9 mp 214-216° dec). N-[B-(3-Indolyl)ethyl]-A 2-3-acetylpiperideine (77) 1 9 A suspension of pyridinium s a l t (76, 5.8 g) and 10% palladium-charcoal (1.2 g) i n ethanol (200 ml) and triethylamine (12.9 ml) was hydrogenated at room temperature and atmospheric pressure; the uptake ceased a f t e r 5 hr (hydrogen uptake 950 ml). F i l t r a t i o n and concentration of the f i l t r a t e under reduced pressure afforded a syrup that was dissolved i n chloroform (50 ml). A f t e r washing with water (2 x 50 ml), the chloroform was dried 115 over potassium carbonate and concentrated under reduced pressure to give the impure tetrahydro ketone as a syrup: v (CHC1 3): 3320 (NH), 1620 TD SIX and 1561 (vinylogous amide) cm - 1. The crude tetrahydro ketone, 77, was immediately used f o r the next reacti o n . T e t r a c y c l i c ketone (78) Impure tetrahydro ketone (77) was treated with IN hydrochloric acid (350 ml) under an atmosphere o f nitrogen on a steam bath f o r 2 hr. A f t e r cooling to 0°, the aqueous phase was b a s i f i e d with potassium carbonate and extracted with chloroform (4 x 50 ml). The combined chloroform extracts were d r i e d over potassium carbonate and concentrated to y i e l d a syrup which was chromatographed on alumina (150 g). The product was eluted with benzene and subsequently c r y s t a l l i z e d from methanol to a f f o r d meedles of the t e t r a c y c l i c ketone, 78 (2.24 g), mp 165-170°. The mother liquors were subjected to isomerizing conditions (2 hr r e f l u x i n 25 ml o f 0.07 M sodium methoxide i n methanol). The product was c r y s t a l l i z e d from methanol to give needles (1.51 g) , mp 163-170°. The combined material was r e c r y s t a l l i z e d from methanol to give pure t e t r a c y c l i c ketone, 78: mp 174-177°; X (log e): max 222 (4.54), 286 (3.89), 291 (3.78) my; v K B r : 3320 (NH), 1683 (C=0) cm"1; in six nmr: x 2.21 ( s i n g l e t , IH, NH), 2.78 (multiplet, 4H, aromatic), 7.77 ( s i n g l e t , 3H, C-CH3) ( L i t 1 9 mp 171-173°). Chloroindolenine (79) of t e t r a c y c l i c ketone t-Butyl hypochlorite (0.0845 g, 0.79 mmole) i n carbon t e t r a c h l o r i d e (15.7 ml) was added dropwise over 30 min to a s t i r r e d s o l u t i o n o f 78 (0.20 g, 0.79 mmole) i n anhydrous methylene chloride (10 ml) containing one drop triethylamine, cooled i n an i c e - s a l t mixture. A f t e r the addition was completed, s t i r r i n g was continued f o r 30 min. The reaction mixture was washed with water (15 ml), dri e d (sodium sulfate) and evaporated to give the crude chloro d e r i v a t i v e , 79, as a yellow viscous o i l (0.254 g): 116 X : 234, 297 my; v (CHC1 3): 1700, 1585 cm'1. The crude product was used d i r e c t l y f o r the next re a c t i o n . Oxindole (80) A s o l u t i o n of the above impure chloroindolenine (79) and potassium hydroxide (0.06 g) i n methanol (12.5 ml) was refluxed f o r 2 hr under an atmosphere o f nitrogen. A h a l f of the solvent was evaporated o f f and the mixture was d i s t r i b u t e d between water and methylene c h l o r i d e . The organic layer was separated and drie d over sodium s u l f a t e . The o i l (0.222 g) obtained a f t e r the solvent was removed was chromatographed on alumina (20 g). Examination o f the f r a c t i o n s showed that the reaction product was a complex mixture. The major component, eluted with benzene-chloroform 1:1, was further p u r i f i e d using preparative t i c ( s i l i c a g e l , ethyl acetate-ethanol 2:1) to a f f o r d the pure compound, 80 (0.045 g). C r y s t a l l i z a t i o n from methanol gave c o l o r l e s s prisms: mp 205-208°; X m a x (log e): 210 (4.44), 253 (3.85), 280 (3.21); v ( n u j o l ) : 3122 (NH), in 3.X 1721 (C=0), 1695 (N-C=0) cm"1; nmr (60 Mc/s): T 1.00 ( s i n g l e t , IH, NH), 2.94 (multiplet, 4H, aromatic), 7.30 ( s i n g l e t , 3H, C-CH 3). Anal. Calcd. f o r C 1 7 H 2 0 N 2 O 2 : C, 71.80; H, 7.09, Found: C, 71.88; H, 7.51. Formation of ethylene ket a l (82) A s o l u t i o n of ketone (78, 1.5 g) ethylene g l y c o l (6 ml), p-toluene-s u l f o n i c acid (1.2 g) and benzene (40 ml) was refluxed f o r 4 hr under an atmosphere of nitrogen. The r e s u l t i n g orange s o l u t i o n was cooled to 0° and slowly poured into 5% aqueous sodium hydroxide (50 ml) s t i l l maintaining a s o l u t i o n temperature o f 0°. The benzene layer was separated and the aqueous phase extracted with a d d i t i o n a l amounts of benzene (3 x 15 ml). The combined benzene extract was dried over sodium s u l f a t e and evaporated to y i e l d an orange residue (1.88 g) which was chromatographed on alumina 117 (100 g). E l u t i o n o f the column with benzene afforded the compound, 82 (1.272 g) which could not be induced to c r y s t a l l i z e : X : 222, 279, v ' max ' ' 290 my; v ( C H C 1 3 ) : 3330 cm'1 (N-H); nmr: T 0.28 ( s i n g l e t , IH, NH), 2.77 (multiplet, 4H, aromatic), 6.07 (multiplet, 4H, ethylene k e t a l ) , 8.62 ( s i n g l e t , 3H, C-CH 3); mass spectrum ( i n t e n s i t y ) : m/e 312 ( 1 0 0 ) , 311 ( 1 0 0 ) . Further e l u t i o n o f the column with chloroform-benzene 1:4 afforded another epimer o f 82 (0.256 g) which was c r y s t a l l i z e d from methanol: mp 187-188°; X (log e): 222 (4.57), 281 (3.91), 290 (3.82) my; v in 3.X TTlclX (CHCI3): 3350 cm"1 (NH); nmr: T -0.03 ( s i n g l e t , IH, NH), 2.75 (multiplet, 4H, aromatic), 5.96 (multiplet, 4H, ethylene k e t a l ) , 8.52 ( s i n g l e t , 3H, C-CH 3). Anal. Calcd. f o r CigH 2 i»N 202: C, 73.04; H, 7.74; N, 8.97. Found: C, 73.17; H, 7.83; N, 8.78. Chloroindolenine (83) The chloroindolenine (93) of the t e t r a c y c l i c ethylene ketal (82, 0.09 g) was formed i n the manner previously described to a f f o r d a yellow o i l : * m a x : 2 5 3 > 292 (sh) my. The crude product was used d i r e c t l y for the next r e a c t i o n . Oxindole (84) A s o l u t i o n of the crude chloroindolenine (83) and potassium hydroxide (0.08 g) in methanol (4 ml) was refluxed for 2 hr under an atmosphere of nitrogen. A h a l f of the solvent was evaporated o f f and the mixture was d i s t r i b u t e d between water" and methylene c h l o r i d e . The organic layer was separated and d r i e d over sodium s u l f a t e . A f t e r removal of the solvent, the o i l was p u r i f i e d using preparative t i c (ethyl acetate-ethanol 2:1) to a f f o r d the oxindole, 84, as an o i l (0.042 g) : v ( C H C 1 3 ) : 3100 (NH), 118 1700 (C=0) cm"1; nmr (60 Mc/s): T 2.72 (multiplet, 4H, aromatic), 6.10 (m u l t i p l e t , 4H, ethylene k e t a l ) , 8.64 ( s i n g l e t , 3H, C-CH 3). T e t r a c y c l i c alcohol (85) 1 9 A s o l u t i o n o f sodium borohydride (0.050 g, 1.3 mmole) i n absolute methanol (10 ml) was added slowly to a s t i r r e d s o l u t i o n o f 78 (0.20 g, 0.7 mmole) i n absolute methanol (10 ml). A f t e r s t i r r i n g for 2 hr at room temperature under an atmosphere of nitrogen, the mixture was d i l u t e d with water (5 ml) and the methanol removed under reduced pressure. The remaining aqueous s o l u t i o n was a c i d i f i e d with 2N hydrochloric acid u n t i l a c i d to litmus and then made basic with potassium carbonate s o l u t i o n . The aqueous s o l u t i o n was extracted with methylene chl o r i d e (3 x 10 ml). The combined extracts were drie d over sodium s u l f a t e and evaporated under reduced pressure to a f f o r d the a l c o h o l , 85, as a white s o l i d (0.204 g). This s o l i d was c r y s t a l l i z e d from methanol: mp 228-230°; * m a x (log e ) : 224 (4.53), 282 (3.87), 290 (3.80) my; v j ^ : 3130, 3030 cm"1; nmr: T 0.20 ( s i n g l e t , IH, NH), 2.78 (multiplet, 4H, aromatic), 8.74 (doublet, J = 6 cps, 3H, -CHOHCH3); high r e s o l u t i o n mass spectrum: Calcd. f o r C17H22N2O: M.W. 270.173. Found: 270.172. Chloroindolenine (86) of t e t r a c y c l i c alcohol (85) t-Butyl hypochlorite (0.124 g, 1.1 mmole) i n carbon t e t r a c h l o r i d e (23 ml) was added dropwise over 30 min to a s t i r r e d s o l u t i o n of 85 (0.28 g, 1.04 mmole) i n anhydrous methylene chloride (80 ml) containing one drop triethylamine, cooled i n an i c e - s a l t mixture. A f t e r the addition was completed, s t i r r i n g was continued f o r 30 min. The reaction mixture was washed with water (20 ml), dried over sodium s u l f a t e and evaporated to give the crude chloro d e r i v a t i v e , 86, as a yellow viscous o i l : A_ : 253, 290 (sh) my. The crude product was used d i r e c t l y f o r the next reaction because i n i t i a l attempts at p u r i f i c a t i o n resulted i n decomposition. 119 Imino ether (87) A s o l u t i o n o f the above crude chloroindolenine, 86, and potassium hydroxide (0.078 g) i n methanol (19 ml) was refluxed f o r 2 hr under an atmosphere o f nitrogen. A h a l f o f the solvent was evaporated o f f and the mixture d i s t r i b u t e d between water and methylene c h l o r i d e . The organic layer was separated and dri e d over sodium s u l f a t e . The o i l (0.321 g) obtained a f t e r removal o f the solvent was chromatographed on alumina (30 g). The major component was eluted with benzene-chloroform 4:1 and further p u r i f i e d using preparative t i c ( s i l i c a g e l , ethyl acetate-ethanol 2:1) to a f f o r d the pure imino ether, 87 (0.099 g). C r y s t a l l i z a t i o n from ether gave f i n e needles: mp 140-143°; X (log e): 213 (4.38), in six 253-258 (3.72) my; v K B r 3300 (OH), 1575 (N=C-0) cm"1; nmr: T 2.82 max (multiplet, 4H, aromatic), 5.94 ( s i n g l e t , 3H, OCH3), 9.29 (doublet, J = 6 cps, 3H, -CHOHCH3); mass spectrum ( i n t e n s i t y ) : m/e 300 (100), 282'. (7), 255 (9), 141 (41), 96 (68); high r e s o l u t i o n mass spectrum: Calcd. f o r C18H2>4N202: M.W. 300.184. Found: 300.186. Anal. Calcd. f o r C 1 8H 2 i 4 N 202: C, 71.97; H, 8.05. Found: C, 71.97; H, 8.05. Adduct (89) A mixture of 87 (0.30 g), methyl cyanoacetate (2 ml) and t r i e t h y l -amine (0.1 ml), i n a sealed tube was heated at 65° for 100 hr. The excess methyl cyanoacetate was d i s t i l l e d o f f using a bath temperature of 60° and a pressure of 0.01 mm. The red residue was chromatographed on alumina (25 g) and the major component (0.172 g) was eluted with petroleum ether-benzene 3:1. C r y s t a l l i z a t i o n from hexane gave the adduct, 89, as flakes: mp 185-188°; X (log e ) : 234 (4.12), 334 (4.32);and X . (log e ) : 258 max min ( 2 . 9 7 ) ; v K B r : 3190 (NH), 2190 (CN), 1680 (COOCH3); nmr: x -1.03 ( s i n g l e t , 120 IH, NH), 2.78 (multiplet, 4H, aromatic), 6.19 ( s i n g l e t , 3H, C00CH 3); mass spectrum ( i n t e n s i t y ) : m/e 323 (30), 97 (100); high r e s o l u t i o n mass spectrum: Calcd. for C 1 9 H 2 i N 3 0 2 : M.W. 323.163. Found: 323.163. Anal. Calcd. f o r C 1 9 H 2 i N 3 0 2 : C, 70.56; H, 6.55; 0, 9.90. Found: C, 70.85; H, 6.81; 0, 9.78. Wolff-Kishner reduction of t e t r a c y c l i c ketone (78) A s o l u t i o n of ketone (78, 1.0 g), 85% hydrazine (20 ml), and potassium hydroxide (3.0 g) i n diethylene g l y c o l (65 ml) was refluxed f o r 1.5 hr (bath temperature 165°) under nitrogen. The condenser was removed and the bath temperature was r a i s e d to 215° while water and excess hydrazine were removed by passing nitrogen through the reaction s o l u t i o n . Heating at 215° was continued f o r 5 hr. A f t e r c o o l i n g , the reaction product was d i l u t e d with water (10 ml) and extracted with methylene chloride (3 x 25 ml). The combined organic extracts were d r i e d over sodium s u l f a t e and evaporated to af f o r d a yellow gum (1.5 g) which was chromatographed on alumina (110 g). E l u t i o n o f the column with benzene afforded the pure t e t r a c y c l i c indole, 93 (0.73 g) which was c r y s t a l l i z e d from methanol: mp 65-95° dec; A (log e): 221 (4.54), 273 (3.87), 2.78 (3.86), 289 (3.76) my; v m a x (CHC1 3): 3450 cm"1 (NH); nmr: T 1.99 ( s i n g l e t , IH, NH), 2.75 (multiplet, 4H, aromatic), 9.02 ( t r i p l e t , 3 = 1 cps, 3H, CH 2-CH 3) ( L i t 6 7 mp 60-100°). Anal. Calcd. f o r C 1 7H 22N 2: C, 80.27; H, 8.72. Found: C, 79.90; H, 9.00. Chloroindolenine (94) t-Butyl hypochlorite (0.05 M i n carbon t e t r a c h l o r i d e , 10 ml) was added dropwise over 30 min to a s t i r r e d s o l u t i o n of 93 (0.114 g) i n anhydrous methylene ch l o r i d e (10 ml) containing one drop of triethylamine cooled i n an i c e - s a l t mixture. A f t e r the addition was completed, s t i r r i n g was 121 continued f o r 30 min. The reaction mixture was washed with water (10 ml), dri e d over sodium s u l f a t e and evaporated to give the crude chloro d e r i v a t i v e as a yellow viscous o i l (0.140 g). P u r i f i c a t i o n by preparative t i c ( s i l i c a g e l , ethyl acetate-ethanol 2:1) afforded a pure sample (0.097 g) of the chloro d e r i v a t i v e , 94. Concentration of a methylene chloride s o l u t i o n of the chloro d e r i v a t i v e afforded a yellow amorphous powder: mp 95-100°; v m a x (CHC1 3): 1588 cm"1 (-N=C-); X m a x (log e): 225 (4.30), 266 (3.29), 293 (3.21) my; nmr: x 2.62 (multiplet, 4H, aromatic), 9.06 ( t r i p l e t , 3H, C H 2 C H 3 ) ; mass spectrum ( i n t e n s i t y ) : m/e 290 (2), 288 (5), 253 (100). Lithium aluminum hydride reduction of chloroindolenine (94) A s o l u t i o n of chloro d e r i v a t i v e (94, 0.005 g) i n ether (1 ml) was treated with excess l i t h i u m aluminum hydride. A f t e r 10 min at room tempera-ture, water was added and the mixture extracted with ether ( 3 x 1 ml). The ether extracts were combined, dri e d over sodium s u l f a t e and concentrated to dryness. Examination by t i c , u l t r a v i o l e t and i n f r a r e d spectrum of the product showed that i t was i d e n t i c a l to the t e t r a c y c l i c indole (93). Imino ether (95) A small chip o f sodium was d i s s o l v e d i n methanol (10 ml) and the s o l u t i o n added to a s o l u t i o n o f the chloroindolenine (94, 0.31 g) i n methanol (10 ml). A f t e r r e f l u x i n g f o r 11 hr under an atmosphere of nitrogen two-thirds of the solvent was evaporated o f f and the mixture d i s t r i b u t e d between water and methylene c h l o r i d e . The organic layer was separated and d r i e d over sodium s u l f a t e . The o i l obtained a f t e r evaporation of the solvent was chromatographed on alumina (15 g). The desired imino ether was eluted with petroleum ether-benzene 2:1 and was subjected to further p u r i f i c a t i o n using preparative t i c on s i l i c a gel (ethyl acetate-ethanol 2:1) to a f f o r d a mixture of two imino ethers, 95 (0.032 g), i n a r a t i o of 4:1 as determined by nmr. This mixture was inseparable using a v a r i e t y 122 of t i c adsorbents and solvents and as a r e s u l t the s p e c t r a l c h a r a c t e r i s t i c s were determined on the mixture: v ( C H C I 3 ) : 1568 cm"1 (-N=C-0); X : max max 215, 254-258, 285 (sh), 292 (sh) my; nmr: T 2.80 (multiplet, 4H, aromatic), 5.92 and 5.97 ( s i n g l e t s , 3H, -0CH 3), 9.50 ( t r i p l e t , 3H, -CH 2CH 3); mass spectrum ( i n t e n s i t y ) : m/e 284 (21), 125 (59), 96 (100). Anal. Calcd. f o r C 1 8H2itN 20: M.W. 284.189. Found: 284.184 (mass spectrometry). Adduct (96) A s o l u t i o n o f imino ether (95, 0.050 g), methyl cyanoacetate (0.5 ml) and triethylamine (0.1 ml) was heated at 76° f o r 120 hr i n a sealed tube. The excess methyl cyanoacetate was d i s t i l l e d o f f using a bath temperature of 80° and a pressure of 0.7 mm. The red residue was chromatographed on alumina (7 g) and the desired adduct was eluted with petroleum ether-benzene 1:1 and further p u r i f i c a t i o n on preparative t i c ( s i l i c a g e l , ethyl acetate-ethanol 2:1) afforded the pure adduct, 96 (0.009 g). C r y s t a l l i z a t i o n from methanol afforded c o l o r l e s s blocks: mp 165-168°; v . (CHC1 3): 3300 (NH), r max 2210 (CN), 1666 (COOCH3) cm"1; X (log e): 236 (4.08), 295 (3.79), 335 (4.30); X m i n (log e): 258 (2.91); nmr: x -1.08 ( s i n g l e t , IH, NH), 2.72 (m u l t i p l e t , 4H, aromatic), 6.18 ( s i n g l e t , 3H, COOCH3), 9.53 ( t r i p l e t , 3H, -CH 2CH 3); mass spectrum ( i n t e n s i t y ) : m/e 351 (13), 125 (50), 124 (45), 96 (100). Anal. Calcd. f o r C 2 1 H 2 5 N 3 O 2 : M.W. 351.195. Found: 351.195 (mass spectrometry). Adduct 96 was also prepared by the following procedure. A mixture of chloroindolenine (94, 0.110 g) methyl cyanoacetate (1 ml) and triethylamine (0.1 ml) i n a sealed tube was heated at 76° f o r 144 hr. The excess methyl cyanoacetate was d i s t i l l e d o f f using a bath temperature of 65° and a pressure of 0.1 mm. The red residue was chromatographed on 123 alumina (5 g) and the desired adduct was eluted with petroleum ether-benzene 1:1 followed by further p u r i f i c a t i o n on preparative t i c ( s i l i c a g e l , ethanol-ethyl acetate 1:2) afforded the pure adduct, 96 (0.008 g). Crys-t a l l i z a t i o n from methanol afforded c o l o r l e s s blocks, mp 165-168°. This adduct was i d e n t i c a l i n a l l respects (mixed mp, t i c , i r , uv, nmr) to that obtained from the reaction of the imino ether, 95, with methyl cyanoacetate. Ex t r a c t i o n of a l k a l o i d s from Vinca minor Linn The following procedure was developed i n order to extract and p u r i f y the a l k a l o i d s o f Vinca minor Linn plants. This procedure was used f o r a l l extractions o f V. minor L. plants and was scaled according to the wet weight o f plants used. Vinca minor Linn plants (9 kg, wet weight) obtained from the gardens of the U n i v e r s i t y of B r i t i s h Columbia were mascerated with methanol i n a Waring Blender, f i l t e r e d and re-mascerated u n t i l the f i l t r a t e was c o l o r l e s s . This green f i l t r a t e (8,000 ml) was concentrated to dryness under reduced pressure and the residue dissolved i n 2N hydrochloric a c i d (4,500 ml). The acid layer was extracted with benzene (2 x 2,000 ml) and the benzene extracts were back extracted with 2N hydrochloric a c i d (2 x 500 ml). The combined aqueous phases were made basic with 15N ammonium hydroxide,taking care that the temperature of the s o l u t i o n d i d not r i s e above 25°,and extracted with chloroform (3 x 2,400 ml). The combined chloroform extracts were washed with water (2,000 ml), dried over sodium s u l f a t e , and concentrated under reduced pressure. The r e s u l t i n g a l k a l o i d residue (13.6 g) was dissolved i n benzene-methylene ch l o r i d e 1:1 (100 ml) and chromatographed on alumina (700 g). The column was eluted successively with petroleum ether 30/60°, benzene, chloroform, and methanol; f r a c t i o n s of 700 ml were taken. The fr a c t i o n s eluted with petroleum ether-benzene 2:1 were combined and subjected 124 to a d d i t i o n a l column chromatography on alumina and f i n a l p u r i f i c a t i o n using preparative t i c on s i l i c a gel (ethyl acetate-chloroform 3:7) to afford quebrachamine (97, 0.002 g), vincadine (98, 0.005 g) and vincaminoreine (99, 0.035 g). Continued e l u t i o n of the column with petroleum ether-benzene 1:1 afforded vincadifformine (102, 0.010 g) and further e l u t i o n with petroleum ether-benzene 1:4 afforded minovine (103, 0.41 g). The i n i t i a l f r a c t i o n s eluted with benzene contained 1,2-dehydroaspidospermidine (101; 1,2-double bond, approx. 0.002 g), but no attempt was made to f i n a l l y p u r i f y t h i s compound. The l a t e r f r a c t i o n s eluted with benzene were combined and c r y s t a l l i z e d from methanol affo r d i n g vincamine (29, 0.65 g).' A l l the 62 above a l k a l o i d s were compared on t i c with authentic samples and i n 68 a d d i t i o n s p e c t r a l comparison was made with l i t e r a t u r e values Ex t r a c t i o n o f a l k a l o i d s from Vinca rosea Linn (Catharanthus roseus G. Don) The following procedure was developed i n order to extract and p u r i f y the a l k a l o i d s o f Vinca rosea Linn p l a n t s . This procedure was used for a l l extractions of V. rosea L. plants and was scaled according to the wet weight of plants used. Vinca rosea Linn plants were obtained from the greenhouse at the University 69 of B r i t i s h Columbia . The plants (300 g, wet weight) were mascerated with methanol i n a Waring Blender, f i l t e r e d and re-mascerated u n t i l the f i l t r a t e was c o l o r l e s s . This green f i l t r a t e (combined volume was 3,000 ml) was evaporated to dryness, the residue taken up i n 2N hydrochloric acid (2,000 ml) and washed with benzene (3 x 1,000 ml). The combined benzene extracts were back extracted with 2N hydrochloric acid (2 x 500 ml). The combined aqueous phases were made basic with 15N ammonium hydroxide and extracted with chloroform (3 x 1,000 ml). The combined chloroform extracts were washed with water (1,000 ml), dried over sodium s u l f a t e and evaporated to give a 125 brown o i l (1.003 g). The o i l was dissolved i n benzene-methylene chloride 1:1 (3 ml) and chromatographed on alumina (100 g). The column was .eluted successively with petroleum ether 30/60°, benzene, chloroform and methanol; fr a c t i o n s o f 100 ml were taken. The l a t e r benzene-petroleum ether 1:1 fr a c t i o n s were combined and c r y s t a l l i z e d from methanol affo r d i n g catharanthine ( 9 , 0 . 0 4 4 g), the benzene f r a c t i o n s were combined and c r y s t a l l i z e d from methanol affording ajmalicine (10, 0.025 g), and the i n i t i a l benzene-chloroform 4:1 fr a c t i o n s were combined and c r y s t a l l i z e d from ether giving vindoline (7, 0.029 g). When required, the hydrochloride s a l t o f catharanthine and vindoline was formed by blowing hydrogen c h l o r i d e gas on the surface o f an ethereal s o l u t i o n o f the a l k a l o i d ; and catharanthine hydrochloride was c r y s t a l l i z e d from methanol, whereas vind o l i n e hydrochloride was c r y s t a l l i z e d from acetone. The hydrochloride s a l t o f ajmalicine was prepared by adding concentrated hydrochloric acid (10 drops) to a concentrated methanolic s o l u t i o n of the a l k a l o i d and c r y s t a l l i z a t i o n of ajmalicine hydrochloride from methanol. 18a-Carbomethoxy(01'tCH3)-4a-dihydrocleavamine (105) and 186-carbomethoxy-(01 **CH3) -4a-dihydrocleavamine (105) A s o l u t i o n of 18a-carbomethoxy-4a-dihydrocleaVamine (105, 0.008054 g), benzene (1 ml), boron t r i f l u o r i d e etherate (0.2 ml) and methanol- 1 MC (0.2 ml, 3.875 x 107 dpm) was refluxed f o r 12 hr and then s t i r r e d at room temperature f o r a further 12 hr. A f t e r pouring i n t o saturated aqueous sodium bicarbonate, the aqueous s o l u t i o n was extracted with methylene chloride (3 x 10 ml). A f t e r drying over sodium s u l f a t e , the organic extract was concentrated to dryness under reduced pressure to a f f o r d an o i l (0.0091 g). This o i l was p u r i f i e d using preparative t i c ( s i l i c a g e l , chloroform) to af f o r d 18B-carbomethoxy(0 1 4CH 3)-4a-dihydrocleavamine (0.005 g, 3.55 x 106 dpm) and 18a-carbomethoxy(0 l uCH 3)-4a-dihydrocleavamine (0.002 g, 1.24 x 106 dpm) 126 as o i l s . Inactive 18B-carbomethoxy-4a-dihydrocleavamine (0,0206 g) was added to the 18B-carbomethoxy(0 1 4CH 3)-4a-dihydrocleavamine and the hydro-chlo r i d e s a l t was formed by blowing gaseous hydrogen chloride upon an ethereal s o l u t i o n and c r y s t a l s (0.009 g, 4.63 x 10 7 dpm/mmole) were obtained from acetone. Inactive 18a-carbomethoxy-4a-dihydrocleavamine (0.0090 g) was added to the 18a-carbomethoxy(0 1 4CH 3)-4a-dihydrocleavamine and the hydrochloride s a l t was formed as above and c r y s t a l s (0.0052 g, 4.62 x 10 7 dpm/mmole) were obtained from acetone. T r i f l u r o a c e t i c a cid- 3H T r i f l u r o a c e t i c anhydride (1.17 g, 5.55 mmole) was added to water- 3H (0.10 g, 5.55 mmole, 100 mcurie/g) using a vacuum t r a n s f e r system. The r e s u l t i n g t r i f l u r o a c e t i c acid- 3H (1.27 g, 0.9 mc/mmole) was stored under an atmosphere of nitrogen at -10° u n t i l required. T r i t i u m l a b e l l e d r a d i o a c t i v e a l k a l o i d s f o r biosynthesis studies The following procedure i s t y p i c a l f o r the formation of a l l the radio-a c t i v e precursors u t i l i z i n g t r i t i u m i n the aromatic portion of the a l k a l o i d molecule. T r i f l u r o a c e t i c acid- 3H (0.5 g, 0.9mc/mmole) was added to tryptamine (41) hydrochloride (0.048 g) using a vacuum t r a n s f e r system. The so l u t i o n was allowed to stand under an atmosphere of nitrogen at room temperature for 24 hr. The t r i f l u r o a c e t i c acid- 3H was removed using a vacuum transf e r system. Concentrated ammonium hydroxide s o l u t i o n (10 ml) was c a r e f u l l y added to the residue and the organic components extracted with d i c h l o r o -methane (10 x 15 ml). The organic extract was washed with water (10 ml), dried over sodium s u l f a t e , and concentrated to dryness under reduced pressure to af f o r d an o i l (0.034 g). This o i l was subjected to column chromatography on alumina (1.0 g) and [ar- 3H]-tryptamine (0.010 g, 6.16 x 10 7 dpm) was eluted with chloroform. Various t i c systems were used 127 i n order to confirm that a pure material was recovered. Feeding experiment no. 1 [ar- 3H]-Carbomethoxycleavamine (106) hydrochloride (0.009078 g, 2.28 x 10 8 dpm/mmole) was dissol v e d i n d i s t i l l e d water and the s o l u t i o n administered to V. rosea L. p l a n t s (wet weight 35 g) by the cotton wick method. A f t e r 8 days under intermittent fluorescent lamp i l l u m i n a t i o n , the plants were extracted to a f f o r d the al k a l o i d s as a brown gum (0.084 g). The a l k a l o i d extract was p u r i f i e d to a f f o r d catharanthine (9) as an o i l . Inactive catharanthine (0.050 g) was added and the catharanthine was c r y s t a l l i z e d to constant a c t i v i t y . The t o t a l a c t i v i t y i n the catharanthine was 800 dpm representing a maximum incorporation o f 0.015%. Another p o r t i o n of the above [ar- 3H]carbomethoxycleavamine hydrochloride was diss o l v e d i n water and allowed to remain i n the open test-tube f o r 8 days. A f t e r made b a s i c , the aqueous s o l u t i o n was extracted with dich l o r o -methane (3 x 10 ml). The combined organic extracts were d r i e d (sodium s u l f a t e ) , evaporated and was p u r i f i e d as above to a f f o r d r a d i o a c t i v e catharanthine (9). The conversion o f carbomethoxycleavamine to catharanthine i n the test-tube was 0.04%. Feeding experiment no. 2 [ar- 3H]-Carbomethoxycleavamine (106) hydrochloride (0.00652 g, 2.31 x I 0 8 dpm/mmole) was dissolved i n d i s t i l l e d water (10 ml) and the so l u t i o n administered to V. rosea L. l e a f discs (15 g) by vacuum i n f i l -t r a t i o n . A f t e r 42 hr under fluorescent lamp i l l u m i n a t i o n , the l e a f discs were extracted and the catharanthine (9) c r y s t a l l i z e d to constant a c t i v i t y as in feeding experiment no. 1. The t o t a l a c t i v i t y i n the catharanthine was 350 dpm representing a maximum incorporation of 0.008%. Feeding experiment no. 3 [ar- 3H]-Carbomethoxycleavamine (106) hydrochloride (0.00207 g, 128 2.31 x 10 8 dpm/mmole) was dissolv e d i n 0.1N a c e t i c acid (0.4 ml) and the s o l u t i o n adminstered to growing V. rosea L. plants (wet weight 139 g) by p l a c i n g f i n e glass c a p i l l a r i e s containing the so l u t i o n into the l e a f veins of the pla n t s . The s o l u t i o n was absorbed into the l e a f veins by t h i s method. A f t e r 6 days under intermittent fluorescent lamp i l l u m i n a t i o n , the plants were extracted and the catharanthine (9) c r y s t a l l i z e d to constant a c t i v i t y as i n feeding experiment no. 1. The t o t a l a c t i v i t y i n the catha-ranthine was 630 dpm or a maximum incorporation o f 0.05%. Feeding experiment no. 5 18B-Carbomethoxy(0 1 HCH3)-4a-dihydrocleavamine (105) hydrochloride (0.00278 g,4.63 x 10 7 dpm/mmole) was dissolved i n d i s t i l l e d water (10 ml) and d i s t r i b u t e d equally among 10 test-tubes. V. rosea L. cuttings (91 g) were inse r t e d i n t o these test-tubes and placed under intermittent fluorescent lamp i l l u m i n a t i o n with the aqueous l e v e l i n the test-tubes maintained with d i s t i l l e d water. A f t e r 7 days, the cuttings were extracted to a f f o r d the al k a l o i d s as a brown gum (0.187 g, 1.37 x 10 5 dpm). The a l k a l o i d extract was p u r i f i e d by preparative t i c (alumina, benzene-chloroform 3:1, and then s i l i c a g e l , ethyl acetate-ethanol 2:1) to a f f o r d coronaridine (107) as an o i l (0.001 g, 50 dpm) representing a maximum incorporation o f 0.015%. Feeding experiment no. 6 18a-Carbomethoxy(0 1 4CH 3)-4a-dihYdrocleavamine (105) hydrochloride (0.00356 g, 4.62 x 10 7 dpm/mmole) was administered to V. rosea L. cuttings as i n feeding experiment no. 5, except the time f o r incorporation was 5 days. A f t e r extraction and p u r i f i c a t i o n , the coronaridine (107) did not contain any r a d i o a c t i v i t y . Feeding experiment no. 12 [ar- 3H]-Tryptamine (41, 0.010 g, 6.16 x 10 7 dpm) was dissolved i n 0.1N a c e t i c a c i d (15 ml) and administered to growing 4-6 month old 129 V. rosea L. plants (wet weight 300 g). The administration was by the cotton wick method, where a wick of cotton s t r i n g was passed through the plant stem about 1 inch above the ground and the ends o f the s t r i n g placed i n a small test-tube near the plant. The test-tubes were f i l l e d with the s o l u t i o n o f [ar- 3H]-tryptamine and a f t e r t h i s s o l u t i o n was absorbed into the p l a n t , further volumes of 0.1N a c e t i c a c i d were added to the t e s t -tubes i n order to wash a l l the [ar- 3H]-tryptamine i n t o the plant. The plants were placed under intermittent fluorescent lamp i l l u m i n a t i o n and watered as required. A f t e r 9 days, the a l k a l o i d s (1.003 g, 1.2 x 10 7 dpm) were i s o l a t e d and p u r i f i e d by chromatography to a f f o r d the pure a l k a l o i d s : catharanthine (9, 0.044 g), ajmalicine (10, 0.025 g), and vindoline (7, 0.029 g). A f t e r c r y s t a l l i z a t i o n o f the free base and then i t s hydrochloride s a l t to constant a c t i v i t y , the following incorporations were obtained: catharanthine (0.01%), ajmalicine (0.4%), and vindoline (0.003%). Feeding experiments no. 11-16, 23, 24, and 26 These feeding experiments were conducted i n a manner s i m i l a r to feeding experiment no. 12. The incorporations into catharanthine (9), ajmalicine (10), and vindoline (7) were obtained by c r y s t a l l i z a t i o n o f the free base and then i t s hydrochloride s a l t to constant r a d i o a c t i v i t y and these r e s u l t s are summarized i n Tables II and IV. Feeding experiment no. 17 [ar- 3H]-Minovine (103, 0.0142 g, 6,25 x 10 6 dpm) was dissolved i n 0.1N a c e t i c a c i d (10 ml) and methanol (0.5 ml) and d i s t r i b u t e d equally among 10 test-tubes. V. minor L. cuttings (45 g) were inserted into these test-tubes and placed under intermittent fluroescent lamp i l l u m i n a t i o n with the aqueous l e v e l i n the test-tubes maintained with d i s t i l l e d water. A f t e r 7 days, the cuttings were extracted to afford the al k a l o i d s as a brown gum (0.094 g, 7.7 x 10 5 dpm). A f t e r p u r i f i c a t i o n , the vincaminoreine 130 (99) was i s o l a t e d as an o i l (0.009 g, 21.8 dpm) which represented a maximum incorporation of 0.0003%. Feeding experiment no. 18 The epimeric mixture of [ar- 3H]-52 hydrochloride (0.0025 g, 1.35 x 10 6 dpm) was dissolved i n d i s t i l l e d water (10 ml) and d i s t r i b u t e d equally among 10 test-tubes. V. minor L. cuttings (46 g) were inserted i n t o these test-tubes and placed under intermittent fluorescent lamp i l l u m i n a t i o n with the aqueous l e v e l i n the test-tubes maintained with d i s t i l l e d water. A f t e r 5 days, the cuttings were extracted to af f o r d the al k a l o i d s as a brown gum (0.155 g, 8.7 x IO 4 dpm). A f t e r p u r i f i c a t i o n , the vincamine (29, 0.05 g) was obtained c r y s t a l l i n e and did not contain any r a d i o a c t i v i t y . The fr a c t i o n s containing quebrachamine (97), vincadine (95), vincaminoreine (99) and minovine (103) also showed a lack of any r a d i o a c t i v i t y . Feeding experiment no. 19 The epimeric mixture of [ar- 3H]-53 hydrochloride (0.00218 g, 2.4 x 10 7 dpm) was dissolved i n d i s t i l l e d water (10 ml) and d i s t r i b u t e d equally among 10 test-tubes. V. minor L. cuttings (41 g) were inserted into these test-tubes and placed under intermittent fluorescent lamp i l l u m i -nation with the aqueous l e v e l i n the test-tubes maintained with d i s t i l l e d water. A f t e r 7 days, the cuttings were extracted and the al k a l o i d s obtained as an o i l (0.171 g, 5.6 x 10 6 dpm). The alka l o i d s quebrachamine (97), vincadine (8), vincaminoreine (99), minovine (103) and vincamine (29) were p u r i f i e d and did not contain any r a d i o a c t i v i t y . Feeding experiment no. 20 The epimeric mixture o f [ar- 3H]-53 hydrochloride (0.00187 g, 2.06 x 10 7 dpm) was dissolved i n ethyl alcohol (3 ml) containing 10% dimethyl sulf o x i d e and painted on the leaves of growing V. rosea L. 131 plants (10.8 g). The plants were placed under intermittent fluorescent lamp i l l u m i n a t i o n and the s o i l watered as necessary. A f t e r 5 days, the plants were extracted to af f o r d the al k a l o i d s as a brown gum (0.034 g, 5.8 x 10 6 dpm) from which catharanthine (9, 0.004 g) was i s o l a t e d . Inactive catharanthine (0.04791 g) was added and a f t e r c r y s t a l l i z a t i o n of the free base and then i t s hydrochloride s a l t , the catharanthine did not contain any r a d i o a c t i v i t y . Feeding experiment no. 21 The epimeric mixture o f [ar- 3H]-53 (0.009 g, 7.5 x 10 7 dpm) was dissolv e d i n ethanol (5 ml) containing 10% dimethyl sulfoxide and painted on the leaves o f growing V. rosea L. plants (385 g). The plants were placed under intermittent fluorescent lamp i l l u m i n a t i o n and the s o i l watered as necessary. A f t e r 6 days, the plants were extracted to a f f o r d the a l k a l o i d s as a brown gum (1.223 g, 3.56 x 10 7 dpm). P u r i f i c a t i o n provided the following a l k a l o i d s : catharanthine (9, 0.044 g), ajmalicine (10, 0.019 g) to which i n a c t i v e ajmalicine (0.0498 g) was added, and vindoline (7, 0.022 g) to which i n a c t i v e vindoline (0.0724 g) was added. A f t e r c r y s t a l l i z a t i o n o f the free base and then i t s hydrochloride s a l t , the above a l k a l o i d s were obtained without any r a d i o a c t i v i t y . Feeding experiment no. 22 DL-Tryptophan-[3- l l +C] (1, 0.0121 g, 5.38 x 10 8 dpm) was dissolved i n ethanol (5 ml) containing 10% dimethyl sulfoxide and painted on the leaves of growing V. rosea L. plants (320 g). The plants were placed under i n t e r -mittent fluorescent lamp i l l u m i n a t i o n and the s o i l watered as necessary. A f t e r 9 days, the plants were extracted to af f o r d the alka l o i d s which were subsequently p u r i f i e d . A f t e r c r y s t a l l i z a t i o n of the free base and then i t s hydrochloride s a l t to constant a c t i v i t y , the following incorporations were obtained: catharanthine (9, 0.009%), ajmalicine (10, 0.003%) and 132 i v i n d o l i n e (7, 0.005%). Feeding experiment no. 25 [ar- 3H]-Adduct (96, 0.014 g, 1.1 x 10 6 dpm) was dissolved i n 0.1N a c e t i c ac i d (3 ml) and methanol (0.2 ml) and d i s t r i b u t e d equally among 9 test-tubes. V. minor L. cuttings (27.4 g) were inserted into these t e s t -tubes and placed under intermittent fluorescent lamp i l l u m i n a t i o n with the aqueous l e v e l i n the test-tubes maintained with d i s t i l l e d water. A f t e r 2 days, the cuttings were extracted to a f f o r d the a l k a l o i d s as a brown gum (0.114 g, 6 x 10** dpm). A f t e r chromatography, the i s o l a t e d minovine (103, 0.003 g) was d i l u t e d with i n a c t i v e minovine (0.040 g) and repeatedly r e c r y s t a l l i z e d u n t i l the minovine did not contain any r a d i o a c t i v i t y . The i s o l a t e d vincamine (29, 0.010 g) was repeatedly r e c r y s t a l l i z e d u n t i l i t d i d not contain any r a d i o a c t i v i t y . Incorporation o f DL-tryptophan-3- l l +C into Vinca minor L. at various time i n t e r v a l s The following procedure i s t y p i c a l o f the manner i n which the experiments were performed. Nine cuttings of Vinca minor Linn (wet weight 16.4 g) were taken from an outdoor garden and immediately placed i n three separate t e s t tubes (three cuttings per tube) containing tryptophan-3- 1 I +C (0.00184 g, 6.37 x 10 7 dpm) i n 0.1N a c e t i c a c i d (1.0 ml) with a few drops of methanol to cause s o l u t i o n . The cuttings were allowed to take up the s o l u t i o n , the tubes being r e f i l l e d as necessary, and a f t e r the s o l u t i o n had been taken nearly to dryness three times, the tube was f i l l e d with s a l i n e s o l u t i o n and l e f t f o r a period of two days, being i r r a d i a t e d with fluorescent daylight-type l i g h t s f o r 36 out of 48 hr. A f t e r 48 hr, the cuttings were extracted to a f f o r d an o i l (0.0334 g, 133 2.52 x 106 dpm) which was chromatographed on alumina (5.0 g) in the following manner. Fraction Solvent Volume (ml) Weight of fraction (mg) 1 petroleum ether (40-60) 30 1.20 2 petroleum ether-benzene 18:7 25 0.65 3 petroleum ether-benzene 18:7 50 0.35 4 petroleum ether-benzene 50 1.65 5 benzene 50 5.40 6 benzene 50 1.90 7 benzene-chloroform 4:1 50 4.30 8 chloroform 50 6.50 9 methanol 20 2.15 Vincaminoreine (99) and vincadine (98) were present in fraction 2, vincadifformine (102) in fraction 3, and minovine (103) in fraction 4. Appropriate aliquots of these fractions were run on a s i l i c a chromatogram sheet developed in chloroform-ethyl acetate 19:1. The sheet was then passed through a calibrated Nuclear-Chicago Actigraph II Model 1039 t i c counter connected to a recorder (Nuclear-Chicago Model 8416) and integrator (Nuclear-Chicago Model 8704). The incorporation of tryptophan into the various alkaloids was calculated and ratios determined. 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Kutney, W.J. Cretney, P. Le Quesne, B. McKague, and E. P i e r s , J . Amer. Chem. S o c , 88, 4756 (1966). 59. These feeding experiments were performed by Dr. W.J. Cretney. 60. J.P. Kutney, W.J. Cretney, J.R. Hadfield, E.S. H a l l , V.R. Nelson, and D.C. W i g f i e l d , J . Amer. Chem. S o c , 90_, 3566 (1968). 61. J.P. Kutney, K.K. Chan, A. F a i l l i , J.M. Fromson, C. Gletsos, and V.R. Nelson, J . Amer. Chem. S o c , 90, 3891 (1968). 62. J. Mokry and I. Kompis, Lloydia, 27, 428 (1964). 63. J.P. Kutney, C. Ehret, V.R. Nelson, and D.C. Wigfie l d , J . Amer. Chem. S o c , 9£, 5929 (1968). 64. A.A. Qureshi and A.I. Scott, Chem. Comm., 945, 948 (1968). 65. "Mark 1 Liq u i d S c i n t i l l a t i o n Systems Instruction Manual", Nuclear-Chicago Company, p. 18, Section I (1966). 66. S. Sugasawa and M. Kirisawa, Pharm. B u l l . Japan, 3, 190 (1955). 67. E. Wenkert and B. Wickberg, J . Amer. Chem. S o c , 84, 4914 (1962). 68. We are g r a t e f u l to Dr. I. Kompis, I n s t i t u t e of Chemistry, B r a t i s l a v a , Czechoslovakia, for providing us with samples of vincadine, vinca-minoreine, vincadifformine and minovine. 69. Appreciation i s g r a t e f u l l y acknowledged to Mr. P. Salisbury f or advice and f o r the c u l t i v a t i o n o f Vinca rosea L. plants. 70. Appreciation i s g r a t e f u l l y acknowledged to Dr. C. Ehret and Dr. D.C. Wigfield f o r t h e i r assistance with t h i s experiment. 

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